Дисертації з теми "Mycobacterium tuberculosis σ Factors"

Tuberculosis is a major threat to human health. About one third of the world's population is latently infected with Mycobacterium tuberculosis . In these cases the bacillus is in a state of low metabolic activity, making eradication difficult with conventional chemotherapy, which targets actively metabolizing organisms. The mechanisms by which M. tuberculosis reactivates to cause disease are currently unknown but a better understanding could greatly improve the treatment of tuberculosis. Resuscitation-promoting factor is a protein first identified in the supernatant of stationary phase cultures of Micrococcus luteus. It is active in picomolar concentrations, increasing the number of culturable M. luteus cells from dormant populations and shortening the lag phase of growth of small inocula. Bioinformatic searches reveal over 40 examples of rpf-ke genes in the high G-C cohort of Gram-positive bacteria, including M. tuberculosis , which contains five rpf gene orthologues. The work presented here investigated aspects of the M. tuberculosis Rpfs. Improvements in solubility of recombinant mycobacterial (M. tuberculosis and M. smegmatis) Rpfs were achieved by manipulating induction times and temperatures during protein expression and by using new hosts and vectors and producing novel fusion proteins. New assays were devised to measure the biological activity of recombinant Rpfs, using ATP bioluminescence of M. luteus cultures. A phage display library for M. tuberculosis was constructed, in an attempt to identify a protein receptor for Rpf. Rpf expression in human infection was investigated for the first time, using immunocytochemistry. Anti-Rpf antibodies were applied to human tissue sections infected with M. tuberculosis. Rpf was found to be located within epithelioid giant cells and in the immediate vicinity of acid-fast bacilli in necrotic centres. The presence of Rpf in human tuberculosis infection demonstrated in this work suggests that Rpfs may have a role in controlling dormancy of the bacilli in human disease.

Thesis (PhD (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2009.
Dissertation presented for the degree of Doctor of Philosophy at Stellenbosch University.
ENGLISH ABSTRACT: This study describes the molecular epidemiology of Mycobacterium tuberculosis strains with the Beijing genotype. This genotype has received clinical prominence due to its global distribution and the hypothesis that these strains have acquired the ability to evade the protective effect of BCG vaccination, spread more readily, acquire drug resistance and cause severe forms of disease. Molecular biological techniques were used in a series of studies to elucidate the genetic evolutionary mechanisms underlying the success of this genotype in Cape Town, South Africa. Using a collection of 40 different markers it was possible for the first time to construct a phylogenetic history of Beijing genotype strains. This phylogeny was characterized by the consecutive evolution of 7 sublineages. Analysis of epidemiological data in relation to these sublineages showed an association between more recently evolved Beijing strains and an increased ability to transmit and cause disease. From these findings it was hypothesized that the pathogenic characteristics of the Beijing genotype were not conserved but rather that strains representative of the different sublineages had evolved unique properties. In order to determine whether these socalled unique properties were associated with either the host population or the genetic background of strains from sublineage 7, a meta-analysis of published Mycobacterial Interspersed Repetitive-Unit (MIRU) typing data (East Asia) was compared with MIRU typing data from the South African strains in the context of their phylogenetic histories. This study showed that Beijing genotype strains in South Africa originated in East Asia following their introduction during the early 18th century. A significant association was observed between the frequency of occurrence of strains from defined Beijing sublineages and the human population from whom they were cultured (p <0.0001). Based on these findings it was proposed that either the host population (South African) had selected for a particular Beijing sublineage (i.e. sublineage 7) or that strains from that sublineage had adapted to be more successful in the South African population. In a subsequent study, using the methodology developed in the above studies, it was shown that strains from the ancestrally positioned lineage (termed “atypical” Beijing genotype) were over-represented in drug resistant isolates in the Eastern Cape region. This contradicts current dogma which suggests that “atypical” Beijing genotype strains are attenuated in their ability to transmit. However, this phenomenon may be ameliorated in immune-compromised patients as review of the clinical records showed that transmission was associated with HIV co-infection. These findings highlight the need to improve tuberculosis control in vulnerable populations as strains which would normally not contribute significantly to the epidemic now become a cause for concern especially if they are associated with drug resistance. To improve our understanding of the evolution of the Beijing genotype, the genomic stability of an additional 27 polymorphic markers were analysed. These markers have recently been proposed as the new standard in molecular epidemiological studies and were based on MIRU-Variable Number Tandem-Repeats (VNTR) sequences. Superimposition of the MIRU-VNTR data onto the phylogenetic tree showed excellent concordance thereby demonstrating that these alleles were largely stable over time. It is currently not known how the alleles that do change could influence pathogenicity. The results of this study also demonstrated discordance between strains defined by IS6110 DNA fingerprinting and those defined by MIRU-VNTR typing thereby demonstrating that these markers evolve independently and at different rates. Furthermore, the MIRU-VNTR typing method was unable to predict transmission of drug resistant strains which contradict previous reports from low incidence settings. This has significant implications for the use of this typing method in high incidence settings. Using an improved PCR-based method it was possible for the first time, to identify the 5 most prominent phylogenetic lineages in primary cultures of adult tuberculosis patients resident in a high HIV/TB co-infection setting. The results of this study showed that 15% of the study population was infected with two or more strains and Beijing genotype strains were over-represented in these mixed infections. Furthermore, drug susceptibility tests showed that one patient was co-infected with both a drug sensitive and a drug resistant strain. Since mixed infections have been implicated in treatment failure, these findings demonstrate the epidemiological importance of detecting mixed infections in vulnerable populations. This PCR-based method was further applied to cultures of paediatric tuberculosis patients to classify strains which spoligotyping was unable to define. The result of this study showed three mixed infections which otherwise would have been missed. In order to determine whether clinical disease presentation of patients infected with strains of the Beijing genotype were different from that of patients infected with non-Beijing genotype strains, clinical and demographic data of these two groups were analysed. This study showed that patients infected with strains of the Beijing genotype were highly infectious as defined by the increased bacterial load in sputum specimens. However, this finding could not be validated by lung pathology according to chest radiographs of infected patients.
AFRIKAANSE OPSOMMING: Hierdie studie beskryf die molekulêre epidemiologie van Mycobacterium tuberculosis rasse met die Beijing genotipe. Hierdie genotipe is van groot kliniese belang weens hul globale verspreiding en die hipotese dat hierdie rasse die vermoë ontwikkel het om die beskermende effek van BCG vaksinasie te vermy, om meer geredelik te versprei, middelweerstandigheid te ontwikkel en erger vorms van siekte te veroorsaak. Molekulêre biologiese tegnieke is gebruik in ‘n reeks studies om die genetiese evolusionêre meganismes onderliggend tot die sukses van hierdie genotipe in Kaapstad, Suid-Afrika te verklaar. Deur ‘n versameling van 40 verskillende merkers te gebruik, was dit moontlik om vir die eerste keer ‘n filogenetiese stamboom van die Beijing ras genotipe te skep. Hierdie filogenie word gekenmerk deur die opeenvolgende evolusie van 7 ras sublyne. Met die analise van epidemiologiese data in verhouding tot hierdie ras sublyne, is ‘n assosiasie tussen die mees onlangs ontwikkelde Beijing rasse en die verhoogde vermoë om te versprei en siekte te veroorsaak, getoon. Vanweë hierdie bevindinge, is ‘n hipotese daargestel dat die patogeniese kenmerke van die Beijing genotipe nie in alle raslyne voorkom nie, maar eerder dat verteenwoordigende rasse van die verskillende sublyne unieke eienskappe deur evolusie ontwikkel het. ‘n Metaanalise van gepubliseerde MIRU tipering data van Oos-Asië is vergelyk met MIRU tipering data van Suid-Afrikaanse rasse in die konteks van hul filogenetiese geskiedenis om te bepaal watter van hierdie sogenoemde unieke eienskappe geassosieer is met die gasheerpopulasie en watter eienskappe geassosieer is met die genetiese agtergrond van die sublyn 7 rasse. Hierdie studie het getoon dat die Beijing ras genotipe van Suid-Afrika hul oorsprong gekry het van Oos-Asië en vir die eerste keer waargeneem is in die vroeë 18de eeu. ‘n Betekenisvolle assosiasie is waargeneem tussen die frekwensie waarteen die rasse van ‘n bepaalde Beijing sublyn voorkom en die menslike populasie van wie hulle geïsoleer is (p < 0.0001). Gebaseer op hierdie bevindinge is dit voorgestel dat die menslike populasie (Suid-Afrikaners) vir ‘n spesifieke Beijing sublyn geselekteer het (bv. Sublyn 7) of dat rasse van hierdie sublyn aangepas het om meer suksesvol te wees in die Suid-Afrikaanse populasie In ‘n daaropvolgende studie is, deur gebruik te maak van die metodiek wat ontwikkel is vir die bogenoemde studies, getoon dat die voorouerlike sublyn (bekend as die“atipiese” Beijing genotipe) die mees verteenwoordigende sublyn was onder middelweerstandige isolate van die Oos-Kaap gebied. Dit is teenstrydig met die bestaande dogma wat bepaal dat die “atipiese” Beijing genotipe rasse hulle vermoë om te versprei verloor het. Hierdie verskynsel kan egter versterk word in immuun inkompetente pasiënte aangesien hersiening van die kliniese rekords aangedui het dat verspreiding geassosieer was met HIV ko-infeksie. Hierdie bevindinge bring die behoefte om TB beheer in vatbare populasies te verbeter, na vore, omrede rasse wat gewoonlik `n onbetekenisvolle bydrae tot die epidemie lewer, nou ‘n rede vir kommer is veral as hulle met middelweerstandigheid geassosieer is. Om ons insig rakende die evolusie van die Beijing genotipe te verbeter, is die genomiese stabiliteit van ‘n addisionele 27 polimorfiese merkers geanaliseer. Daar is onlangs voorgestel dat hierdie merkers, wat gebaseer is op MIRU-VNTR volgordes,die nuwe standaard vir molekulêre studies is. Die MIRU-VNTR data is op die filogenetiese boom geplaas en het uitstekende ooreenstemming getoon wat die allele se stabiliteit oor tyd gedemonstreer het. Dit is tans nie duidelik hoe van die allele wat wel verander, die patogenisiteit beïnvloed nie. Die resultate van die studie wys ook onenigheid tussen rasse wat deur IS6110 DNA tipering gedefinieer is en dié wat deur MIRU-VNTR tipering gedefinieer is. Dit impliseer dus dat die evolusie van merkers onafhanklik van mekaar plaasvind en teen verskillende tempos. Verder was die MIRU-VNTR tipering metode nie in staat om verspreiding van middelweerstandige rasse te voorspel nie, wat teenstrydig is met vorige verslae waar lae insidensie omgewings bestudeer is. Dit het noemenswaardige implikasies vir die gebruik van hierdie tipering metode in hoë insidensie omgewings. ‘n Verbeterde PKR-gebaseerde metode is vir die eerste keer gebruik om die 5 mees prominente filogenetiese sublyne in primêre kulture van volwasse tuberkulose pasiënte van ‘n hoë MIV/TB ko-infeksie omgewing, te identifiseer. Die resultate van hierdie studie het gewys dat 15% van die studiepopulasie geïnfekteer is met twee of meer rasse en dat die Beijing genotipe ras die meeste voorgekom het in gemengde infeksies. Verder het middelweerstandige toetse gewys dat een pasiënt geïnfekteer was met beide ‘n middelsensitiewe en ‘n middelweerstandige ras. Gemengde infeksies is al vantevore gekoppel aan onsuksesvolle behandeling en dus demonstreer hierdie bevindinge die epidemiologiese belang van die opsporing van gemengde infeksies in vatbare populasies. Hierdie PKR-gebaseerde metode is verder gebruik om rasse wat voorkom in kulture van pediatriese pasiënte, wat spoligotipering nie kon klassifiseer nie, te klassifiseer. Die resultate het drie gemengde infeksies gewys wat sonder die PKR-gebaseerde metode, nie geïdentifiseer sou gewees het. Om te bepaal of die kliniese beeld van pasiënte wat geïnfekteer is met rasse van die Beijing genotipe verskil van dié van pasiënte wat geïnfekteer is met rasse van die nie-Beijing genotipe, is die kliniese en demografiese data van die twee groepe pasiënte geanaliseer. Hierdie studie wys dat pasiënte wat geïnfekteer is met rasse van die Beijing genotipe hoogs aansteeklik is (gedefinieer op grond van hoë bakteriële lading in sputum monsters). Hierdie bevindinge kon egter nie met behulp van long patologie op borskas X-strale bevestig word nie.

Tuberculosis (TB) remains the leading cause of death due to a single infectious agent with more than 1.5 million people killed each year. In 2018, the World Health Organization (WHO) estimated that one third of the world’s population was infected with Mycobacterium tuberculosis (Mtb), the pathogen responsible for the disease.In 2000, EthR, a mycobacterial transcriptional repressor, was identified as a key modulator of ethionamide (ETH) bioactivation. ETH is one of the main second-line drugs used to treat drug-resistant strains and it is a prodrug that is activated in Mtb by the mono-oxygenase EthA and then inhibits InhA, an enzyme involved in the mycolic acid biosynthesis. In 2009, it was demonstrated that co-administration of ETH with the drug-like inhibitors of EthR was able to boost ETH activity by a factor three in a mouse-model of TB-infection, thus validating EthR protein as a target for a new therapeutic strategy. The first part of this thesis deals with the validation and deep characterization of the solved EthR-ligand structures based on all analysis of how each ligand bind to the EthR. In this section, based on the study of both co-crystal structures and the physicochemical properties of the ligands, we have rationalized the information currently available and understood the interaction of all EthR inhibitors in order to lead to more effective inhibitor design.More recently, another mycobaterial repressor, denoted EthR2, was identified as a putative target that appears to be functionally comparable to EthR (then the locus has been termed EthA2/EthR2, due to its similarity to the EthA/EthR locus). Furthermore, a spiroisoxazoline family of small-molecules, generically denoted as SMARt, has been identified as effective ligand of EthR2. However, according to the data present in the literature, this spiroisoxazoline family can also bind to the former EthR. In order to investigate this proposition, I have solved these small molecules in complex with EthR and compared their binding interactions to the EthR2 protein as well. The opportunity for the design small-molecules is capable of targeting both repressors, thereby opening the way to a dual-target approach.Finally, the third part of this thesis is devoted to the mycobacterial transcriptional factor MabR (Rv2242). Several studies identified this protein as a regulatory transcription factor of the fatty acid synthase II operon, which is mainly responsible for the mycolic acid biosynthesis in Mtb. I therefore purified to homogeneity and characterized the MabR protein as well as I determined the crystal structure of its C-terminal part. Finally, the functional role of MabR is largely discussed, and the way on how to interfere with its DNA binding ability is commented with respect to our results.
Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie)
info:eu-repo/semantics/nonPublished

A Tuberculose (Tb) à uma doenÃa que tem desafiado a humanidade desde a antiguidade. Acreditava-se que com as novas tecnologias, as doenÃas infecto-contagiosas seriam facilmente controladas e banidas. A realidade, porÃm tem-se apresentado de modo diferente; apesar dos avanÃos no conhecimento da tuberculose e tecnologia disponÃvel para seu controle, o quadro atual encontra-se muito distante das metas estabelecidas pelos governos. à essencial o estudo dos fatores associados à tuberculose pulmonar, uma vez que eles estÃo presentes na primo-infeccÃÃo, na recidiva e na Tb pulmonar multi-resistente. O conhecimento de dados relacionados a tuberculose na populaÃÃo em geral e em grupos de risco especÃficos para Tb sÃo elementos fundamentais para avaliar a realidade epidemiolÃgica do nosso meio possibilitando uma organizaÃÃo adequada das atividades preventivas e assistenciais. OBJETIVO: Investigar os fatores associados ao diagnÃstico de Tb, a freqÃÃncia e as caracterÃsticas da tuberculose pulmonar entre sintomÃticos respiratÃrios atendidos na rotina de trÃs serviÃos de saÃde de Fortaleza. MÃTODOS: Estudo transversal de natureza quantitativa realizado por meio da aplicaÃÃo de um questionÃrio em pacientes atendidos em trÃs unidades de saÃde de Fortaleza. Os entrevistados foram divididos em pacientes com desfecho de tuberculose e desfecho nÃo tuberculose. Foi estudado a influÃncia dos fatores sÃcio demogrÃficos, condiÃÃes de moradia, fatores comportamentais, antecedentes relacionados a infecÃÃo por Tb e variÃveis clÃnicas para o desfecho tuberculose. RESULTADOS: A freqÃÃncia de Tb pulmonar entre sintomÃticos respiratÃrios da amostra estudada foi 41,2%, mas esse dados nÃo podem ser extrapolados, os fatores sÃcio demogrÃficos e clÃnicos independentes associados ao desfecho Tb foram tosse, febre e perda de peso considerando o nÃvel de seis por cento de significÃncia. O principal mÃtodo auxiliar utilizado para diagnÃstico de Tb presumida nos pacientes das unidades estudadas em Fortaleza foi a radiografia de tÃrax. O perfil de sensibilidade de Mycobacterium tuberculosis mostrou 8,82 por cento de cepas MDR e sensibilidade a isoniazida, rifampicina, etambutol e streptomicina de 88,23 por cento nas amostras realizadas, nÃo podendo ser extrapolado. Se excluÃdos todos os TSA provenientes do hospital de Messejana e considerado somente os TSA dos postos de saÃde, o valor da resistÃncia reduz para um caso de MDR para 28 TSA realizados representando 3,6% da amostra, a maioria dos pacientes com Tb pulmonar tiveram BAAR positivo somente uma cruz, o que pode significar tempo de doenÃa nÃo tÃo longo.
Tuberculosis (TB) is a disease that has challenged mankind since antiquity. It was believed that with the new technology, infectious diseases would be easily tracked and banned. The reality has been presented differently, despite advances in knowledge of tuberculosis and technology available for its control, the current picture is far from the targets set by governments. It is essential to the study of factors associated with pulmonary tuberculosis, since they are present in the prime-infeccÃÃo in relapse and multi-drug resistant pulmonary TB. Knowledge of data related to TB in the general population and specific groups at risk for TB are crucial to assess the epidemiological reality of our environment allowing proper organization of prevention and care activities. OBJECTIVE: To investigate factors associated with the diagnosis of TB, the frequency and characteristics of pulmonary tuberculosis with respiratory symptoms in three routine health services in Fortaleza. Cross-sectional study of quantitative accomplished through the application of a questionnaire in patients from three health units of Fortaleza. Respondents were divided on outcome of patients with tuberculosis and non tuberculosis outcome. We studied the influence of sociodemographic factors, housing conditions, behavioral factors, antecedents related to TB infection and clinical outcome for tuberculosis. RESULTS: The rate of pulmonary TB patients with respiratory symptoms among the study sample was 41.2%, but this data can not be extrapolated, the socio demographic and clinical outcome independently associated with TB were cough, fever and weight loss considering the level of six percent of significance. The main method used to assist diagnosis of TB in patients suspected of units studied in Fortaleza was the chest radiograph. The susceptibility profile of Mycobacterium tuberculosis showed 8.82 percent of MDR strains and sensitivity to isoniazid, rifampin, ethambutol and streptomycin from 88.23 percent in the samples taken and can not be extrapolated. If all TSA excluded from the hospital and found only Messejana TSA health posts, the resistance value reduces to a case of MDR 28 to TSA made, representing 3.6% of the sample, most patients with pulmonary TB had AFB positive only a cross, which may mean duration of disease not so long.

Mycothiol (MSH), a low-molecular- weight thiol, is a primary reducing agent and essential for the survival of mycobacteria. The full pathway of MSH biosynthesis and detoxification includes various promising drug targets. Several metalloenzymes are involved in this pathway, such as a deacetylase (MshB) and mycothiol S-conjugate amidase (MCA). MshB catalyzes the deacetylation of GlcNAc-Ins to form GlcN-Ins and acetate. Mycothiol S-conjugate amidase (MCA) cleaves the amide bond of mycothiol S-conjugates of various drugs and toxins. The identification of the native co-factor is critical for the design of potent and effective inhibitors. Therefore, in this study, we identified the possible native co-factors of MshB and MCA from M. smegmatis and M. tuberculosis. To reach our aim, we used a pull-down method to rapidly purify halo-MshB and halo-MCA under anaerobic conditions. Our data indicates that the metal bound to MshB and MCA anaerobically purified from E. coli grown in minimal medium is mainly Fe(II), while proteins purified under aerobic conditions contain bound Zn (II) and Fe(II) that varies with the metal content of the medium. For a further clarification of the metal ion preferences of MshB and MCA, we determined the MshB and MCA affinity for Zn(II) to be in the picomolar range and Ms MshB affinity for Fe(II) in nanomolar range. These results indicate that MshB and MCA can be found bound with either iron or zinc and this is independent to their affinities for these metal ions.
Master of Science

Mycobacterium tuberculosis (Mtb), la bactérie responsable de la tuberculose (TB), et le virus de l'immunodéficience humaine (VIH-1), l'agent du syndrome de l'immunodéficience acquise (SIDA), accélèrent leurs progressions mutuelles chez les patients co-infectés. Alors que de nombreuses données cliniques rapportent une augmentation de la charge virale dans les sites anatomiques co-infectés, les mécanismes qui en sont responsables restent insuffisamment décrits. Mtb cible principalement les macrophages. Nous émettons l'hypothèse que l'infection des macrophages par Mtb créé un microenvironnement propice à la réplication du VIH-1 au niveau des sites co-infectés. Pour le montrer, j'ai utilisé un modèle in vitro précédemment établi par mes équipes (le cmMTB - pour " conditioned media of Mtb-infected macrophages "). Celui-ci permet de mimer un environnement tuberculeux, par la différenciation et l'activation des macrophages vers un profil " M(cmMTB) ", largement retrouvé dans les poumons lors d'une tuberculose active. En rejoignant le laboratoire, j'ai participé à l'étude des mécanismes responsables de l'augmentation de la réplication virale dans le contexte de co-infection, en utilisant ce modèle. Nous avons trouvé que les M(cmMTB) forment de nombreux nanotubes (ponts intercellulaires), leur permettant de transférer plus de virus d'un macrophage à l'autre, et conduit à une forte augmentation de la production virale. L'objectif principal de ma thèse a donc été d'identifier, dans un contexte tuberculeux, de nouveaux facteurs impliqués dans l'augmentation de la réplication du VIH-1 dans les macrophages. Pour cela, une analyse transcriptomique des M(cmMTB) a été réalisée, révélant deux facteurs essentiels : le récepteur Siglec-1 et les interférons de type I (IFN-I) via STAT1. Dans un premier temps, j'ai étudié le rôle de Siglec-1 dans la synergie entre Mtb et le VIH-1 dans les macrophages. D'abord, j'ai montré que son expression de surface était augmentée par le cmMTB, de façon dépendante des IFN-I. Ensuite, j'ai établi que l'abondance des macrophages alvéolaires exprimant Siglec-1 chez les primates non-humains co-infectés avec Mtb et le virus de l'immunodéficience simienne corrélait avec la sévérité de la pathologie, et était associée à la signalisation des IFN-I, via l'activation de STAT1. De plus, j'ai identifié une nouvelle localisation de Siglec-1 le long d'un sous-type de nanotubes.[...]
Mycobacterium tuberculosis (Mtb), the bacteria causing tuberculosis (TB), and the human immunodeficiency virus type 1 (HIV-1), the etiological agent of acquired immunodeficiency syndrome (AIDS), act in synergy to exacerbate the progression of each other in co-infected patients. While clinical evidence reveals a frequent increase of the viral load at co-infected anatomical sites, the mechanisms explaining how Mtb favours HIV-1 progression remain insufficiently understood. Macrophages are the main target for Mtb. Their infection by the bacilli likely shapes the microenvironment that favours HIV-1 infection and replication at sites of co-infection. To address this issue, I took advantage of an in vitro model mimicking the TB-associated microenvironment (cmMTB, "conditioned media of Mtb-infected macrophages") previously established in the laboratory; a model that renders macrophages susceptible to intracellular pathogens like Mtb. Upon joining the team, I participated in the study on how Mtb exacerbates HIV-1 replication in macrophages, using this model. We found that cmMTB-treated macrophages (M(cmMTB)) have an enhanced ability to form intercellular membrane bridges called tunneling nanotubes (TNT), which increase the capacity of the virus to transfer from one macrophage to another, leading to the exacerbation of HIV-1 production and spread. The principal objective of my PhD thesis was to identify novel factors that are involved in the exacerbation of HIV-1 replication in macrophages in the context of tuberculosis. To this end, a transcriptomic analysis of M(cmMTB) was conducted, and revealed two key factors: the Siglec-1 receptor and type I interferon (IFN-I)/STAT1 signaling. The first part of my PhD thesis dealt with the characterization of Siglec-1 as a novel factor involved in the synergy between Mtb and HIV-1 in macrophages. First, I demonstrated that its increased expression in M(cmMTB) was dependent on IFN-I. Second, in Mtb and simian immunodeficiency virus co-infected non-human primates, I established a positive correlation between the abundance of Siglec-1+ alveolar macrophages and the pathology, associated with the activation of the IFN-I/STAT-1 pathway. [...]

The RNA polymerase binding protein A (RbpA) is a 13 kDa protein, encoded by the gene Rv2050, that was shown to be essential for the growth and survival of the important human pathogen Mycobacterium tuberculosis. Although is not clear yet why RbpA is essential in M. tuberculosis, significant progress has been made in the characterization of the protein. For instance, it was shown that RbpA binds to the β-subunit of the RNA polymerase (RNAP) and activates transcription. Interestingly, it was reported that RbpA can enhance the transcription activity of the RNAP containing the primary σ-subunit σ[superscript A] but does not have any detectable effect if the RNAP is associated with the alternative σ-subunit σ[superscript F]. Moreover, it was also shown that RbpA might influence the response of M. tuberculosis to the current frontline anti-tuberculosis drug rifampicin. The research project described in this thesis contributes to the ongoing efforts to characterize RbpA by providing the structure of the protein and identifying the principle σ-subunit σ[superscript A], and the principle-like σ-subunit σ[superscript B], as interaction partners. The solution structure of RbpA reveals the presence of a central structured region and highly dynamic N- and C- termini. Both termini are involved in the formation of a tight complex with the σ-subunit but only the C-terminal region appears to be essential for this interaction. The finding that RbpA also binds to the RNAP σ-subunit suggests new possibilities for the mechanism of action used by RbpA to activate transcription. Furthermore, preliminary data obtained using a ΔRv2050 conditional mutant strain of M. tuberculosis suggest that the interaction with the σ-subunit is essential for the functionality of RbpA.

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The tuberculosis continues to represent a severe worldwide public health issue, particularly in developing countries. Besides early diagnosis and therapy, one of the great challenges for its control is the scarce knowledge available about the transmission mechanisms and the related risks. The retrospective cohort described in this study aims to evaluate the risk of Mycobacterium tuberculosis infection, measured by tuberculin skin testing, in contacts of patients with cavitary and noncavitary pulmonary tuberculosis. Identification and screening of index cases between july/2003 and december/2007 was performed based on analysis of two databases: National information system of disease notification and TB notes. Only index cases, with age above 18 years old, living at Cariacica, Serra, Vila Velha and Vitoria cities were screened. Demographic, clinical and laboratory informations of the index cases and contact informations were captured from the medical charts. Bacteriological confirmation based on mycobacterial cultures was captured from TB notes database. among the 1662 index cases identified, 320 met the inclusion criteria: 154 (48,1%) with cavitary disease (C group) and 166 (51,9%) with non-cavitary disease (NC group) based on chest xray. among these 320 index cases, 1257 contacts were identified. most cases of C and NC groups denied previous contact with tuberculosis: 70,2% and 60,1%, respectively. index cases reported cough ranging between 0 and 980 days; mean of 93,5 and median of 60 days; 25 and 75 percentis were 30 and 90 days, respectively. period of cough, which represent time of disease, was higher in the c group (p=0,01, OR 1,82, IC 95%:2,8-13,5) when the threshold was 60 days. higher number of positive sputum smear patients was observed in the C group (p <0,00, OR=5,86, IC 95%: 2,8-13,5). among the 1257 enrolled contacts, 555 (44%) were contacts of the C group and 702 (56%) of the NC group patients. both groups were similar regarding gender, age and chest xray images. however, more reactors to tuberculin skin testing (PPD ≥ 10 mm) were found among contacts of C group: 48% versus 40,6% (p= 0,009, OR1,35, IC 95% 1,07-1,7 ). Taking into account images observed in chest xray, M tuberculosis infection rate was 1,7 higher among contacts of cavitary in comparison with non-cavitary patients. After logistic regression analysis this association was statistically significant for two variables: period of cough ≥ 30 days (p=0, 0007OR=3, 20, IC 95%: 1, 5-8.6,) and positivity of sputum smears (p=0, 0039 OR=2, 47, IC 95: 1, 24-4, 81). Positivity of sputum smears was also related to the presence of cavitary disease. Index cases included in C group had 5, 86 more chance of having positive sputum smear (IC 95%: 2, 8-13, 5). Using infection prevalence rate of 30% or more as the threshold for transmission analysis, the three following variables were related to infection transmission to contacts: period of cough, cavitary disease and positive sputum smear. It was concluded that: a period of cough ≥30 days was associated with TB infection; smear positivity was associated with a higher chance of infection among contacts. The association between cavitary disease and TB infection transmission to contacts was marginally statistically significant.
Um dos principais desafios para o controle da tuberculose (TB) é ainda o pouco conhecimento disponível sobre os mecanismos intrínsecos de sua transmissão e os graus de risco associados a eles. Este estudo, uma coorte retrospectiva, teve por objetivo avaliar o risco de infecção por Mycobacterium tuberculosis, medido pela prova tuberculínica, em contatos de pacientes com tuberculose pulmonar cavitária e não cavitária. A identificação e a seleção dos casos índices foram realizadas por meio da análise de dois bancos de dados: Sistema de Informação de Agravos de Notificação (SINAN) e TB notes. O período abrangido foi de julho de 2003 a dezembro de 2007. Foram selecionados indivíduos residentes em quatro municípios da Região Metropolitana de Vitória (Cariacica, Serra, Vila Velha e Vitória). De todos os casos índices com idade superior a 18 anos notificados no SINAN, obteve-se os prontuários médicos para análise dos dados demográficos, clínicos e laboratoriais e as informações dos respectivos contatos. A confirmação bacteriológica por cultura dos pacientes foi obtida no banco de dados TB-notes. Dos 1662 casos índices identificados, 320 preencheram os critérios de inclusão do estudo 154 (48,1%) com doença cavitária (grupo C) e 166 (51,9%) sem doença cavitária (grupo NC) à radiografia do tórax. Os 320 casos índices geraram 1.257 contatos. A maioria dos casos do grupo C e NC não tinha história epidemiológica de contato prévio com a doença 70,2% e 60,1%, respectivamente. O tempo de tosse variou de 0-980 dias, com média de 93,5 dias (± 130 dias), e mediana, de 60 dias. Os percentis 25 e 75 foram, respectivamente, de 30 e 90 dias. O tempo de tosse, que, na prática, retrata o tempo de doença, foi maior nos pacientes do grupo C (p=0,01, RC 1,82, IC 95%: 1,01-3,04), quando o ponto de corte foi de 60 dias. Houve uma maior concentração de pacientes com baciloscopia positiva no grupo C, o que representa uma diferença estatisticamente significativa (p <0,00, RC=5,86, IC 95%: 2,8-13,5). Dos 1.257 contatos arrolados, 555 (44%) eram contatos do grupo C, e 702 (56%) do grupo NC. Os dois grupos de contatos foram semelhantes em relação ao gênero, idade e resultado da radiografia do tórax. Houve, entretanto, diferença na prevalência de positividade da prova tuberculínica (≥10mm), que foi maior nos contatos do grupo C: 48% versus 40,6% do grupo NC (p= 0,009, RC 1,35, IC95% 1,07-1,7). Os casos índices com doença cavitária infectaram 1,7 vezes mais seus contatos do que os casos índices sem cavidade à radiografia do tórax (IC 95%: 0.94-3.08, p=0, 061). Após análise de regressão logística essa associação mostrou-se estatisticamente significativa para as variáveis: tempo de tosse superior a 30 dias (RC=3,20, IC 95%: 1,5-8.6, p=0, 0007) e positividade da baciloscopia do escarro (RC=2,47, IC 95: 1,24-4,81 p=0, 0039). A positividade da baciloscopia do escarro também esteve associada à presença de doença cavitária. Os casos índices incluídos nessa categoria possuíam 5,86 mais chance de apresentarem exame direto do escarro positivo (IC95%: 2,8-13,5). Essas três variáveis (tempo de tosse, doença cavitária e baciloscopia positiva do escarro) estiveram relacionadas à transmissão da infecção aos contatos, quando se utilizou, como ponto de corte para análise da transmissão, a presença de infecção igual ou superior a 30% nos contatos dos respectivos casos índices. Concluímos que o tempo de tosse superior a 30 dias e a baciloscopia positiva estão fortemente associados à transmissão por M.tuberculosis. Esta associação teve significância limítrofe para doença cavitária.

Interferon regulatory factor-8 (Irf-8), a hematopoietic transcriptional regulator, controls myeloid-cell proliferation and coordinates innate and adaptive host immune responses. Mice from the BXH-2 recombinant inbred strain carry an endogenous R294C mutation in Irf-8. This loss-of-function mutation induces clonal infiltration of undifferentiated Mac-1+/Gr-1 + granulocytic precursors in BXH-2 mice, extramedullary hematopoiesis, and splenomegaly similar to those seen in human chronic myeloid leukemia. It also renders the host permissible to the otherwise avirulent Mycobacterium bovis (BCG), and negatively affects survival or recovery of these mice to other infectious pathogens. Here, we generated a polyc1onal anti-Irf-8 antibody to better characterize the effects of the R294C mutation on Irf-8 protein expression, stability, and inducibility in hematopoietic and non-hematopoietic tissues. We found that mutant Irf-8C294-expressing tissues consistently displayed reduced Irf-8 abundance compared to their wild-type counterparts in both primary splenocytes and following transfection into heterologous cells, presumably due to decreased stability or increased rate of degradation of the mutant isoform. Results also indicate that native Irf-8 is also expressed in the heart, and to a lesser extent, in the kidneys. Since neither of these organs is well-known to be associated with hematopoietic or immune functions, this finding strengthens the possibility that Irf-8 may exert additional regulatory functions in other cellular contexts. Taken together, our study provides a better understanding about the molecular features of the mutant Irf-8 C294 protein and contributes to a growing body of evidence in support of Irf-8 expression in non-hematopoietic tissues.

Despite Africa accounting for ~20% of the global cattle population, prevalence estimates and related risk factors of bovine tuberculosis (bTB), caused by Mycobacterium bovis, are still poorly quantified in many countries across the continent. Control of bTB in Africa is difficult due to poor monitoring of cattle movements and limited abattoir surveillance. Also M. bovis is zoonotic and risk factors for transmission include living in close contact with cattle and consumption of unpasteurised milk. Cattle keeping is integral to some rural populations in Cameroon and understanding the epidemiology of bTB in cattle populations is important both to bovine and public health. Detection of bTB in cattle is difficult due to variability of immune responses to M. bovis infection. The interferon-γ (IFN-γ) assay maybe useful to estimate bTB prevalence and identify bTB risk factors in Cameroon. However its performance can vary at different stages of bTB pathogenesis and in different cattle populations. Recently Fasciola hepatica co-infections have been reported to suppress IFN-γ responses in M. bovis infected cattle but the potential effect with F. gigantica co-infections on bTB prevalence estimates in Cameroon is unknown. An abattoir study was conducted in Cameroon to assess the performance of the IFN-γ assay. In 2012-13; 2064 slaughtered cattle were sampled from Bamenda abattoir (North West Region; NWR) and Ngaoundere abattoir (Vina Division; VD). Individual animal data was collected from routine meat inspection including identification of bTB and Fasciola pathology. Cattle were also tested for bTB using the IFN-γ assay and an M. bovis antibody ELISA. In the absence of a gold-standard diagnostic, the IFN-γ assay was compared to other diagnostic tests to assess agreement and identify factors that affected performance of the assay. Agreement between IFN-γ assay, TB lesion identification and an M. bovis antibody ELISA was poor-moderate, probably partly related to differences in immune response detected. A presence of Fasciola gigantica also increased the odds of false negative IFN-γ assay results. On further investigation co-infected cattle had increased odds of TB lesions and reduced IFN-γ responses that potentially could lead to ~20% reduction in test sensitivity. In an attempt to take into account the potential impact of F. gigantica, when estimating bTB prevalence, an antibody ELISA was developed to detect the exposure in live cattle. To highlight the awareness of disease in cattle-rearing communities, estimate prevalence and identify risk factors of bTB in cattle populations; two cross-sectional studies were conducted in 2013. A stratified clustered cross-sectional study of pastoral cattle herds, in the NWR and the VD, sampled 1448 pastoral cattle reared by 100 pastoralists. A smaller cross-sectional study sampled 60 dairy cattle from 46 small-holder co-operative dairy farmers. Individual animal data and herd-level data were collected and animals were screened by both the single comparative intradermal skin test (SCITT) and IFN-γ assay. Awareness of zoonotic TB was low yet consumption of raw milk was high in cattle-keeping communities highlighting the need for accurate bTB prevalence estimates. Despite the high awareness of the clinical presentation of bTB, clinical signs identified by pastoral herdsmen were not associated with cattle being bTB positive. The SCITT was used to compare two manufacturers cut offs for the IFN-γ assay, ≥0.05 and ≥0.1, and highlighted that these two diagnostics may detect different populations of bTB positive cattle. Using the IFN-γ assay at ≥0.1, bTB prevalence was highest in dairy cattle (21.67%) and was also present in pastoral cattle in the NWR and VD (11.33% and 6.55% respectively). Importantly, as F. gigantica is endemic in Cameroon and its influence could mean the true prevalence of bTB could be higher. Female pastoral cattle were at lower odds of being IFN-γ assay positive potentially due to immunosuppressive factors had lower odds of disease. Husbandry practices also decreased the odds of being IFN-γ assay positive such as drinking from streams, antelope and contact with herds at grazing. Age increased the odds of pastoral cattle being IFN- assay positive potentially being a confounder to chronicity of bTB and other co-infections may influence IFN-γ responses. Dairy cattle herds had different risk factors for being IFN- positive likely due to differences in husbandry practices. Considering the potential risk to public health of M. bovis this thesis highlights the extent of bTB across two major cattle keeping regions in Cameroon and the public health risk in cattle-rearing communities. Furthermore the relationship between Fasciola co-infection and IFN- responses to M. bovis described has potential implications for bTB diagnosis in cattle populations where the parasite is present across the globe.

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The HIV infection and the tuberculosis (TB) are together the main causes of death for infectious agents in the world, being approximately 13 million people infected with both causative agents. The HIV-infected individuals show increased susceptibility for active tuberculosis, being the imunosupression caused by the virus the main risk factor for the development for active TB. This study investigated the profile of Mycobacterium tuberculosis drug resistance and evaluated the factors related to the development of TB in HIV infected patients who were treated at HSJ, which is a reference hospital in infectious diseases in Fortaleza, CearÃ, Brazil. During the period of July 2003 until June 2006, 208 patients with clinical and/or laboratorial diagnosis of micobacteriosis or tuberculosis and infection for the HIV were taken care in this hospital. The TB/HIV co-infected patients were older (average: 40 years, p= 0.0025) than the control group, being approximately 4 years older. The majority of the patients were male (80.8%, p= 0.0005) and they had shown a risk to develop TB of 43.0% greater compared to the female sex. The risk to develop the TB disease was two times higher in the patients with lower educational levels (less than eight years of schooling) and they represented 85.9% of the patients (p=0.000). The history of previous contact with TB patients and previous treatment for TB were also found to be risk factors for TB. The imunosupression level was higher in TB-HIV coinfected patients being the CD4+ lymphocytes count average of 169 cells/mm Â(p=0.000) and the viral load logarithm average of 4,36 (p=0.0013). The clinical forms most frequents were pulmonary (45.7%), extrapulmonary (28.4%) and disseminated (25.9%). Of the samples that were submitted to identification, 81.2% were M. tuberculosis and 12.8% were not M. tuberculosis. The resistance frequency to one drug and multi-resistance were the same (5.9%). Overall, these observations are important for establishing political strategies of public health to improve the conditions at the regional level.
A infecÃÃo pelo vÃrus HIV e a TB estÃo entre as principais causas de morte por agentes infecciosos no mundo, aproximadamente 13 milhÃes de pessoas estÃo infectadas com ambos agentes causadores. Os indivÃduos infectados pelo HIV apresentam susceptibilidade aumentada para tuberculose ativa, sendo a imunossupressÃo determinada pelo vÃrus o principal fator de risco para o desenvolvimento da doenÃa tuberculose. Este trabalho investigou o perfil de resistÃncia do Mycobacterium tuberculosis e os fatores epidemiolÃgicos relacionados ao desenvolvimento da doenÃa em pacientes co-infectados pelo HIV atendidos no Hospital SÃo Josà de DoenÃas Infecciosas (HSJ), o qual à referÃncia em doenÃas infecciosas em Fortaleza, Estado do CearÃ, Brasil. Foram analisados, no perÃodo de julho de 2003 atà junho de 2006, 208 pacientes com diagnÃstico clÃnico e/ou laboratorial de micobacteriose ou tuberculose e infecÃÃo pelo HIV atendidos no HSJ. Os pacientes co-infectados TB/HIV apresentavam idade superior aos pacientes do grupo controle (mÃdia de 40 anos, p= 0,0025), tendo aproximadamente 4 anos a mais. O sexo masculino apresentou maioria significante (80,8%, p= 0,0005) e teve risco 43,0% superior ao sexo feminino de desenvolver TB. O risco de adquirir TB foi duas vezes maior nos pacientes com escolaridade baixa, que representavam 85,9% dos pacientes (p=0,000). A histÃria de contato prÃvio com pacientes com TB e de tratamento prÃvio para TB foram considerados fatores de risco para TB nesta populaÃÃo. O nÃvel de imunossupressÃo foi maior nos pacientes co-infectados TBHIV com contagem mÃdia de linfÃcitos T CD4+ de 169 cels/mm (p=0,000), assim como o logaritmo da carga viral com mÃdia de 4,36 (p=0,0013). As formas clÃnicas mais freqÃentes foram: pulmonar (45,7%), extrapulmonar (28,4%) e disseminada (25,9%). Das amostras que foram realizadas identificaÃÃo, 81,2% foram identificadas como M. tuberculosis e 12,8% como nÃo M. tuberculosis. A freqÃÃncia de resistÃncia a uma droga e multi-resistÃncia foram iguais (5,9%). Esses resultados fornecem informaÃÃes importantes para o estabelecimento de estratÃgias de polÃticas de saÃde pÃblicas mais adequadas a nÃvel regional.

SUMMARY Aim of the study. To evaluate the possibilities of control of the children tuberculosis in Kaunas County. Objectives. 1. To evaluate the epidemiological situation of the children tuberculosis in Kaunas County in 1996 – 2003. 2. To evaluate the problems of early diagnostics and treatment of the children tuberculosis. 3. To propose the possible means of control of the children tuberculosis. Methods. Study objects were the patients with active tuberculosis treated in the Department of Children Pulmonology of Kaunas II hospital during 1996–2003 and general practitioners (GP) working in Primary Health Care Centers (PHCC). All the files of 538 patients treated with active tuberculosis during the analyzed period and 199 questionnaires answered by GP were analyzed. 79% of children treated were from Kaunas County. The statistical analysis was done with SPSS software for data acquisition and analysis. Results. The contact with adult persons with TM positive tuberculosis was the main factor defining children tuberculosis (TB) cases (45,0%). The number of children treated with TM positive tuberculosis increased from 2,0% to 13,1% (p <0,001) during 1998–2003. Only 64,3% of GP in their PHCC had the possibility to make tuberculin skin test (TST) and X-rays of the chest. The diagnosis of TB was verified during the prophylactic check up in 74,2% of cases in 2000, this number decreased to 27,5% (p <0,001) due to the disturbed supply of tuberculin in 2001. Only 26,1% of GP got the... [to full text]

Bovine tuberculosis (TB) is a contagious neglected zoonosis of cattle that is prevalent but under-investigated in Cameroon, hence this study was designed to assess the epidemiology of bovine TB in cattle, risks for M. bovis infection in cattle and humans; and public health implications of zoonotic bovine TB in the highlands of Cameroon. A retrospective study of meat inspection records (1994 – 2010) was done to estimate the prevalence of TB lesions in slaughtered cattle in the North West region. The prevalence of bovine TB and anti-bovine TB antibodies in live cattle based on tuberculin skin tests (2 surveys) and immune-chromatographic assays respectively were carried out in the Western and Adamawa highlands of Cameroon. The performance of the tuberculin tests for bovine TB diagnosis in cattle using various tuberculin skin test cut-off points against the detection of anti-bovine TB antibodies (hypothesised risks of exposure) was compared. Suspected TB lesions from slaughtered cattle and infected human sputa were cultured on Lowentein – Jesen and Middlebrook 7H9 media to isolate mycobacteria agents for molecular genotyping using genomic deletion analysis and spoligotyping. Risk factors for exposure and transmission of zoonotic bovine TB infection of cattle and cattle professionals, and its public health significance were determined using structured questionnaires. Seventeen years of meat inspection record revealed that suspect TB lesions were identified in 599 of 129,165 slaughtered cattle at the Bamenda abattoir. The lungs and associated lymph nodes (over 60%) were the most affected tissues. Other results showed that the prevalence of anti-bovine TB antibodies in cattle in the study regions was 37.17%. Chi square statistics revealed that irrespective of the tuberculin test cut-off value (P<0.05; χ2>48), strong associations existed between the detection of anti-bovine TB antibodies and disease status. A 95% confidence interval analysis of the comparative cervical tuberculin tests revealed that the prevalence rates were 4.67% – 7.15%, 12.02% – 15.67% and 20.56% – 24.98% at the ≥ 4mm, ≥ 3mm and ≥ 2mm cut-off points, respectively. Overall, the best test performance was realised at ≥ 3-mm, though the ≥ 2-mm cut-off point predicted more positive reactors. Age, sex, breed and husbandry practices served as significant (P<0.05) risks to the prevalence and exposure of bovine TB in cattle. The feedbacks from cattle professionals suggested that there was high possibility of cattle to cattle and cattle to human transmission of bovine TB such as intimate and repeated animal / animal and animal / human interactions, consuming unpasteurised milk and eating raw meat. Genomic deletion analysis of cultured isolates showed evidence of M. tuberculosis from cattle and M. bovis from human while spoligotyping identified five cattle M. bovis strains; and four spoligotype patterns that had not been previously described anywhere. The study has important epidemiological and public health implications requiring prompt and decisive actions from the Cameroonian authority towards controlling zoonotic bovine TB in both humans and animals. A multidisciplinary approach is needed for further collaborative research and effective control strategies such as enhancing the awareness of people to this deadly disease through continuous education, proper food handling and personal hygiene, healthy husbandry practices and maintenance of the environment.

Macrophages infected with a heavy burden of M.tb Erdman undergo a cell death that initially resembles apoptosis but quickly transitions to necrosis. Unlike the previously reported TNF dependent apoptosis induced by avirulent Mycobacterium [1], this form of macrophage cell death is not microbicidal [2]. Microbicidal effects are observed however, when the heavily infected macrophage encounters an uninfected naïve macrophage. My studies describe in part, the crosstalk between the uninfected and infected macrophage that results in the killing of the intracellular M.tb Cell contact between the two cell populations is not necessary for this killing of bacilli to occur and the soluble “signal” of communication between the two cell populations is transferrable, without naïve macrophages present, to newly infected cells also resulting in the reduced viability of the bacilli. We have found that when the IL-1 receptor is absent in the naïve macrophage population that the co-culture antimycobacterial effect is abrogated, suggesting that IL-1 released by the infected dying macrophage is critical for naïve macrophages to respond in a way that results in the decrease in mycobacterial viability. The signaling between the two cell population ultimately converges on activation of iNOS in the infected cell however ROS appears not to be involved.

Adaptation to external environmental conditions is essential for the survival of a bacterial cell. Bacteria have thus evolved multiple mechanisms to sense environmental stimuli and to couple this information into an appropriate cellular response. The cellular response, in its simplest sense involves a change in the cellular content which is achieved by regulating gene expression. Gene expression in bacteria is primarily regulated at the first step, also referred to as transcription initiation. Extensive studies on this mechanism over the past two decades provide a molecular picture of this process. The main enzyme in this process is the DNA dependent RNA polymerase (RNAP). The RNAP enzyme however lacks a specificity factor that dictates which genes are to be selectively expressed. Selectivity is enforced by a dissociable subunit, the sigma (σ) factor. σ factors have specificity determinants that allow recognition of promoter sequences in the DNA. The sequence features on DNA include the –10 elements (Pribnow box), –35 elements, the extended -10 region, the spacing between –10 and –35 elements and the upstream sequence to –35 promoter element. σ factors recognize a few or several of these promoter sequence features thereby providing an efficient mechanism for RNAP recruitment at a specific promoter. σ factors substantially differ in sequence and structural elements. The principal σ factor of the σ70 family has several domains. This includes specific domains that interact with –10 and –35 promoter elements and the sequence between these two promoter elements. The N-terminal domain of σ factors of the σ70 family also encodes regions enabling auto-regulation of this initiation factor. A defining feature that distinguishes σ70 members from other σ factors (σ38 and σ54) is that σ70 does not require ATP for its activity. The number of σ factors in a bacterial cell varies across species. For example Streptococcus pneumonia has one σ factor, Lactococcus lactis has two, Haemophilus influenza has four while Mycobacterium tuberculosis has thirteen σ factors. The exceptions are Streptomyces coelicolor with 65and Sorangium cellulosum with 109 σ factors. The number of σ factors in a bacterial cell is suggested to be correlated with the diverse environmental conditions encountered by the bacterium and the genomic size. The focus of work reported in this thesis is on M. tuberculosis σ factors. There are large variations in the number of σ factors in different mycobacterial species. While M. leprae has two, M. tuberculosis has thirteen σ factors. This variation in the number of σ factors has widely been believed to aid rapid signal transduction of environmental stimuli into changes in gene expression. While other transcription factors enable the recruitment of RNAP to specific promoter sequences, repressors that abrogate transcription and effectors that modulate transcription also dictate transcription levels. The intra cellular levels of a σ factor is often the primary determinant of the expression profile. A specific group of σ factors referred to as Extra Cytoplasmic Function (ECF) σ factors, govern the cellular response to specific environmental stimuli. The ECF family of σ factors has been shown to be regulated by diverse mechanisms. These include transcriptional, translational and post translational control by protein – protein interactions. The focus of this thesis was to understand the regulatory mechanism that involves σ factor interacting proteins. ECF σ factors that are governed by protein – protein interactions are often co-expressed with a regulator protein, the anti-σ factor. In several cases, they are a part of the same operon. This ensures similarity in the expression levels of σ factors and their cognate anti-σ factors. The selective dissociation of an inactive σ factor from a σ/anti-σ complex is also effected by diverse mechanisms. These include structural changes in response to environmental stimuli, conformational changes brought about by the binding of metabolites or by targeted proteolysis of anti-σ factors. The cellular concentration of an activated σ factor is thus often subject to the rate at which it is released from an inactive complex. The notion of specific σ/anti σ factor pairs has recently been challenged with a suggestion that a σ factor could also make non-specific interactions with other anti-σ factors. This finding has implications for the widely accepted model for bacterial gene expression that relies on a cellular estimate of free active σ factors. In this model, referred to as the partitioning of σ factor space model of bacterial transcription, σ factors compete for a limited pool of apo-RNAP and recruit the enzyme to target promoter elements. Two aspects of the mechanism that governs σ factor activity were explored in the course of the studies reported in this thesis. The first is recognition of a particular stress by structurally similar anti-σ factors. This is described in the second chapter of this thesis. The relative sensitivity of redox sensors plays an important role in providing a calibrated response to environmental stimuli and cellular homeostasis. This cellular machinery plays a crucial role in the human pathogen M. tuberculosis as it encounters diverse microenvironments in the host. The redox sensory mechanism in M. tuberculosis is governed by two component and one component systems, alongside ECF σ factors. ECF σ factors that govern the cellular response to redox stimuli are negatively regulated by forming a complex with proteins called zinc associated anti – σ factors (ZAS). ZAS proteins release their cognate σ factor in response to oxidative stress. The relative sensitivity of the ZAS sensors to redox processes dictates the concentration of free ECF σ factors in the cell. However, factors governing the redox threshold of these sensors remain unclear. The molecular characterization of three σ factor/ZAS pairs - σL/RslA, σE/RseA and σH/RshA using a combination of biochemical, biophysical and electrochemical techniques revealed the differences in redox sensitivity in these proteins despite apparent structural similarity. This finding can potentially rationalize the hierarchy in the activation of the cognate ECF σ factors under oxidative stress. Put together, the study described in chapter 2 provides a basis for examining sequence and conformational features that modulate redox sensitivity within the confinement of a conserved structural scaffold. The other aspect that was examined in the course of this study, described in chapter 3 of this thesis, was on crosstalk between σ/anti σ factors. In this study, we evaluated the affinity and specificity for σ/anti-σ factor interaction. We then analyzed the conformational determinants that enforce fidelity in σ/anti-σ interactions. The experimental data that we obtained on interactions between cognate and non-cognate σ/anti-σ pairs provide a template to evaluate tolerance between specific and non-specific interactions. The results from this analysis suggest non-cognate interactions are feasible. These interactions are likely to govern the extent to which different σ factors are activated in response to a particular environmental stimulus. It appears likely that this mechanism could provide a route to bacterial survival, perhaps allowing phenotypic diversity that help circumvent an environmental insult. Another aspect we addressed in the course of this work was the evaluation of specific targeting of a prokaryotic transcription initiation factor. Peptide ligands of a M. tuberculosis σ factor, targeting the RNAP-σ interface were evaluated biochemically and biophysically. A sixteen residue long helical peptide from the core RNAP that could interact with σ4 region and suitably designed control peptides are validated in vitro. Consistent findings from in silico and in vitro observations validated the design strategy that can also be extended across σ factors. This study suggests that designed peptide binders can be explored as chemical tools for studying the regulation of transcription. The fifth chapter of this thesis provides a summary of the findings from the three research themes described in this thesis. This thesis has three appendices. The first two report projects that were discontinued as they were not feasible from the perspective of structural characterization. While Appendix I reports preliminary studies on hemagglutinin-antibody complex, Appendix II describes structural studies on Escherichia coli toxin–anti toxin pairs to evaluate a conformational rationale for protein–protein interactions. Appendix III is a compilation of sequence and structural data that was used for the bioinformatics analysis presented in chapter 3. Put together the studies described in this thesis reveal exquisite adaptation strategies employed by M. tuberculosis to ensure survival under diverse micro-environments in the host.

Regulation of transcription in prokaryotes is primarily governed at the transcription initiation step. This feature has been extensively characterized in model prokaryotes notably Escherichia coli and Bacillus subtilis. Transcription initiation was initially thought to be governed primarily by initiation factors that recruit the RNA polymerase (RNAP) enzyme to initiate expression of given gene. Recent studies reveal multiple mechanisms at play including additional protein factors that can modulate gene expression. Nonetheless, understanding transcription factors is key to rationalize the nuanced changes in prokaryotic gene expression in response to diverse environmental stimuli. This is particularly relevant in the case of the human pathogen, Mycobacterium tuberculosis, especially due to the ability of this bacterium to survive in the host, often for several decades prior to the onset of the disease. Transcription initiation factors, also called σ factors in prokaryotes, are diverse in size and sensory/regulatory mechanisms. Indeed, the number of alternate σ factors vary substantially from six in E. coli to more than 118 in Plesiocystis pacifica. The large number of alternative σ factors has been suggested to be correlated with the diversity of micro-environments experienced by a bacterial cell. Studies on several prokaryotic σ factors reveal common features in these proteins that was not evident earlier due to poor sequence conservation. A central theme that emerges from these studies is that a minimalistic architecture of two domains can recognize promoter DNA and recruit the RNAP enzyme to initiate transcription. Additional domains are required when certain promoter elements are missing or to enable a specific, context dependent regulatory mechanism. The work reported in this thesis was influenced by previous studies in this laboratory and elsewhere on M. tuberculosis σ factors. While these studies revealed multiple features of transcription initiation, several aspects of this mechanism, including some classes of σ factors remain to be examined. The focus of this study was to examine an under-explored sub-group of σ factors, classified as the ECF41 sub-group. This sub-group has an additional domain at the Carboxy-terminus that has been hypothesised to influence σ factor activity. Towards this goal, M. tuberculosis σJ was examined. Previous studies suggested a role for this σ factor in modulating the response to hydrogen peroxide stress. An intriguing feature based on sequence analysis was that neither did this extra-cytoplasmic function σ factor have an anti-σ factor that can respond to oxidative stress nor was it directly associated with a mechanism to sense oxidative stress. The specific goal of the research described here was to understand the structural and mechanistic features that govern σJ activity. This thesis is organized as follows- The first chapter provides a brief introduction to prokaryotic transcription and regulatory mechanisms that govern this process. This chapter also has the literature necessary to phrase the problem in characterizing this family of proteins with particular reference to the unique physiology of Mycobacterium tuberculosis. A summary of the previous work is provided in this chapter to place the current study in context of previous studies and highlight the lacunae in our understanding of the transcription mechanism in M. tuberculosis. Chapter two describes the structural characterization of M. tuberculosis σJ by single-crystal X-ray diffraction. The poor sequence similarity of σJ to known σ factors precluded efforts to obtain phase information by molecular replacement methods. Here we also describe the steps that were essential to obtain diffraction quality crystals and the subsequent steps to account for pseudo-merohedral twinning, an imperfection that could have potentially been a limitation for structure determination. The crystal structure of σJ provide an example of successful phase determination with data collected on near-perfectly twinned crystals using single-wavelength anomalous dispersion. Chapter three describes computational efforts to understand the regulatory mechanisms of M. tuberculosis σJ. Classical Molecular Dynamics (MD) simulations were performed to understand the role of a C-terminal SnoaL_2 domain in this transcription factor. The MD simulations suggest that the C-terminal SnoaL_2 domain limits inter-domain movements between σJ2 (the pribnow box binding domain) and σJ4 (the -35 promoter element binding domain) and confers a compact three domain organization to this protein. The biochemical and functional characterization of M. tuberculosis σJ is described in chapter four. This includes in vitro studies on σJ and cognate promoter DNA interactions performed using Surface Plasmon Resonance (SPR) and Electrophoretic Mobility Shift Assays (EMSA). The ex vivo reporter based experiments to examine the effect of SnoaL_2 domain on σJ activity are also described. Spectroscopic studies on σJ interactions with a small molecule limonene-1,2-epoxide suggested a potential novel role for the SnoaL_2 domain in σJ. Chapter five summarizes the work on M. tuberculosis σJ reported in this thesis. We note that this study opens up a new perspective to understand σ factors. In particular, M. tuberculosis σJ suggests that the domain organization is likely to be retained in ECF41 sub-group of σ factors. This study also hints at broader implications in the distinction between one-component systems and transcription factors. Bioinformatic analysis suggest that observations similar to that noted in M. tuberculosis σJ are likely to be more widespread across diverse phyla than currently acknowledged. This thesis has three annexures. Annexure-I summarizes experimental details of the work performed on the M. tuberculosis σ/anti-σ factor complex σH/RshA. Annexure-II summarizes experimental details and strategies that could not be incorporated in the main body of this thesis. Annexure-III describes a short project performed on a bi-domain protein tyrosine phosphatase PTP99A.

Regulation of transcription in prokaryotes is primarily governed at the transcription initiation step. This feature has been extensively characterized in model prokaryotes notably Escherichia coli and Bacillus subtilis. Transcription initiation was initially thought to be governed primarily by initiation factors that recruit the RNA polymerase (RNAP) enzyme to initiate expression of given gene. Recent studies reveal multiple mechanisms at play including additional protein factors that can modulate gene expression. Nonetheless, understanding transcription factors is key to rationalize the nuanced changes in prokaryotic gene expression in response to diverse environmental stimuli. This is particularly relevant in the case of the human pathogen, Mycobacterium tuberculosis, especially due to the ability of this bacterium to survive in the host, often for several decades prior to the onset of the disease. Transcription initiation factors, also called σ factors in prokaryotes, are diverse in size and sensory/regulatory mechanisms. Indeed, the number of alternate σ factors vary substantially from six in E. coli to more than 118 in Plesiocystis pacifica. The large number of alternative σ factors has been suggested to be correlated with the diversity of micro-environments experienced by a bacterial cell. Studies on several prokaryotic σ factors reveal common features in these proteins that was not evident earlier due to poor sequence conservation. A central theme that emerges from these studies is that a minimalistic architecture of two domains can recognize promoter DNA and recruit the RNAP enzyme to initiate transcription. Additional domains are required when certain promoter elements are missing or to enable a specific, context dependent regulatory mechanism. The work reported in this thesis was influenced by previous studies in this laboratory and elsewhere on M. tuberculosis σ factors. While these studies revealed multiple features of transcription initiation, several aspects of this mechanism, including some classes of σ factors remain to be examined. The focus of this study was to examine an under-explored sub-group of σ factors, classified as the ECF41 sub-group. This sub-group has an additional domain at the Carboxy-terminus that has been hypothesised to influence σ factor activity. Towards this goal, M. tuberculosis σJ was examined. Previous studies suggested a role for this σ factor in modulating the response to hydrogen peroxide stress. An intriguing feature based on sequence analysis was that neither did this extra-cytoplasmic function σ factor have an anti-σ factor that can respond to oxidative stress nor was it directly associated with a mechanism to sense oxidative stress. The specific goal of the research described here was to understand the structural and mechanistic features that govern σJ activity. This thesis is organized as follows- The first chapter provides a brief introduction to prokaryotic transcription and regulatory mechanisms that govern this process. This chapter also has the literature necessary to phrase the problem in characterizing this family of proteins with particular reference to the unique physiology of Mycobacterium tuberculosis. A summary of the previous work is provided in this chapter to place the current study in context of previous studies and highlight the lacunae in our understanding of the transcription mechanism in M. tuberculosis. Chapter two describes the structural characterization of M. tuberculosis σJ by single-crystal X-ray diffraction. The poor sequence similarity of σJ to known σ factors precluded efforts to obtain phase information by molecular replacement methods. Here we also describe the steps that were essential to obtain diffraction quality crystals and the subsequent steps to account for pseudo-merohedral twinning, an imperfection that could have potentially been a limitation for structure determination. The crystal structure of σJ provide an example of successful phase determination with data collected on near-perfectly twinned crystals using single-wavelength anomalous dispersion. Chapter three describes computational efforts to understand the regulatory mechanisms of M. tuberculosis σJ. Classical Molecular Dynamics (MD) simulations were performed to understand the role of a C-terminal SnoaL_2 domain in this transcription factor. The MD simulations suggest that the C-terminal SnoaL_2 domain limits inter-domain movements between σJ2 (the pribnow box binding domain) and σJ4 (the -35 promoter element binding domain) and confers a compact three domain organization to this protein. The biochemical and functional characterization of M. tuberculosis σJ is described in chapter four. This includes in vitro studies on σJ and cognate promoter DNA interactions performed using Surface Plasmon Resonance (SPR) and Electrophoretic Mobility Shift Assays (EMSA). The ex vivo reporter based experiments to examine the effect of SnoaL_2 domain on σJ activity are also described. Spectroscopic studies on σJ interactions with a small molecule limonene-1,2-epoxide suggested a potential novel role for the SnoaL_2 domain in σJ. Chapter five summarizes the work on M. tuberculosis σJ reported in this thesis. We note that this study opens up a new perspective to understand σ factors. In particular, M. tuberculosis σJ suggests that the domain organization is likely to be retained in ECF41 sub-group of σ factors. This study also hints at broader implications in the distinction between one-component systems and transcription factors. Bioinformatic analysis suggest that observations similar to that noted in M. tuberculosis σJ are likely to be more widespread across diverse phyla than currently acknowledged. This thesis has three annexures. Annexure-I summarizes experimental details of the work performed on the M. tuberculosis σ/anti-σ factor complex σH/RshA. Annexure-II summarizes experimental details and strategies that could not be incorporated in the main body of this thesis. Annexure-III describes a short project performed on a bi-domain protein tyrosine phosphatase PTP99A.

A distinctive feature of host-pathogen interactions in the case of Mycobacterium tuberculosis is the asymptomatic latent phase of infection. The ability of the bacillus to survive for extended periods of time in the host suggests an adaptive mechanism in M. tuberculosis that can cope with a variety of environmental stresses and other host stimuli. Extensive genomic studies and analysis of knock-out phenotypes revealed elaborate cellular machinery in M. tuberculosis that ensures a rapid cellular response to host stimuli. Prominent amongst these are two-component systems and σ factors that exclusively govern transcription re-engineering in response to environmental stimuli. M. tuberculosis σK is a σ factor that was demonstrated to control the expression of secreted antigenic proteins. The study reported in this thesis was geared to understand the molecular basis for σK activity as well as to explore conditions that would regulate σK activity. Transcription in bacteria is driven by the RNA polymerase enzyme that can associate with multiple σ factors. σ factors confer promoter specificity and thus directly control the expression of genes. The association of different σ factors with the RNA polymerase is essential for the temporal and conditional re-engineering of the expression profile. Environment induced changes in expression rely on a subset of σ factors. This class of σ factors (also referred to as Class IV or Extra-cytoplasmic function (ECF) σ factors) is regulated by a variety of mechanisms. The regulation of an ECF σ factor activity at the transcriptional, translational or posttranslational steps ensures fidelity in the cellular concentration of free, active ECF σ factors. In general, ECF σ factors associate with an inhibitory protein referred to as an anti-σ factor. The release of a free, active σ factor from a σ /anti-σ complex is thus a mechanism that can potentially control the cellular levels of an active σ factor in the cell. M. tuberculosis σK is associated with a membrane bound anti-σK (also referred to as RskA) (Said-Salim et al., Molecular Microbiology 62: 1251-1263: 2006). The extracellular stimulus that is recognized by RskA remains unclear. However, recent studies have suggested the possibility of a regulated proteolytic cascade that can selectively degrade RskA and other membrane associated anti-σ factors. The goal of the study was to understand this regulatory mechanism with a specific focus on the M. tuberculosis σK/RskA complex. The structure of the cytosolic σK/RskA complex and the associated biochemical and biophysical characteristics revealed several features of this /anti-σ complex that were hitherto unclear. In particular, these studies revealed a redox sensitive regulatory mechanism in addition to a regulated proteolytic cascade. These features and an analysis of the M. tuberculosis σK/RskA complex vis-à-vis the other characterized σ/anti σfactor complexes are presented in this thesis. This thesis is organized as follows- Chapter 1 provides an overview of prokaryotic transcription. A brief description of the physiology of M. tuberculosis is presented along with a summary of characterized factors that contribute to the pathogenecity and virulence of this bacillus. The pertinent mechanistic issues of σ/anti-σ factor interactions are placed in the context of environment mediated changes in M. tuberculosis transcription. A summary of studies in this area provides a background of the research leading to this thesis. Chapters 2 and 3 of this thesis describe the structural and mechanistic studies on the σK/RskA complex. The crystal structure of the σK/RskA complex revealed a disulfide bond in domain 4 (σK4). σK4 interacts with the -35 element of the promoter DNA. The disulfide forming cysteines were seen to be conserved in more than 70% of σK homologs, across both gram-positive and gram-negative bacteria. The conservation of the disulfide-forming cysteines led us to further characterize the role of this disulfide in σK/RskA interactions. These were examined by several biochemical and biophysical experiments. The redox potential of these disulfide bond forming cysteine residues were consistent with the proposed role of a sensor. The crystal structure and biochemical studies thus suggest that M. tuberculosis σK is activated under reducing conditions. Chapter 4 of this thesis describes the progress made thus far in the structural and biochemical characterization of an intra-membrane protease, M. tuberculosis Rip1 (Rv2869c). This protein is an essential component of the proteolytic cascade that selectively cleaves RskA. The proteolytic steps that govern the selective degradation of an anti-σ factor were first characterized in the case of E. coli σE (Li, X. et al. Proc. Natl. Acad. Sci. USA, 106:14837-14842, 2009). This cascade is triggered by the concerted action of a secreted protease (also referred to as a site-1 protease) and a trans-membrane protease (also referred to as a site-2 protease). M. tuberculosis Rip1 was demonstrated to be bona-fide site 2 protease that acts on three anti-σ factors viz., RskA, RslA and RsmA (Sklar et al., Molecular Microbiology 77:605-617; 2010). To further characterize the role of Rip1 in the proteolytic cascade, this intra-membrane protease was cloned, expressed and purified for structural, biochemical and biophysical analysis. The preliminary data on this membrane protein is described in this chapter. The conclusions from the studies reported in this thesis and the scope for future work in this area is described in Chapter 5. Put together, the σK/RskA complex revealed facets of σ/anti-σ factor interactions that were hitherto unrecognized. The most prominent amongst these is the finding that an ECF σfactor can respond to multiple environmental stimuli. Furthermore, as seen in the case of the σK/RskA complex, the σ factor can itself serve as a receptor for redox stimuli. Although speculative, a hypothesis that needs further study is whether these features of the σK/RskA complex contribute to the variable efficacy of the M. bovis BCG vaccine. In this context it is worth noting that σK governs the expression of the prominent secreted antigens- MPT70 and MPT83. The studies reported in this thesis thus suggest several avenues for future research to understand mycobacterial diversity, immunogenicity and features of host-pathogen interactions. The appendix section is divided into two subparts- Appendix 1 of the thesis is a review on peptidase V. This is a chapter in The Handbook of Proteolytic enzymes (Elsevier Press, ISBN:9780123822192). Appendix 2 of the thesis includes technical details and an extended materials and methods section.

A distinctive feature of host-pathogen interactions in the case of Mycobacterium tuberculosis is the asymptomatic latent phase of infection. The ability of the bacillus to survive for extended periods of time in the host suggests an adaptive mechanism in M. tuberculosis that can cope with a variety of environmental stresses and other host stimuli. Extensive genomic studies and analysis of knock-out phenotypes revealed elaborate cellular machinery in M. tuberculosis that ensures a rapid cellular response to host stimuli. Prominent amongst these are two-component systems and σ factors that exclusively govern transcription re-engineering in response to environmental stimuli. M. tuberculosis σK is a σ factor that was demonstrated to control the expression of secreted antigenic proteins. The study reported in this thesis was geared to understand the molecular basis for σK activity as well as to explore conditions that would regulate σK activity. Transcription in bacteria is driven by the RNA polymerase enzyme that can associate with multiple σ factors. σ factors confer promoter specificity and thus directly control the expression of genes. The association of different σ factors with the RNA polymerase is essential for the temporal and conditional re-engineering of the expression profile. Environment induced changes in expression rely on a subset of σ factors. This class of σ factors (also referred to as Class IV or Extra-cytoplasmic function (ECF) σ factors) is regulated by a variety of mechanisms. The regulation of an ECF σ factor activity at the transcriptional, translational or posttranslational steps ensures fidelity in the cellular concentration of free, active ECF σ factors. In general, ECF σ factors associate with an inhibitory protein referred to as an anti-σ factor. The release of a free, active σ factor from a σ /anti-σ complex is thus a mechanism that can potentially control the cellular levels of an active σ factor in the cell. M. tuberculosis σK is associated with a membrane bound anti-σK (also referred to as RskA) (Said-Salim et al., Molecular Microbiology 62: 1251-1263: 2006). The extracellular stimulus that is recognized by RskA remains unclear. However, recent studies have suggested the possibility of a regulated proteolytic cascade that can selectively degrade RskA and other membrane associated anti-σ factors. The goal of the study was to understand this regulatory mechanism with a specific focus on the M. tuberculosis σK/RskA complex. The structure of the cytosolic σK/RskA complex and the associated biochemical and biophysical characteristics revealed several features of this /anti-σ complex that were hitherto unclear. In particular, these studies revealed a redox sensitive regulatory mechanism in addition to a regulated proteolytic cascade. These features and an analysis of the M. tuberculosis σK/RskA complex vis-à-vis the other characterized σ/anti σfactor complexes are presented in this thesis. This thesis is organized as follows- Chapter 1 provides an overview of prokaryotic transcription. A brief description of the physiology of M. tuberculosis is presented along with a summary of characterized factors that contribute to the pathogenecity and virulence of this bacillus. The pertinent mechanistic issues of σ/anti-σ factor interactions are placed in the context of environment mediated changes in M. tuberculosis transcription. A summary of studies in this area provides a background of the research leading to this thesis. Chapters 2 and 3 of this thesis describe the structural and mechanistic studies on the σK/RskA complex. The crystal structure of the σK/RskA complex revealed a disulfide bond in domain 4 (σK4). σK4 interacts with the -35 element of the promoter DNA. The disulfide forming cysteines were seen to be conserved in more than 70% of σK homologs, across both gram-positive and gram-negative bacteria. The conservation of the disulfide-forming cysteines led us to further characterize the role of this disulfide in σK/RskA interactions. These were examined by several biochemical and biophysical experiments. The redox potential of these disulfide bond forming cysteine residues were consistent with the proposed role of a sensor. The crystal structure and biochemical studies thus suggest that M. tuberculosis σK is activated under reducing conditions. Chapter 4 of this thesis describes the progress made thus far in the structural and biochemical characterization of an intra-membrane protease, M. tuberculosis Rip1 (Rv2869c). This protein is an essential component of the proteolytic cascade that selectively cleaves RskA. The proteolytic steps that govern the selective degradation of an anti-σ factor were first characterized in the case of E. coli σE (Li, X. et al. Proc. Natl. Acad. Sci. USA, 106:14837-14842, 2009). This cascade is triggered by the concerted action of a secreted protease (also referred to as a site-1 protease) and a trans-membrane protease (also referred to as a site-2 protease). M. tuberculosis Rip1 was demonstrated to be bona-fide site 2 protease that acts on three anti-σ factors viz., RskA, RslA and RsmA (Sklar et al., Molecular Microbiology 77:605-617; 2010). To further characterize the role of Rip1 in the proteolytic cascade, this intra-membrane protease was cloned, expressed and purified for structural, biochemical and biophysical analysis. The preliminary data on this membrane protein is described in this chapter. The conclusions from the studies reported in this thesis and the scope for future work in this area is described in Chapter 5. Put together, the σK/RskA complex revealed facets of σ/anti-σ factor interactions that were hitherto unrecognized. The most prominent amongst these is the finding that an ECF σfactor can respond to multiple environmental stimuli. Furthermore, as seen in the case of the σK/RskA complex, the σ factor can itself serve as a receptor for redox stimuli. Although speculative, a hypothesis that needs further study is whether these features of the σK/RskA complex contribute to the variable efficacy of the M. bovis BCG vaccine. In this context it is worth noting that σK governs the expression of the prominent secreted antigens- MPT70 and MPT83. The studies reported in this thesis thus suggest several avenues for future research to understand mycobacterial diversity, immunogenicity and features of host-pathogen interactions. The appendix section is divided into two subparts- Appendix 1 of the thesis is a review on peptidase V. This is a chapter in The Handbook of Proteolytic enzymes (Elsevier Press, ISBN:9780123822192). Appendix 2 of the thesis includes technical details and an extended materials and methods section.

Mycobacterium tuberculosis has one ribosomal RNA operon. The survival of this bacillus thus depends on a transcription mechanism that can effectively couple gene expression to changes in the environment. σ factors are transcription proteins that bind to the RNA polymerase (RNAP) and dictate gene expression. Extra Cytoplasmic Function σ factors (ECF) are a subset of σ factors that coordinate environment-induced changes in transcription. The environment specific binding of ECF σ factors to the RNAP presents an effective mechanism for the bacillus to modulate gene expression. ECF σ factors, in turn, are regulated by their interaction with an anti-σ factor. The active σ factor is released from this complex upon specific cellular or environmental stimuli. The aim of this study was to understand the structural and mechanistic aspects of σ factor activation. Towards this goal, two ECF σ factors, σC and σL, were examined. Structural and biophysical studies on M. tuberculosis σC provided a novel insight into ECF σ factor regulation. Inter-domain interactions in σC were sufficient to occlude the DNA recognition regions even in the absence of an interacting protein. The structure of M. tuberculosis σL in complex with the anti-σ factor RslA provides a structural basis to rationalize the release of active σL under oxidative stress. The other chapters of this thesis include a description of the structure and biochemical features of a hypothetical protein Rv2704 that is co-transcribed with the primary σ factor σA. In an effort to understand the collaboration-competition-redundancy model of prokaryotic σ factors, we performed a computational analysis of this system compiling experimental data from the E. coli and B. subtilis model systems. These results are also presented in this thesis. Put together, the structural and biochemical characteristics of the σ factors presented in this thesis suggest substantial variations in the regulatory mechanisms of the M. tuberculosis σ factors when compared to the canonical E. coli or B. subtilis model systems. This thesis is organized as follows: Chapter 1: The introductory chapter of this thesis is organized to frame the pertinent mechanistic issues involved in the σ factor-regulatory protein interactions in the context of the underlying biology of M. tuberculosis. The first part of this chapter provides an overview of σ factors and a summary of the classification of these proteins and their roles in different prokaryotes. The latter part of this chapter is a summary of the pathogen M. tuberculosis in terms of its genetic composition, gene expression as well as aspects of virulence and pathogenecity. Chapter 2: This chapter describes the characterization of the ECF σ factor, σC. Here we report the structure of an ECF σ factor σC from M. tuberculosis. σC is essential for the lethality of M. tuberculosis in a mouse model of infection. Our studies suggest that M. tuberculosis σC differs from the canonical ECF σ factors as it has an N-terminal domain comprising of 126 amino acids that precedes the σC2 and σC4 domains. In an effort to understand the regulatory mechanism of this protein, the crystal structures of the σC2 and C4 domains of σC were determined. These promoter recognition domains are structurally similar to the corresponding domains of E. coli σA despite the low sequence similarity. Fluorescence experiments using the intrinsic tryptophan residues of σC2 as well as surface plasmon resonance measurements reveal that the σC2 and σC4 domains interact with each other. Mutational analysis suggests that the Pribnow box-binding region of σC2 is involved in this inter-domain interaction. Interactions between the promoter recognition domains in M. tuberculosis σC are thus likely to regulate the activity of this protein even in the absence of an anti-σ factor. Chapter 3 provides an account of the regulatory features of the ECF σ factor, σL. ECF σ factors are often regulated by their interactions with an anti-σ factor that can sense diverse environmental stimuli. Transcriptional responses to changes in the oxidation state are particularly important for M. tuberculosis as it adapts to the environment of the host alveoli and macrophages. Here we demonstrate that the protein RslA binds Zinc and can sequester σL in a reducing environment. Our data suggests that the cytosolic domain at the N-terminus of RslA alone is involved in binding σL. Under oxidizing conditions, the σL/RslA complex undergoes substantial conformational rearrangements that coincide with the release of the Zinc cofactor. In the absence of Zinc, the affinity of RslA for σL reduces by ca 8 fold compared to the holo form. The CXXC motif of RslA acts as a redox sensor. In response to oxidative stimuli, the proximal cysteines in this motif can form a disulfide bond with the release of the bound Zn2+ ion. This observation could be rationalized based on the crystal structure of the σL4/RslA complex. Put together, RslA is a distinct variant of the Zinc binding anti-σ factor (ZAS) family. The structural and biophysical parameters that control σL/RslA interactions demonstrate how variations in the rate of Zinc release and associated conformational changes in RslA could regulate the release of free σL in a measured response to oxidative stress. Chapter 4 is based on the biochemical and structural characterization of a hypothetical protein Rv2704. The gene for M. tuberculosis Rv2704 is located in the same operon as the principal σ factor σA. The biochemical and structural features of Rv2704 were thus examined to identify its role, if any, in the regulation of σA. This protein is a trimer in solution and adopts a chorismate mutase-like fold. The crystal structure reveals that Rv2704 is a member of the functionally diverse YjgF family of proteins. The important structural differences between Rv2704 and other YjgF proteins lie in the arrangement of secondary structural elements and the putative functional clefts between the subunit interface. Although Rv2704 does not interact with σA in vitro, the structural similarities to the YjgF family suggests that this protein could interact with a variety of metabolites, potentially influencing its function. Chapter 5 of this thesis is based on a computational analysis of σ factors. Four conformational segments of σ factors, referred to as σ1, σ2, σ3 and σ4 interact with specific regions of promoter DNA. ECF σ factors are a subset of σ factors that coordinate environment-induced transcription. ECF σ factors are minimalist σ factors with two DNA binding domains viz., σ2 and σ4 that recognize the –10 and –35 promoter elements and are unable to interact with either upstream-activating regions or the extended –10 element of the promoter. There are several ECF σ factors in a typical bacterium often characterized by substantial overlap in function. Here we present an analysis of B. subtilis ECF σ factors and their cognate promoters to understand functional overlap and redundancy in this class of proteins. As expected, conserved bases in the –10 element appear more critical for promoter selectivity than the –35 element. However, we note distinct conformational features in the –35 promoter interaction with the helix-turn-helix (HTH) motif when compared to a data-set of known HTH-DNA complexes. Furthermore, we note differences in –35 element interaction between σ factors that act alone and those that overlap in function. The σ factor promoter interactions were then examined vis-à-vis the estimated cellular concentration of these proteins and their affinity to bind the core RNAP. Put together, this analysis suggests that while the cellular protein concentration dictates the choice of an ECF σ factor to form a complex with the RNAP, conformational features of the –35 element serve to select potential collaborative members, a subset of which eventually initiate transcription. Collaborative arrangements and functional redundancy in ECF σ factors are thus possible within the limits placed by these two parameters. Chapter 6 is a summary of the work reported in this thesis and the conclusions that can be drawn based on these studies. The appendix section of this thesis comprises of technical details that were not included in the main text of this thesis. Appendix I describes the initial characterization of the M. tuberculosis σD/anti-σD complex. Appendix II provides the experimental protocols as well as some of the supplementary data to the work reported in Chapters 2-5 of this thesis.

Mycobacterium tuberculosis has one ribosomal RNA operon. The survival of this bacillus thus depends on a transcription mechanism that can effectively couple gene expression to changes in the environment. σ factors are transcription proteins that bind to the RNA polymerase (RNAP) and dictate gene expression. Extra Cytoplasmic Function σ factors (ECF) are a subset of σ factors that coordinate environment-induced changes in transcription. The environment specific binding of ECF σ factors to the RNAP presents an effective mechanism for the bacillus to modulate gene expression. ECF σ factors, in turn, are regulated by their interaction with an anti-σ factor. The active σ factor is released from this complex upon specific cellular or environmental stimuli. The aim of this study was to understand the structural and mechanistic aspects of σ factor activation. Towards this goal, two ECF σ factors, σC and σL, were examined. Structural and biophysical studies on M. tuberculosis σC provided a novel insight into ECF σ factor regulation. Inter-domain interactions in σC were sufficient to occlude the DNA recognition regions even in the absence of an interacting protein. The structure of M. tuberculosis σL in complex with the anti-σ factor RslA provides a structural basis to rationalize the release of active σL under oxidative stress. The other chapters of this thesis include a description of the structure and biochemical features of a hypothetical protein Rv2704 that is co-transcribed with the primary σ factor σA. In an effort to understand the collaboration-competition-redundancy model of prokaryotic σ factors, we performed a computational analysis of this system compiling experimental data from the E. coli and B. subtilis model systems. These results are also presented in this thesis. Put together, the structural and biochemical characteristics of the σ factors presented in this thesis suggest substantial variations in the regulatory mechanisms of the M. tuberculosis σ factors when compared to the canonical E. coli or B. subtilis model systems. This thesis is organized as follows: Chapter 1: The introductory chapter of this thesis is organized to frame the pertinent mechanistic issues involved in the σ factor-regulatory protein interactions in the context of the underlying biology of M. tuberculosis. The first part of this chapter provides an overview of σ factors and a summary of the classification of these proteins and their roles in different prokaryotes. The latter part of this chapter is a summary of the pathogen M. tuberculosis in terms of its genetic composition, gene expression as well as aspects of virulence and pathogenecity. Chapter 2: This chapter describes the characterization of the ECF σ factor, σC. Here we report the structure of an ECF σ factor σC from M. tuberculosis. σC is essential for the lethality of M. tuberculosis in a mouse model of infection. Our studies suggest that M. tuberculosis σC differs from the canonical ECF σ factors as it has an N-terminal domain comprising of 126 amino acids that precedes the σC2 and σC4 domains. In an effort to understand the regulatory mechanism of this protein, the crystal structures of the σC2 and C4 domains of σC were determined. These promoter recognition domains are structurally similar to the corresponding domains of E. coli σA despite the low sequence similarity. Fluorescence experiments using the intrinsic tryptophan residues of σC2 as well as surface plasmon resonance measurements reveal that the σC2 and σC4 domains interact with each other. Mutational analysis suggests that the Pribnow box-binding region of σC2 is involved in this inter-domain interaction. Interactions between the promoter recognition domains in M. tuberculosis σC are thus likely to regulate the activity of this protein even in the absence of an anti-σ factor. Chapter 3 provides an account of the regulatory features of the ECF σ factor, σL. ECF σ factors are often regulated by their interactions with an anti-σ factor that can sense diverse environmental stimuli. Transcriptional responses to changes in the oxidation state are particularly important for M. tuberculosis as it adapts to the environment of the host alveoli and macrophages. Here we demonstrate that the protein RslA binds Zinc and can sequester σL in a reducing environment. Our data suggests that the cytosolic domain at the N-terminus of RslA alone is involved in binding σL. Under oxidizing conditions, the σL/RslA complex undergoes substantial conformational rearrangements that coincide with the release of the Zinc cofactor. In the absence of Zinc, the affinity of RslA for σL reduces by ca 8 fold compared to the holo form. The CXXC motif of RslA acts as a redox sensor. In response to oxidative stimuli, the proximal cysteines in this motif can form a disulfide bond with the release of the bound Zn2+ ion. This observation could be rationalized based on the crystal structure of the σL4/RslA complex. Put together, RslA is a distinct variant of the Zinc binding anti-σ factor (ZAS) family. The structural and biophysical parameters that control σL/RslA interactions demonstrate how variations in the rate of Zinc release and associated conformational changes in RslA could regulate the release of free σL in a measured response to oxidative stress. Chapter 4 is based on the biochemical and structural characterization of a hypothetical protein Rv2704. The gene for M. tuberculosis Rv2704 is located in the same operon as the principal σ factor σA. The biochemical and structural features of Rv2704 were thus examined to identify its role, if any, in the regulation of σA. This protein is a trimer in solution and adopts a chorismate mutase-like fold. The crystal structure reveals that Rv2704 is a member of the functionally diverse YjgF family of proteins. The important structural differences between Rv2704 and other YjgF proteins lie in the arrangement of secondary structural elements and the putative functional clefts between the subunit interface. Although Rv2704 does not interact with σA in vitro, the structural similarities to the YjgF family suggests that this protein could interact with a variety of metabolites, potentially influencing its function. Chapter 5 of this thesis is based on a computational analysis of σ factors. Four conformational segments of σ factors, referred to as σ1, σ2, σ3 and σ4 interact with specific regions of promoter DNA. ECF σ factors are a subset of σ factors that coordinate environment-induced transcription. ECF σ factors are minimalist σ factors with two DNA binding domains viz., σ2 and σ4 that recognize the –10 and –35 promoter elements and are unable to interact with either upstream-activating regions or the extended –10 element of the promoter. There are several ECF σ factors in a typical bacterium often characterized by substantial overlap in function. Here we present an analysis of B. subtilis ECF σ factors and their cognate promoters to understand functional overlap and redundancy in this class of proteins. As expected, conserved bases in the –10 element appear more critical for promoter selectivity than the –35 element. However, we note distinct conformational features in the –35 promoter interaction with the helix-turn-helix (HTH) motif when compared to a data-set of known HTH-DNA complexes. Furthermore, we note differences in –35 element interaction between σ factors that act alone and those that overlap in function. The σ factor promoter interactions were then examined vis-à-vis the estimated cellular concentration of these proteins and their affinity to bind the core RNAP. Put together, this analysis suggests that while the cellular protein concentration dictates the choice of an ECF σ factor to form a complex with the RNAP, conformational features of the –35 element serve to select potential collaborative members, a subset of which eventually initiate transcription. Collaborative arrangements and functional redundancy in ECF σ factors are thus possible within the limits placed by these two parameters. Chapter 6 is a summary of the work reported in this thesis and the conclusions that can be drawn based on these studies. The appendix section of this thesis comprises of technical details that were not included in the main text of this thesis. Appendix I describes the initial characterization of the M. tuberculosis σD/anti-σD complex. Appendix II provides the experimental protocols as well as some of the supplementary data to the work reported in Chapters 2-5 of this thesis.

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2014
Tuberculosis is a pathological manifestation of the infection by Mycobacterium tuberculosis in humans. Facultative intracellular parasites, like M. tuberculosis, evolved mechanisms to subvert the proper functioning of their host cells to their benefit. In this thesis we token advantage of the characteristics of the fluorescent proteins to develop methodologies based on fluorimetry to quantify the macrophage internalization and the number of intracellular mycobacteria. The application of these techniques allowed: 1) to demonstrate that the GFP expressing mycobacteria may be used to test the effects of new drugs namely pyrazinoic acid esters against M. tuberculosis growth in liquid cultures. However GFP is not a good fluorophore to quantify intracellular mycobacteria. Instead we found tdTomato the election fluorophore to this purpose 2) To determine the effect of 120 genes in the host vesicular traffic during macrophage infection with M. tuberculosis. We identified 10 genes, 8 lead to a reduction and 2 an increase of macrophage M. tuberculosis burden. 3) To demonstrate that a reduction in macrophage internalization was the cause for the reduction in intracellular M. tuberculosis observed in some of the genes identified in 2) and the modulation of internalization by the microRNA miR-142-3p. Additionally, we studied the effect of Rab GTPases silencing in exosome secretion. MicroRNAs (miR) are small non-coding RNA molecules that regulate gene expression. Previous studies in our laboratory shown that miR-142-3p was up-regulated during early stages of M. tuberculosis macrophage infection. MiR142-3p binds to Wasl gene controlling the expression of N-wasp protein involved in signal transduction of membrane receptors and actin cytoskeleton. N-wasp silencing reduces M. tuberculosis internalization by macrophage. These studies demonstrate that M. tuberculosis regulates its internalization through N-Wasp. Taken together our results demonstrate the effect of specific host factors in the survival and/or internalization of M. tuberculosis by macrophages. Tuberculosis bacilli apparently modulate some of them. The development of new molecules in formulations that inhibit resistant M. tuberculosis strains is another strategy for the eradication of this pathogen from humans.
A tuberculose (TB) é a manifestação patológica da infecção do Mycobacterium tuberculosis no seu hospedeiro: os humanos. Esta doença afecta os humanos há milénios e constitui ainda um problema de saúde pública a nível global. A reemergência da TB após decadas de declínio resulta sobretudo da co-infecção com o vírus da imunodeficiência humana e do aparecimento de estirpes de M. tuberculosis resistentes aos antibióticos. O M. tuberculosis é um parasita intracelular facultativo, cujo principal nicho de sobrevivência são os macrófagos: as células do sistema imune cuja função é a destruição dos microrganismos. Após fagocitado pelo macrófago, o M. tuberculosis subverte o normal funcionamento do seu hospedeiro. Os fagossomas contendo o bacilo da tuberculose não maturam para fagolisossomas, não fundindo com endossomas tardios ou lisossomas, protegendo o bacilo dos factores microbicidas do macrófago. Apesar de o fagossoma estar bloqueado num estádio precoce da sua maturação este funde com outras vesículas do macrófago hospedeiro, fornecendo à micobactéria recursos para a sobrevivência e replicação. Em particular, multiplas publicações sugerem que diferentes proteínas do hospedeiro reguladoras do tráfego vesicular ou do citoesqueleto da actina poderão desempenhar um papel relevante na sobrevivência deste patogénio. Contudo, os mecanismos através dos quais é estabelecida esta subversão não são conhecidos. O desenvolvimento de metodologias de larga escala para triagem no estudo dos factores do hospedeiro envolvidos na infecção de macrófagos, pelo M. tuberculosis, têm sido refreados pelas características únicas desta micobactéria. A única metodologia disponível para a quantificação de micobactérias intracelulares é a contagem de unidades formadoras de colónias (UFC), uma técnica laboriosa e dispendiosa. Nesta tese aproveitamos as características das proteínas fluorescentes para desenvolvermos metodologias para a quantificação da internalização e sobrevivência de micobactérias em macrófagos e em culturas líquidas. O desenvolvimento dessas técnicas permitiu: 1) Desenvolver uma metodologia baseada nas propriedades das proteínas fluorescentes para a determinação da sobrevivência de M. tuberculosis intracelularmente em macrófagos infectados. 2) Determinar o efeito de cerca de 120 genes humanos do tráfego vesicular na carga do M. tuberculosis em macrófagos humanos. 3) Estudar o efeito dos genes validados no ponto 2) e do micro RNA mIR143-3p na internalização do M. tuberculosis por macrófagos humanos. 4) Adicionalmente foi estudado o efeito do silenciamento de Rab GTPases na secreção de exosomas. De forma a desenvolver um método alternativo ao UFC compatível com a possibilidade de rastreamento em larga escala da sobrevivência de micobactérias aproveitamo-nos das características das proteínas fluorescentes com o objectivo de monitorizar a infecção de macrófagos pelo M. tuberculosis. Numa primeira fase culturas líquidas de M. tuberculosis expressando GFP foram sujeitos à acção microbicida de vários antibióticos e determinadas as suas concentrações mínimas inibitórias por fluorimetria. Posteriormente foram utilizados vários esteres derivados do ácido pirazinóico em M. tuberculosis expressando GFP por fluorimetria tendo-se quantificado a sua actividade biológica. Todos os compostos apresentaram uma maior actividade biológica que a pirazinamida ou o ácido pirazinóico. Compostos com maior comprimento da cadeia linear do álcool e maior lipofilia estão positivamente correlacionados com a actividade biológica. Apesar de adequado para a quantificação da inibição de crescimento de M. tuberculosis em culturas líquidas, a proteína GFP não se demonstrou propicia para a quantificação de micobactérias intracelulares. A quantificação por fluorimetria de M. tuberculosis intracelulares expressando a proteína fluorescente vermelha tdTomato, demonstrou-se uma boa alternativa ao UFC. Adicionalmente, a quantificação da intensidade da fluorescência de macrófagos humanos THP-1 expressando GFP, permitiu quantificar tanto o número como a viabilidade dos macrófagos. O estabelecimento de uma metodologia de rastreamento em larga escala para a quantificação de M. tuberculosis intracelular, permitiu-nos proceder ao silenciamento sistemático de cerca de 120 genes do tráfego vesicular humano e quantificar o seu efeito na carga de M. tuberculosis em macrófagos. Os candidatos obtidos no rastreamento foram posteriormente validados quanto à sua expressão genética. Identificámos 10 genes com efeito na carga intracelular de M. tuberculosis em macrófagos, em 2 genes, foi observado um aumento e em 8 genes, uma redução da carga de M. tuberculosis em macrófagos humanos. Os candidatos foram maioritariamente proteínas Rab, proteínas reguladoras e efectoras de Rab e proteínas do tráfego vesicular não classificadas. De forma a quantificar o contributo da internalização nos resultados observados no rastreamento dos 120 genes, desenvolvemos uma metodologia baseada em citometria de fluxo. Determinamos que, para micobactérias virulentas e avirulentas, a grande maioria dos genes testados são, provavelmente, reguladores positivos da internalização. Estas observações justificaram e confirmaram alguns dos resultados obtidos no rastreamento genómico e propõem um papel activo das micobactérias na modulação da sua internalização por macrófagos humanos. De forma a validar biologicamente alguns dos candidatos, estes foram silenciados novamente e foi quantificada a carga micobacteriana em macrófagos utilizando a metodologia convencional, as unidades formadoras de colónias. Com os resultados observados ficou demonstrado que a proteína Rab7a induz um aumento da carga macrofágica de M. tuberculosis. O silenciamento de Rab34 e Sintaxina 4 induz uma redução da carga de M. tuberculosis em macrófagos humanos sendo esta redução, possivelmente devida à redução da internalização nestas células. Os micro RNAs (miR), recentemente descritos, são uma nova classe de moléculas que controlam eventos pós-transcripcionais durante a expressão genética. Os miR ligam-se a regiões especificas do RNA mensageiro (RNAm) de uma proteína bloqueando a tradução proteica e/ou levando à degradação do RNAm. Os miRs controlam selectivamente a expressão de proteínas de diferentes vias metabólicas das células. Estudos feitos no nosso laboratório demonstraram que o miR-143-3p está sobre-expresso nos estádios iniciais da infecção de M. tuberculosis em macrófagos. O miR-143-3p liga-se ao mRNA do gene Wasl controlando a expressão da proteína N-Wasp, um transdutor de receptores membranares e do citoesqueleto de actina. O silenciamento da N-Wasp reduz a internalização de M. tuberculosis pelos macrófagos. Estes resultados demonstram a regulação de N-Wasp pelo M. tuberculosis para controlar a sua internalização. Os exossomas são vesículas com um diâmetro de 30 a 100 nm que são secretadas por diversos tipos de células. Os exossomas desempenham um papel importante na sinalização entre células e em macrófagos têm um papel de apresentação de antigénios. Os exossomas secretados por macrófagos infectados por M. tuberculosis são pró-inflamatórios in vitro e in vivo. No laboratório do Dr. Amigorena foi desenvolvida uma metodologia para quantificação de exossomas através de pérolas de latex conjugadas dom CD63 e a utilização de citometria de fluxo. Em colaboração com o Dr Ostrwsky, do laboratório do Dr Amigorena, Instituto Curie, Paris, França, efectuei um rastreio de larga escala para determinação dos genes do tráfego intracelular envolvidos na secreção de exossomas em células HeLa B6H4. Nesse estudo a proteína Rab27a e Rab27b foram envolvidas na secreção de exossomas em células humanas. Era nosso objectivo determinar se tal se verificava em macrófagos humanos, no contexto da infecção do M. tuberculosis, não foi possível por razões alheias à nossa vontade. Conjuntamente os dados apresentados nesta tese demonstram o efeito de factores específicos do hospedeiro que influenciam a sobrevivência e/ou internalização do M. tuberculosis por macrófagos, alguns deles aparentemente modulados pelo bacilo da tuberculose. Em particular, os dados sugerem que o M. tuberculosis desenvolveu um mecanismo específico de invasão dos macrófagos alveolares, provavelmente, para estabelecer o seu nicho replicativo sem activar em demasia o sistema imune do hospedeiro. Estes factores do hospedeiro, e em especial as proteínas que as codificam ou regulam são potenciais alvos para o desenvolvimento de drogas orientadas para a modulação do hospedeiro de forma a inibir ou controlar a infecção por M. tuberculosis. O desenvolvimento de novas drogas em formulações que inibam estirpes de M. tuberculosis resistentes é outra estratégia de erradicação deste patogénio nos humano.
Fundação para a Ciência e a Tecnologia (FCT)

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) has infected one third of the world’s population and continues to claim more lives annually than any other infectious disease agent. A significant proportion of individuals carry latent TB infection (LBTI) which is characterized by the absence of clinical symptoms and it has been postulated that the tubercle bacilli are in a dormant-like state during this type of infection. Resuscitation promoting factors (Rpfs) are cell wall hydrolases which cleave glycosidic bonds within the peptidoglycan (PG), a mechanism thought to result in reactivation of bacteria from the state of dormancy. M. tuberculosis encodes five rpf-like homologues which are collectively dispensable for growth but are required for reactivation from dormancy in vitro, and for virulence in the mouse model of infection. LTBI thus poses a huge threat to the global burden of active disease. The purpose of this study was to further investigate the biological roles of Rpfs by assessing the effects of rpf gene deletion in M. smegmatis, a model organism used for TB research. M. smegmatis encodes four rpf-like genes designated rpfA, rpfB, rpfC and rpfE, and deletion mutants that lack one or more of these genes were constructed by allelic exchange mutagenesis. M. smegmatis mutant strains that lack either rpfA or rpfB display no significant differences in growth both on solid and in liquid medium when compared to wild type. However, loss of rpfA resulted in bacterial clumping during stationary-phase growth in broth culture and changes in cell morphology. Moreover; the rpfA rpfB double mutant and its derivative strain lacking rpfC, rpfA rpfB rpfC displayed a ca. 2-4 log increase in susceptibility to erythromycin and vancomycin. Furthermore, unusual colonial morphologies with reduced serpentine cording and smooth peripheries were observed for these multiple mutants. Cell surface defects and cell distortions were evidenced as wrinkle-textured, bent cells and polar tip bulges for the abovementioned mutants. The multiple deletion strains also displayed a defect in biofilm formation revealing an inability for the mutants to form complex cell-cell interactions. Collectively, the data are suggestive of a loss of bacterial cell wall integrity due to rpf gene deletion and it is proposed that rpfA, in combination with other genes, is largely responsible for cell wall integrity maintenance. Our data indicate that these factors play an important role in cell growth and division and therefore represent an untapped source of novel targets for anti-tubercular drug discovery.

博士
慈濟大學
醫學科學研究所
101
The annual incidence of new pulmonary tuberculosis cases in Eastern Taiwan was significantly higher than average incidence across Taiwan. In the first chapter, the theme of the research is to explore Mycobacterium tuberculosis (Mtb) outbreaks amongst hosts. Take advantage of the molecular tools combined spoligotying and mycobacterial interspersed repetitive unit-variable tandem-repeat (MIRU-VNTR), two PCR-based techniques, we may identify Mtb genotype and explore the multi drug-resistant tuberculosis (MDRTB) transmission in Eastern Taiwan. The results showed a 61.6% cluster ratio between MDRTB infected subjects in Eastern Taiwan, especially amongst indigenous groups. MDR strains might cause widespread transmission contrasted with the previous view that MDR-TB was less infectious. In chapter 2, we intend to investigate if diabetes mellitus (DM) is correlated with the infection of drug-resistant tuberculosis strains. Also, we stratified the subjects according to their tuberculosis treatment history. The host immunity of diabetic patients may be poor compare to those without diabetes; more diabetic subjects are supposed to be re-treated for tuberculosis infection. Irrespective of new or previously treated status, DM was found to be significantly associated with isoniazid-related resistant strains of tuberculosis both in new and re-treated subjects, which showed an adjusted odd ratio: 6.76 compared to those without DM. In this retrospective investigation, we did not find the significant association between DM and MDRTB, which may be attributed to the limited sample size. In chapter 3, we animal model was utilized to investigate defense mechanism of host against tuberculosis infection via a molecular perspective. Indoleamine 2,3-dioxygenase 1 (IDO1) is as a rate limiting enzyme in tryptophan metabolism and could exert a tolerogenic function in regulating the immune system. So far, no evidence proved that IDO1 can exert a killing effect against M. tuberculosis in vivo despite the fact that IDO1 own antimicrobial property against some pathogenic microbes. In this thesis, relevant experiments were conducted majorly upon its immuno-regulatroy effect in the mice model. IDO1 interacts with regulatory T cell by a direct contact fashion and is crucial in specifying to the development of a Foxp3 positive regulatory lineage during the CD4+ T cell lineage differentiation; in addition, tryptophan metabolites were reportedly to participate in the development of Th1 and Th17 cell differentiation. The mechanisms how IDO1 gene regulate the immune homeostasis in tuberculosis infection were less documented. In our preliminary data, tuberculosis infection might lead to distinct difference in mortality between IDO1 knockout mice and their counterparts with C57BL/6 background. Thus far we have shown that enzymatic degradation of tryptophan might be restored by alternative pathways in later stage of Mtb infection in IDO1-/- mice. However, its immune modulating ability may not be replaced and contributed to the distinct mortality phenotypes. Moreover, different doses of Mtb antigens exposure might act significantly in host’s immune homeostasis, which has shown in our study to influence the mortality between IDO1-/- and WT mice. In 1600CFU Mtb CFU infection via aerosol route, there was an earlier mortality in IDO1 -/- mice; a persistent higher proportion of neutrophil influx into the lungs, which was assumed at least to be link with Th17 lineage specification and IL-17a chemo-attraction. Also, the mortality was suspected to be partially associated with the IL-10 production of neutrophils. In this study, we also investigated the IDO2 response to Mtb infection.

The mechanism of prokaryotic transcription has been characterized primarily in the classic system, Escherichia coli, and cannot be confidently extended to include other prokaryotic species, such as those of the Actinobacteria phylum. Actinobacteria represents a diverse group of Gram-positive species that range from soil dwellers to obligate pathogens, such as Mycobacterium tuberculosis (Tb). These species encode RNA polymerase (RNAP) binding proteins that are not present in model organisms, and therefore present a unique lens through which the basic mechanism of transcription can be further explored outside of model systems. In addition, these mechanisms of transcriptional regulation can be studied in the context of M. Tuberculosis pathogenesis. The model we use for tuberculosis is Mycobacterium Smegmatis, a homologue, which has a faster doubling time and is only Biosafety level 1. Within Actinobacteria, notable conserved RNAP binding proteins include RNA polymerase binding protein A (RbpA) and CarD. RbpA is specific to Actinobacteria, binding the β subunit of RNAP and primary σ factors. CarD binds to the β subunit and associates with DNA. Both proteins are upregulated upon exposure to stress, and have implications in the initiation of rRNA transcription. Each is proposed to stimulate the formation of transcriptionally competent RNAP-holoenzyme open promoter complexes, and CarD is thought to act as a global transcriptional regulator. RbpA and CarD are believed to be essential in M. Tuberculosis and M. Smegmatis. Recent structural analyses of RbpA and CarD suggest the two proteins may share a region of similarity that could compete for binding to the β subunit, and brings into question whether the two proteins are capable of coordinately modulating transcription or antagonize each other's activity. This was investigated through purification of CarD and RbpA and in vitro studies performed with [α-32P] Uridine triphosphate used to measure the level of transcription. These experiments led to the conclusion that RbpA and CarD are able to associate with the same RNAP and have an additive stabilizing action on the polymerase. Whether or not RbpA is an essential protein was also investigated genetically, and by using a Tetracycline on/off system. Sigma factors play an important role in transcription due to their ability to recognize promoter regions and initiate transcription. One connection that we have preliminary data for, through DNA pull downs, is that sigF binds rRNA promoters, and CarD and RbpA are both studied in the context of rRNA transcription. Therefore sigF is another factor that could be regulating rRNA transcription, possibly during stress. SigF is also the sigma factor that responds to oxidative stress, and CarD is involved in oxidative stress. Sigma F is a member of a family of 13 different sigma factors that are preset in M. Tuberculosis. There are two different types of sigma factors: primary, which are essential for normal growth, and alternative, which are typically expressed during differing environmental conditions. Sigma F has been shown to be upregulated during oxidative stress, which is why it was of particular interest to us. To investigate the roles of sig F, we exposed sig F deletion mutants and wild type strains to oxidative stress and measured ribosomal RNA production by reverse transcription quantitative real time PCR. It was concluded that sigF is a probable suppressor of rRNA when exposed to oxidative stress.

Submitted in fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry, Durban University of Technology, Durban, South Africa, 2017.
In drug discovery and development of anti-tubercular therapeutics, it is necessary to study the physiology and genetics of the molecular mechanisms present in the Mycobacterium tuberculosis. The virulence of M. tuberculosis is attributed to its unique genome, which contains a high frequency of glycine-rich proteins and genes involved in the metabolism of the fatty acids. Consequently, the presence of a diversity of the pathogenic pathways such as acid tolerance and drug resistance mechanisms in M. tuberculosis makes the treatment of Tuberculosis (TB) challenging. However, the molecular basis of the virulence factors involved in the pathogenesis is not fully understood. Accordingly, the current study focuses on better understanding of the pathogenic proteins present in this bacterium using available computational techniques. In South Africa, there is an alarming increase in the drug-resistant TB in HIV co-infected patients, which is one of the biggest challenges to the current anti-tubercular therapies. An extensive literature search showed that the mutations in the virulent proteins of M. tuberculosis resulted in the development of drug tolerance in the pathogen. The molecular and genetic studies identified frequently occurring point mutations associated with the drug resistance in proteins of M. tuberculosis. Despite the efforts, TB infection is still increasing because different pathogenic pathways in the bacterial system are still undiscovered. Therefore, this study involves an in silico approach aimed at the identification of novel drug resistance implicated point mutations. The site- directed mutations leading to the development of resistance against four first-line drugs (Ethambutol, Isoniazid, Rifampicin, and Streptomycin) were studied extensively. In the primary investigation, pathogenic mutational landscapes were classified in the sequences of the studied proteins. The effects of these mutations on the stability of the proteins were studied using diverse computational techniques. The structural basis of the point mutations with the highest destabilizing effects was analyzed using the principles of the Density Functional Theory (DFT), molecular docking and molecular dynamics (MD) simulation studies. The varied conformational behavior resulted from these predicted substitutions were compared with the experimentally derived mutations reported in the literature. The outcome of this study enabled the identification of the novel drug resistance-associated point mutations which were not previously reported. Furthermore, a detailed understanding of the conformational behavior of diverse virulent proteins present in M. tuberculosis was also generated in this study. Literature study showed that inside the host’s macrophage cells, the virulent proteins such as isocitrate lyase, lipase lipF, magnesium transporter MgtC, porin protein OmpATb, a protein of two component systems PhoP, Rv2136c and Rv3671c have an established role in the development of the acid tolerance. On the other hand, information regarding their role in the acid resistance is scarce. Accordingly, the structural basis of their role in acid resistance was analyzed using constant pH based MD simulations. In the studied proteins, the lipF and PhoP showed highest structural stability in highly acidic conditions throughout the course of MD simulations. Therefore, these proteins may play a primary role in the process of resistance. In addition to these pathogenic proteins, there is a need to identify new undiscovered virulent proteins in the genome of M. tuberculosis, which increases the efficiency of the current therapy. The knowledge generated by the analyses of the proteins involved in resistance and pathogenic mechanisms of M. tuberculosis forms the basis for the identification of new virulence factors. Therefore, an in silico protocol was used for the functional annotations and analyses of the virulence characteristics. M. tuberculosis contains 1000 Hypothetical Proteins (HPs), which are functionally uncharacterized proteins and their existence was not validated at the biochemical level. In this study, the sequences of the HPs were extensively analyzed and the functions of 662 HPs were successfully predicted. Furthermore, 483 HPs were classified in the category of the enzymes, 141 HPs were predicted to be involved in the diverse cellular mechanisms and 38 HPs may function as transporters and carriers proteins. The 307 HPs among this group of proteins were less precisely predicted because of the unavailability of the reliable functional homologs. An assessment of the virulence characteristics associated with the 1000 HPs enabled the classification of 28 virulent HPs. The structure of six HPs with highest predicted virulence score was analyzed using molecular modelling techniques. Amongst the predicted virulent HPs, the clone for Rv3906c purchased from the DNASU repository because of the ease of its availability. The gene of Rv3906c was isolated and cloned into a pET-21c expression vector. The analyses of the nucleotide sequence showed that Rv3906c gene (500 bp) encodes a 169 amino acid protein of molecular weight 17.80 kDa (~18.0 kDa). The sequence analyses of Rv3906c showed that the HPs showed high similarities with pullulanase, a thermophilic enzyme. The stability profile at different temperatures for Rv3906c generated using MD simulations showed that Rv3906c maintained its structural identity at higher temperatures. It is expected that this study will result in the design of better therapeutic against the infection of M. tuberculosis, as novel undiscovered virulence factors were classified and analyzed in addition to the conformational profiles of the virulent proteins involved in the resistance mechanisms.
M

碩士
國立臺灣大學
微生物學研究所
107
Human tuberculosis, also known as "the white plague", mainly results from Mycobacterium tuberculosis infection which was found in early human history. Due to the long incubation period and drug resistance of M. tuberculosis, the researches of diagnosis and treatment in tuberculosis still remain a considerable development space. Generally, many studies on M. tuberculosis and tuberculosis use Mycobacterium marinum with higher safety and faster growth rate as a model in laboratory works. M. marinum shows high conservation with M. tuberculosis in genetic relationship and the type seven secretion system, which is critical for virulence. In our study, first we used five constructed M. marinum ESX-1 gene deletion mutants: ∆espE, ∆espF, ∆espG, ∆espH and ∆eccA1 to investigate the role of the type seven secretion system ESX-1 in NLRP3 inflammasome activation. After infecting THP-1 differentiated macrophages with these deletion mutants respectively, we found ∆espG induced less IL-1β in cell supernatant through enzyme-linked immunosorbent assay (ELISA). Our data from Western blot also showed ∆espG reduced activated caspase-1 secretion in infected macrophages. Additionally, THP-1 cells pretreated with NLRP3 inhibitor before infected with wildtype M. marinum showed the difference in secretion of IL-1β, while ∆espG-infected cells did not show this pattern. The data could indirectly demonstrate that M. marinum induces IL-1β through activating NLRP3 inflammasomes, and espG may involve in NLRP3 inflammasome activation. Subsequently, we constructed the M. tuberculosis espG1 deletion mutant and the espG1 complemented strains, infected THP-1 cells with M. tuberculosis, and analyzed the protein expression level of IL-1β and caspase-1 in cell lysate and supernatant. Although M. tuberculosis espG1 deletion mutant reduced the secretion of IL-1β significantly, the complemented strain did not repair the function of espG1 completely. In view of the previous studies that ESAT-6 in M. tuberculosis is involved in activation of NLRP3 inflammasomes, we used Western blot to analyze whether the difference in IL-1β secretion is related to the expression of ESAT-6 in M. marinum espG and M. tuberculosis espG1. According to the results, the ESX-1 major virulent proteins, ESAT-6 and CFP-10, showed substantial decrease in both M. marinum ∆espG and M. tuberculosis ∆espG1::pMN437 supernatants respectively, but were increased in bacterial lysates. Thus, we suggested that M. marinum espG gene and M. tuberculosis espG1 gene are involved in secretion of ESAT-6 and CFP-10.

Tuberculosis is a re-emerging global health problem. Bacille Calmette-Guerin (BCG), the available vaccine against the disease, is only effective short term and is associated with adverse reactions clinically. The development of new effective vaccines will require an understanding of virulence, immunogenic factors and the beneficial immune responses induced in the human host. My thesis investigates phoP and whiB3, two genes associated with virulence and immunogenicity in Mycobacterium tuberculosis. Study of PhoP in a natural phoP mutant, BCG-Prague, and in the clinically safe BCG-Japan, shows that over-expression of PhoP increases the immunogenicity of these vaccine strains. In addition, I found that WhiB3 impacts carbon metabolism in BCG-Birkhaug and BCG-Sweden, although the effect of this on virulence in vivo is still unclear. The characterization of genes involved in virulence and immunogenicity allows us to develop novel approaches for improving the efficacy of BCG, which has important implications for future TB vaccine development.

Tese de mestrado. Biologia (Microbiologia Aplicada). Universidade de Lisboa, Faculdade de Ciências, 2013
A tuberculose é uma doença infecciosa que tem acompanhado historicamente a Humanidade. Caracteriza-se tipicamente por uma infecção pulmonar, manifestando-se através de tosse persistente, febre e especialmente por uma fraqueza generalizada. Apesar da sua ancestralidade, é ainda hoje a doença infecciosa responsável pelo maior número de mortes a nível mundial. O agente etiológico da tuberculose é uma bactéria, Mycobacterium tuberculosis, descrita pela primeira vez por Robert Koch no século XIX. Pertence à ordem Actinomycetales e à família das Mycobacteriaceae; são bacilos, Gram-positivos, e apresentam por norma um crescimento lento e uma membrana com uma composição invulgar, rica em lípidos particulares como o lipoarabinomanano. M. tuberculosis é um patogénio particularmente resistente por se encontrar extremamente adaptado ao hospedeiro humano, e o problema agrava-se com a actual epidemia do vírus de imunodeficiência humana (VIH). Indivíduos infectados com VIH não só têm uma probabilidade maior de desenvolver tuberculose, como apresentam morbilidades mais elevadas, sendo a tuberculose a principal causa de morte entre doentes de VIH. Outro dos obstáculos mais significativos é a resistência a antibióticos, que nas últimas décadas se tem tornado mais proeminente em M. tuberculosis. Várias ocorrências de resistência aos antibióticos de primeira e segunda linha foram já documentados, e o surgimento de estirpes multirresistentes representa um sério problema de saúde pública. Continua a não existir uma vacina eficaz para substituir a BCG (Bacilo Calmette-Guérin, estirpe atenuada do agente da tuberculose bovina Mycobacterium bovis), que está em uso desde 1921, e cuja eficácia na protecção contra a tuberculose pulmonar em adultos é manifestamente insuficiente. Muita da dificuldade em combater a tuberculose está também relacionada com a sua prevalência e persistência. Segundo a Organização Mundial de Saúde, um terço da população mundial pode ser portadora de M. tuberculosis no seu estado latente, ou seja, sem desenvolver doença activa. Ainda que a doença não seja contagiosa neste estado, a sua presença no hospedeiro pode vir a manifestar-se de forma oportunista em casos de enfraquecimento imunitário, como por VIH, drogas imunossupressivas, doença concomitante ou simplesmente pelo envelhecimento. Por estas razões a investigação no combate à tuberculose está cada vez mais a dirigirse no sentido de compreender os sistemas de imunidade do hospedeiro e a sua relação com este patogénio. A pedra angular da interacção patogénio-hospedeiro é a fagocitose. É um processo de imunidade inata altamente conservado evolutivamente através do qual células eucarióticas são capazes de internalizar e digerir partículas externas ou microorganismos, eliminandoos. Certas células do sistema imunitário humano servem-se deste processo para neutralizar patogénios invasores, denominando-se células fagocitárias, ou fagócitos, entre os quais se destacam os macrófagos. M. tuberculosis é tipicamente inalado por aerossóis e nos pulmões entra em contacto com os macrófagos alveolares residentes, que oferecem a resposta fagocitária inicial. Da fagocitose fazem parte várias etapas. Tem início pela internalização das partículas, que consiste na captura e envolvimento do alvo numa vesícula endocítica. Esta é depois transportada pelo citoplasma, via elementos do citoesqueleto, e vai fundindo com outras vesículas endocíticas. Quando o grau de maturação é compatível funde-se com lisossomas, cujo conteúdo enzimático assegura uma degradação proteolítica do alvo. A este processo dá-se o nome de biogénese do fagolisossoma, e nele inicia-se a acção de proteases denominadas catepsinas, bem como óxidos nítricos e superóxidos de efeito bactericida. Depois de digerido o conteúdo fagocitado, os antigénios resultantes serão utilizados para despoletar uma resposta imunitária adquirida, levada a cabo por células T. Quando a partícula a fagocitar é o M. tuberculosis, este é capaz de subverter os mecanismos de defesa do fagócito, conseguindo sobreviver e multiplicar-se no interior da célula hospedeira. Sendo complexo o arsenal molecular de que o fagócito dispõe para combater invasores, também o é o modo como esta micobactéria o evita. O M. tuberculosis consegue impedir a maturação do fagossoma, controlando o hospedeiro através de processos que ainda não são totalmente compreendidos. Sabe-se, porém, que influencia a função de proteínas de tráfego vesicular e transporte membranar, bem como as referidas catepsinas. Sendo estes alguns dos alvos da sua virulência, tornam-se também importantes alvos de investigação biomédica, pois podem ajudar a combater a estratégia de infecção de M. tuberculosis, fortalecendo as defesas do hospedeiro. A fase de internalização das bactérias por macrófagos é um ponto crítico na infecção, uma vez que representa um dos primeiros contactos entre o patogénio e o hospedeiro, e é através dela que têm início todos os restantes mecanismos. Interessa pois compreender como é regulada a internalização, e que influência terá M. tuberculosis nessa regulação. Para estudar devidamente factores de internalização, é necessário em primeiro lugar definir o modelo de internalização de partículas inertes para estabelecer o que ocorre na fagocitose como fenómeno abrangente. Criámos um desenho experimental que nos permitiu explorar um grande número de variáveis. Fizemos variar o tipo de micobactéria: destruída no fagócito (caso de M. smegmatis) versus sobrevivente intracelular (caso de M. tuberculosis), o tempo, a multiplicidade de infecção (MOI, representa o número de bactérias por cada macrófago) e o tipo de macrófago. Para este trabalho foram utilizados três tipos de macrófagos: macrófagos derivados de monócitos humanos (HMDM), macrófagos humanos da linha celular THP-1 e macrófagos de ratinho da linha celular J774. Utilizámos também três espécies dentro do género Mycobacterium: M. tuberculosis, M. bovis BCG e M. smegmatis, sendo as duas últimas consideradas não-patogénicas. Obtendo os valores de internalização ao longo de três pontos no tempo (30 minutos, 1 hora e 4 horas) tornou-se possível traçar perfis de internalização para cada combinação de variáveis. As estirpes à nossa disposição expressavam proteína verde fluorescente (GFP) por meio de um plasmídeo replicativo, o que permitiu que os macrófagos que internalizaram micobactérias pudessem ser identificados em citometria de fluxo através da fluorescência no comprimento de onda do verde. Armados desse desenho experimental e de um protocolo de infecção eficiente que permitiu testar várias variáveis em simultâneo, descrevemos curvas de internalização para as várias combinações de macrófago-micobactéria. Destes resultados foi possível chegar à conclusão esperada de que a internalização é fortemente influenciada pelo tempo e pela multiplicidade de infecção, ou seja, estes factores condicionam fortemente a resposta fagocitária dos macrófagos. Tornou-se também evidente que os HMDM são os agentes fagocitários mais eficientes de entre os macrófagos testados, tendo sido capazes de atingir elevados níveis de internalização mais rapidamente que os restantes. Uma observação que se nos revelou pertinente foi a de que ao fim de apenas 30 minutos, todos os tipos de macrófagos foram capazes de realizar uma considerável internalização das micobactérias na amostra. Põe-se assim em causa muito do background experimental em micobactérias, que prevê infecções de duração igual ou superior a 3 horas. Este nosso trabalho sugere que infecções mais breves são não apenas funcionais, mas até preferíveis para o estudo de reguladores das fases iniciais de fagocitose, como é o caso da internalização. Infecções demasiado prolongadas podem levar à perda desta informação e a uma alteração do estado fisiológico dos macrófagos, particularmente com cargas bacterianas elevadas. Foi também importante observar que as diferentes espécies de Mycobacterium apresentam diferenças ao nível da internalização. M. smegmatis foi internalizada mais rapidamente e em maiores quantidades, seguida por M. bovis BCG e esta por M. tuberculosis. Ora, a esta ordem de internalização corresponde a ordem de inversa de patogenicidade, ou seja, com o aumento da patogenicidade diminui a internalização. Levanta-se imediatamente a sugestão de que a diminuição da internalização será um dos processos de virulência de M. tuberculosis. Parece haver uma contradição, pois sendo M. tuberculosis um patogénio intracelular esperar-se-ia que promovesse a sua internalização o mais possível. No entanto, uma carga microbiana demasiado elevada sobre as células do hospedeiro leva a processos de morte celular, privando assim M. tuberculosis do seu habitat. Força-se então um equilíbrio cuja regulação importa compreender. Para investigar o papel de factores do hospedeiro que influenciem a internalização, realizámos uma triagem sobre 71 proteínas envolvidas em tráfego vesicular e na maturação do fagossoma, a maioria pertencente aos grupos das Rab guanosina trifosfatases (Rab GTPases), catepsinas e cistatinas. Pelo silenciamento de cada uma das proteínas individualmente torna-se possível avaliar a sua contribuição para o processo de internalização, ou seja, descobrir a sua função por redução ou ausência-de-função. O silenciamento individual das proteínas foi feito por intermédio de bibliotecas lentivirais contendo plasmídeos que codificam para um pequeno RNA de interferência (hairpin), complementar ao RNA mensageiro de um gene específico. Graças a essa complementaridade o RNA mensageiro é destruído, impedindo-se assim a produção da proteína correspondente. Assim construímos uma linha knockdown de macrófagos THP-1, a qual submetemos a ensaios semelhantes aos que desenvolvemos na primeira parte dos trabalhos. Na abordagem experimental, as variáveis de tempo e MOI foram restringidas a apenas 1 hora e uma MOI de 10, pois estes valores representam um estímulo mais moderado e permitem assim realçar as eventuais diferenças provocadas pelos silenciamentos. Utilizámos nesta fase apenas M. tuberculosis e M. smegmatis na qualidade de controlo não-patogénico. Nestas condições, a grande maioria dos silenciamentos provocou um decréscimo na internalização de micobactérias, o que levou a que as suas proteínas-alvo fossem classificadas como potenciais reguladores positivos da internalização. Aplicando limites para aquilo que deveria ser considerada uma diferença significativa face aos controlos nãosilenciados, 26 proteínas emergiram como reguladores, sendo por isso denominados os hits da triagem. Este é um número surpreendentemente elevado, tendo em conta que as bibliotecas lentivirais não foram desenhadas especificamente para ensaios de internalização. Destes hits destacam-se especialmente aqueles que se manifestaram apenas em infecções com uma das micobactérias, pois permitem inferir uma relação directa com a patogenicidade. É o caso da catepsina L, identificada como hit apenas para M. smegmatis. Considerando que a ausência desta proteína levou a que M. smegmatis fosse internalizado em menor nível e, tendo presente a correlação demonstrada entre baixa internalização e patogenicidade, pode dizer-se que nesta situação M. smegmatis se comportou como uma micobactéria patogénica. Daqui se retira que o silenciamento da expressão de catepsina L pode ser uma das vias através das quais M. tuberculosis manifesta a sua patogenicidade. Três outras proteínas demonstraram um papel regulatório na internalização apenas em infecções com M. tuberculosis: Rab37, Rab38 e catepsina W. Desta última são conhecidas funções na regulação da citotoxicidade de linfócitos, mas não é claro o seu mecanismo de acção. No caso de Rab37 e Rab38, este trabalho representa a primeira associação destas à infecção por M. tuberculosis. Tratando-se de uma triagem abrangente, só será possível definir o modo de funcionamento destas proteínas com novas experiências no futuro. Os restantes 22 hits não são menos importantes; apesar de conhecida a sua associação à tuberculose, demonstraram pela primeira vez ser reguladores positivos da internalização. Há limitações ao estudar infecções in vitro, pois as conclusões a retirar não são necessariamente igualmente válidas para o contexto real de infecções por M. tuberculosis. As diferenças acentuam-se se considerarmos que neste trabalho experimental se simulou um cenário de uma primo-infecção, ou seja, o primeiro contacto entre M. tuberculosis e células humanas, sendo este um cenário artificial que não corresponde à maioria dos casos de tuberculose em humanos. É uma concessão necessária para a investigação experimental e apesar de tudo não impede que estas descobertas tenham potencial terapêutico. As proteínas aqui identificadas poderão servir para controlar mais eficazmente a infecção, se aprofundado o conhecimento sobre a sua função. Esta tese apresenta pois uma nova abordagem experimental, na forma de infecções breves e eficientes e de novos intervenientes nos primeiros passos do equilíbrio dinâmico entre patogénio e hospedeiro.
Mycobacterium tuberculosis is a very successful pathogen that is able to survive and multiply inside the host’s immune cells, thus triggering the active disease of tuberculosis or alternatively entering into an asymptomatic latent state. Its success lies on a powerful subversion of the fundamental innate immunity process of human-host cells: phagocytosis. M. tuberculosis is known to resist destruction by phagocytes through a complex molecular sabotage of phagosome maturation, an integral part of the phagocytic process. On the other hand, internalization, the first and arguably most important step of phagocytosis, has not been so thoroughly investigated. In this work we outline a method for quantifying internalization of green fluorescent mycobacteria through flow cytometry. With it we provide profiles of internalization-over-time for J774, THP-1 and human monocyte-derived macrophages (HMDM), which were infected with M. tuberculosis, M. bovis BCG and M. smegmatis. From these experiments we gathered that HMDM are the most efficient in internalizing mycobacteria, that there is considerable internalization even after short time periods such as 30 minutes and also that M. tuberculosis is internalized to a lesser extent than its non-pathogenic counterparts. It is suggested that M. tuberculosis manipulates its own internalization. In order to decipher which are the host factors targeted by this subversive strategy, we performed an internalization-based screening on knockdown THP-1 macrophages. Protein knockdowns were achieved by shRNA interference from lentiviral libraries targeting a total of 71 proteins. Using the previously designed method, we identified 26 putative positive regulators of internalization including a large number of Rab GTPases and cathepsins, proteins associated with vesicular trafficking and proteolysis in the mature phagosome. Through this novel approach we were able to quantify rates of internalization, which were previously only empirically estimated, and use them as a model to highlight new potential therapeutic targets in the ongoing battle against tuberculosis.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Doctor of Philosophy February 2013
Mycobacterium tuberculosis is presented with environmental host assaults that damage its DNA during infection. Tubercle bacilli possess mechanisms to protect against moststresses imposed by the host, including genotoxic stress. However, tolerance of DNA lesions that have escaped the normal repair processes requires the function of specialist DNA polymerases that can introduce mutations during translesion synthesis (replication by-pass), thus leading to damage-induced mutagenesis. Mycobacteria employ a novel DNA polymerase, DnaE2, for DNA damage tolerance and induced mutagenesis. DnaE2 belongs to the C-family of DNA polymerases, which are known to replicate DNA with high fidelity, and has been implicated in virulence and the emergence of rifampicin resistance of M. tuberculosis in vivo. In this study, DnaE2 was shown to function in the same pathway as two accessory proteins, ImuB and ImuA’, for damage tolerance and induced mutagenesis in mycobacteria. In this system, DnaE2 performs the polymerase function in translesion synthesis whereas ImuB is a cryptic Y-family DNA polymerase that lacks critical active site residues. It contains a β-clamp binding motif that allows interaction with the β-clamp and presumably enables DnaE2 and ImuA’ to access the replication fork. ImuB has a C-terminal region extending from the β-clamp binding motif which contains disordered regions that allow the interaction with other proteins and is important for function. ImuA’ is also essential for damage tolerance and induced mutagenesis but its function remains unknown. This protein is structurally similar to Escherichia coli RecA protein in the N-terminus and the middle domain, but it has a distinct C-terminus that was shown to be important for the interaction with ImuB. The essential replicative, C-family polymerase, DnaE1, was shown to be upregulated in response to DNA damage and was also shown to interact with ImuB. To explore the possibility that other proteins are involved in this pathway, ImuB was Cterminally tagged for use as bait in pull-down experiments in M. smegmatis. However, introduction of the tag disrupted ImuB function, further reinforcing the importance of the Cterminal region of ImuB for the function of this protein, presumably via protein-protein interactions. In contrast, a variant of ImuA’ which was N-terminally tagged was shown to retain functionality; however, experiments using this protein as a bait for pull-down proved to be unsuccessful. Proteomic analysis of wild type M. smegmatis, a dnaE2 deletion mutant and complemented derivative was carried out on cells exposed to the same conditions as used in the pull-down assay. Base excision repair (BER) components were identified in this analysis, but did not detect ImuB and ImuA’, suggesting that the levels of expression of these proteins were comparatively lower under the conditions tested resulting in failure of the pull-down experiment. Finally, numerous attempts were made to express and purify recombinant forms of ImuB and ImuA’ in E. coli for use in structural studies. Both proteins were expressed in the soluble and insoluble fractions; however the levels of soluble protein were low, and as a result, purified protein preparations could not be obtained.

Two mechanisms, intrinsic and factor-dependent, have evolved for accomplishing the termination of transcription in eubacteria. In this thesis, the first chapter is an introduction to the topic that presents what is known about the mechanisms of termination. The properties of the primary and secondary ‘players’- intrinsic terminators, Rho protein, rho-dependent terminators, RNA polymerse and Nus factors - are presented and the known mechanisms by which termination functions are discussed. In Chapter 2, a detailed analysis of intrinsic terminators – their differential distribution, similarity and divergence - has been penned. The database, compiled using the program GeSTer (Genome Scanner for Terminators), comprises ~2000 sequences and is one of the largest of its kind. Furthermore, analyzing the data from over 700 bacteria reveals how different species have fine-tuned intrinsic terminators to suit their cellular needs. Non-canonical intrinsic terminators emerge to be a significant fraction of the observed structures. The conserved structural features of identified intrinsic terminators are discussed and the relationship between the two modes of termination is assessed. Chapter 3 deals with the importance of transcription termination in regulating horizontally acquired DNA. The results show that genomic islands are scarce in intrinsic terminators and thus constitute most likely sites for Rho-dependent termination. Plausible reasons for why such a scenario has evolved are discussed and a generally applicable model is presented. Chapters 4 and 5 focus on Rho protein from Mycobacterium tuberculosis. In silico identification of M. tuberculosisgenes that rely on MtbRho-dependent termination is followed by experimental validation. The data show that Rho-dependent termination is the predominant mechanism in this species.MtbRho is a majorly expressed protein that governs termination of protein-coding and non-protein coding genes. Further, MtbRho can productively interact with RNA that has considerable secondary structure. Such interactions cause conformational changes in the enzyme. Given that MtbRho has to function with a GC-rich transcriptome, the altered properties could have evolved for optimal function. Taken together, the thesis extends our current understanding of both modes of termination. The importance of non-canonical intrinsic terminators in mycobacteria and other organisms is discussed. The unusual function of Rho and its predominant role in mycobacteria is elucidated. Finally, the inter-relationship between the two modes of termination is also discussed.

Two mechanisms, intrinsic and factor-dependent, have evolved for accomplishing the termination of transcription in eubacteria. In this thesis, the first chapter is an introduction to the topic that presents what is known about the mechanisms of termination. The properties of the primary and secondary ‘players’- intrinsic terminators, Rho protein, rho-dependent terminators, RNA polymerse and Nus factors - are presented and the known mechanisms by which termination functions are discussed. In Chapter 2, a detailed analysis of intrinsic terminators – their differential distribution, similarity and divergence - has been penned. The database, compiled using the program GeSTer (Genome Scanner for Terminators), comprises ~2000 sequences and is one of the largest of its kind. Furthermore, analyzing the data from over 700 bacteria reveals how different species have fine-tuned intrinsic terminators to suit their cellular needs. Non-canonical intrinsic terminators emerge to be a significant fraction of the observed structures. The conserved structural features of identified intrinsic terminators are discussed and the relationship between the two modes of termination is assessed. Chapter 3 deals with the importance of transcription termination in regulating horizontally acquired DNA. The results show that genomic islands are scarce in intrinsic terminators and thus constitute most likely sites for Rho-dependent termination. Plausible reasons for why such a scenario has evolved are discussed and a generally applicable model is presented. Chapters 4 and 5 focus on Rho protein from Mycobacterium tuberculosis. In silico identification of M. tuberculosisgenes that rely on MtbRho-dependent termination is followed by experimental validation. The data show that Rho-dependent termination is the predominant mechanism in this species.MtbRho is a majorly expressed protein that governs termination of protein-coding and non-protein coding genes. Further, MtbRho can productively interact with RNA that has considerable secondary structure. Such interactions cause conformational changes in the enzyme. Given that MtbRho has to function with a GC-rich transcriptome, the altered properties could have evolved for optimal function. Taken together, the thesis extends our current understanding of both modes of termination. The importance of non-canonical intrinsic terminators in mycobacteria and other organisms is discussed. The unusual function of Rho and its predominant role in mycobacteria is elucidated. Finally, the inter-relationship between the two modes of termination is also discussed.

Genus Mycobacterium comprises a large number of species including many pathogens such as Mycobacterium leprae, Mycobacterium abscessus and Mycobacterium tuberculosis (Mtb), the last one is the causative agent of the fatal disease tuberculosis. The unique features of the deadly organism viz, slow growing, tough cell wall, latency and resistance to various drugs demand a systematic understanding of many essential molecular processes including transcription. Studies have been undertaken to understand several aspects of transcription in mycobacteria which revealed its machinery to be conserved with other eubacteria? However, many facets of transcription in mycobacteria and regulation are different. The transcription regulators and them regulation are the basic counter stones which govern gene expression. The present study is aimed to understand better the mechanistic regulation of transcription of important housekeeping functions, DNA gyrase and also to obtain further insights into the role of transcription elongation factor Gre. Chapter 1 of the thesis provides a general introduction of the bacterial transcription machinery, associated transcription regulators and their regulation. It covers the description of the central player- the RNA polymerase (RNAP) followed by each step of the transcription initiation, elongation and various factors involved in their regulation. Finally, an overview of the emerging information on several aspects of mycobacterial transcription is discussed emphasizing on RNAP, promoter architecture, and its regulation. In Chapter 2, the studies are directed to understand the mechanism for topology-dependent regulation of Mtb Gyrase. The gyrase is encoded by two genes gyrB and gyrA which form a bicistronicity operon in Mtb and harbor multiple promoters. The principal promoter PgyrB1 drives the transcription of the dicistron and the weaker divergent promoter PgyrR is engaged in transcription in the opposite direction. The divergent and overlapping PgyrR show decrease in activity when the PgyrB1 was induced upon relaxation of the genome by a phenomenon termed relaxation stimulated transcription (RST). PgyrR plays a role in the fine tuning of gyr gene expression by reiterative transcription (RT), a regulatory mechanism hitherto not described in Mtb. In vitro transcription assays show that RT at PgyrR is dependent on the negatively supercoiled status of the DNA. The principal promoter PgyrB1 is also regulated by DNA topology but does not exhibit RT. It is elucidated that the RNAP binding is efficient at PgyrB1 when the DNA is relaxed whereas binding to PgyrR is preferred when DNA is supercoiled. Thus, a collaboration between RST and RT govern the regulation of gyr operon; the differential topology sensitivity of the overlapping promoters determines and dictate the efficiency of transcription initiation at gyr promoters. In addition, this study suggests a new mechanism of RST distinct from the one observed for other bacteria, such as E. coli or M. smegmatis. Chapter 3 describes studies that have been carried out to delineate the mechanism underlying the differential function of transcription regulator MtbGreA and its homolog Rv3788 (MtbGfh1). MtbGreA binds to RNAP and induces the intrinsic transcript cleavage activity of RNAP thereby allowing RNAP to resume transcription from paused and arrested sites. In spite of having Gre like domains, MtbGfh1 does not stimulate RNA cleavage. Instead, it inhibits transcription by binding to RNAP. Homology modeling and docking data suggest that Gre and MtbGfh1 bind to RNAP in a different orientation. MtbGreA coordinate with the Mg2+ present in the catalytic center of the RNAP while MtbGfh1 was observed to be facing away from Mg2+ Swapping of a stretch of residues from the N-terminus of MtbGreA into MtbGfh1 acquire GreA like transcript cleavage stimulatory activity and enhance promoter clearance for MtbGfh1. Bioinformatics analysis and biochemical assays demonstrate the significance of a stretch of residues in the N-terminus of MtbGreA and MtbGfh1 for their functions. Also, the orientation of the MtbGreA and MtbGfh1 while binding to RNAP is a crucial determinant in governing their respective function. Being the general inhibitor of transcription, overexpression of MtbGfh1 led to the appearance of tiny colonies and slow growth of cells suggesting its regulatory role to maintain the physiology of Mtb. In Chapter 4, the influence of perturbation of GreA level on Mtb growth and physiology has been studied. Mtb contains a single Gre protein (Rv1080c), unlike many other bacteria where both GreA and GreB are present. Further, the GC-rich genome of Mtb may pose an additional challenge to the transcribing RNAP. Hence the role of GreA could be essential to maintain high fidelity of transcription and RNAP distribution in Mtb genome. To validate this, the conditional knockdown strain of MtbGreA was generated. GreA depleted strain exhibited slow growth and caused phenotypical changes in Mtb cells. Moreover, the occupancy of RNAP on the promoter and gene body of candidate gene tested was found to be disrupted upon MtbGreA depletion, suggesting the regulatory role of GreA in modulating Mtb physiology.

Dissertação de Mestrado em Ciências da Saúde
In Mozambique, bovine tuberculosis (BTB) is a serious problem for livestock development; however, surveillance and control programs are not applied consistently and systematically. This disease has direct economic repercussions on livestock due to lower productivity of infected animals, as well as the increased rejection of carcasses at the slaughterhouses. Due to the direct economic repercussions and indirect consequences for human health, knowing the precise distribution of the disease throughout the country is essential to define an effective control strategy and reduce BTB incidence and spread. Although the prevalence in free-ranging wildlife species is unknown, non-systematic surveys have reported a wide variation of BTB prevalence in cattle in different regions of the country and even in distinct herds of the same region. In the Govuro district the BTB’s prevalence levels previously obtained are hardly intercomparable, ranging from 1.49% in one region, using single intradermal tuberculin test (SITT) in caudal fold, to 61.94%) in another region where the SITT was applied in the middle neck region. This latter prevalence level represents one of the highest for BTB in the country and represents a serious risk for the transmission of the disease to humans. However, it should be considered that, a positive result using SITT may be related with cross-reactivity with environmental mycobacteria. We conducted a cross-sectional study in the Govuro district in order to determine the prevalence of BTB in cattle and identify associated risk factors. To access the disease burden in the district, a representative sample of the cattle population from all livestock areas (n=14) was defined using the Epicalc 2000 statistical software (Brixton Books v.1 2); assuming an expected prevalence of 10% with a standard error precision of 0.05 (5%). To compensate for noncompliance (animals selected for tuberculin test but failing the reading day), 20% more animals were targeted at testing. A total of 1136 cattle from 289 different owners were submitted to the more accurate test, the single comparative intradermal tuberculin test (SCITT). Their body condition scores, sex, age and breed were recorded to obtain a better understanding of BTB risk factors. Data analysis was performed using a logistic regression model with binary outcome and livestock area as random effect. The overall prevalence was estimated at 39.6% [95% with confidence interval (CI): 36.8-42.5] and, with the exception of the Luido area (animals from two private farmers), BTB reactors were found in all studied areas with quite diverse prevalence rates. SCITT results showed that 137 (12%; 95% CI: 10.3-14.1) out of 1136 cattle tested reacted positive to avian PPD. From 289 cattle raisers with tested cattle, 192 (66.4%; 95% CI: 60.8- 71.6) had positive cattle. Age was found to be the main individual risk factor; animals older than 4 years were more likely to be positive reactors (45.4% vs 21.9%; OR 2.9, 95% CI: 42.1 – 49.0). Females (37.2%; OR 1.1, 95% CI: 33.9 – 40.7) and animals in good body condition (35.5%; OR 35, 95% CI: 32.0 – 39.1) were associated with lower prevalence’s, although no significant differences were found. Most of the cattle included in the present study are Zebu crossbred and Landim local breed. Zebu crossbred showed a significantly higher prevalence of BTB than the Landim local breed (49.6% vs 35.6%; OR 0.7, 95% CI: 45.4 – 53.9). Thirteen out of 67 positive blood samples were BOVIGAMTM positive. This study showed a high prevalence of BTB in the Govuro district. The findings reveal an urgent need for intervention with an implementation of effective, area-based, control measures in order to reduce prevalence and incidence of the disease and prevent its spread to the human population. Data also points that further studies on the isolation and molecular characterization of the predominant strains lineages that cause tuberculosis (TB) in cattle and humans should be conducted in order to assess the potential transmission of BTB from cattle to humans.
Em Moçambique, a tuberculose bovina (BTB) representa um sério problema para o desenvolvimento da pecuária, no entanto, os programas de vigilância e controle não são aplicados de forma consistente e sistemática. Esta doença tem repercussões económicas diretas na pecuária devido à baixa produtividade dos animais infetados, bem como o aumento da rejeição de carcaças ao nível dos matadouros. Devido às repercussões económicas diretas e consequências indiretas para a saúde humana, é de particular relevância conhecer a distribuição precisa da doença em todo o país de modo a definir uma estratégia de controlo eficaz e consequentemente reduzir as taxas de incidência e propagação da doença. Pesquisas não sistemáticas têm demonstrado uma grande variação da prevalência da doença em bovinos em diferentes regiões do país e até mesmo em rebanhos distintos de uma mesma região, porém a prevalência da doença nas espécies selvagens permanece desconhecida. Estudos anteriormente desenvolvidos em momentos diferentes no distrito de Govuro obtiveram taxas de prevalências dificilmente comparáveis; utilizando a tuberculinização simples na prega caudal (1.49%) e na tábua do pescoço (61.94%). Esta última representa uma das mais altas taxas de prevalência de BTB encontrada no país. No entanto, é de grande relevância considerar que, um resultado positivo obtido a partir do SITT pode estar relacionado com reatividade cruzada com micobactérias ambientais. Com o objetivo de determinar a prevalência da BTB em bovinos e identificar os fatores de risco associados à ocorrência da doença, desenvolveu-se um estudo transversal, no distrito de Govuro. Uma amostra representativa da população bovina pertencente a todas as áreas de produção do distrito (n=14) foi definida a partir do programa estatístico Epicalc 2000 (Brixton Books v.1 2), utilizando uma prevalência estimada de 10% e uma precisão de desvio padrão de 0.05 (5%). De modo a reduzir o efeito das incomparências (animais tuberculinizados que não comparecem no dia da leitura), foram testados 20% a mais do tamanho da amostra pré determinada. Um total de 1136 animais, pertencentes a 289 proprietários foi submetido a um teste de diagnóstico mais preciso, o teste tuberculínico comparativo (SCITT). Dados relativos a condição corporal, sexo, idade e raça foram registrados para obter uma melhor compreensão dos fatores de risco associados à doença. Os dados foram analisados utilizando um modelo de regressão logística com desfecho binário e a área de pecuária como efeito aleatório. A prevalência global estimada foi de 39.6% [95% com intervalo de confiança (CI): 36.8 – 42.5]. Com exceção da área de pecuária Luido (animais pertences a dois criadores privados) todas as restantes áreas estudadas apresentaram reatores positivos. Os resultados do SCITT mostraram que 137 (12%; 95% CI: 10.3 – 14.1) dos 1136 animais testados, reagiram positivamente ao PPD aviário. Dos 289 criadores incluídos no estudo, 192 (66.4%; 95% CI: 60.8 – 71.6) possuem animais positivos. A idade foi o principal fator de risco individual. Animais com mais de quatro anos mostraram-se mais propensos a ser reatores positivos (45.4% vs 21.9%; OR 2.9, 95% CI: 42,1 – 49,0). Animais do sexo feminino (37.2%; OR 1.1, 95% CI: 33.9 – 40.7) e com boa condição corporal (35.5%; OR 35, 95% CI: 32.0 – 39.1) foram associados a prevalências mais baixas, embora não tenham sido encontradas diferenças significativas. A maioria do gado incluído no presente estudo pertence à raça Zebu mestiços e raça local Landim. A raça mestiça Zebu apresentou uma prevalência de BTB significativamente maior em relação a raça Landim (49.6% vs 35.6%; OR 0,7, 95% CI: 45.4 – 53.9). Treze das 67 amostras de sangue positivas reagiram positivamente ao BOVIGAMTM. O presente estudo demonstrou uma alta prevalência de BTB no distrito de Govuro. Esses resultados revelam uma necessidade urgente de intervenção com implementação de medidas de controlo eficazes, baseados na área, de modo a reduzir a prevalência e incidência da doença e consequentemente evitar a sua propagação para o homem. Sugere-se que novos estudos sobre o isolamento e caracterização molecular das estirpes predominantes em bovinos e humanos sejam desenvolvidos de modo a avaliar a transmissão de BTB do gado bovino para o homem.
The laboratory work presented in this thesis was done in the Laboratory of Serology of Veterinary Faculty, Eduardo Mondlane University, Maputo, Mozambique and at the Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Braga, Portugal. The financial support to perform this study was conceded by the Integrated Control of Neglected Zoonosis (ICONZ) – Africa Project and Fundação para a Ciência e a Tecnologia (FCT).

The genus mycobacterium has more than 120 species of bacteria; one being M. tuberculosis (Mtb), the etiological agent of tuberculosis (TB). During infection, the host mounts a heightened immune response to contain the spread of the pathogen. Facilitating the response mediated by the host, PRRs (Pattern Recognition Receptors), upon their interaction with cognate ligands set forth a plethora of signaling cascades that effectuate responses like inflammation, apoptosis or autophagy to eradicate mycobacteria from the system. The early responses against Mtb are brought about by macrophages, dendritic cells (DCs) and neutrophils whereas the effector function of CD4+ and CD8+ T cells are critical for suppression of mycobacterial infection. Tumor necrosis factor (TNF)-alpha, Cytokines like interleukin (IL)-23, IL-12 and interferon (IFN)-gamma are secreted by type I T helper (Th1) subset of CD4+ T cells that provides protective immunity against pathogenic mycobacteria. Inspite of these extreme measures taken by the host to contain the infection, afflicted individuals are unable to eliminate the pathogen. Over one-third of the world’s population is infected with Mtb, which is a testimony of its success as a pathogen. This can be attributed to the various immune evasion strategies it employs like inhibition of phagosome-lysosome fusion, inhibition of antigen processing and presentation, suppression of pro-inflammatory cytokines, shifting the immune response towards Th2 type suppression of RNI and ROS, inhibition of apoptosis/autophagy and induction of regulatory T cells (Tregs) etc. Further, the emergence of MDR/XDR strains and co-infections has compounded the graveness of the disease. Understanding the mechanism underlying such immune evasion strategies will provide effective check on pathogenesis of mycobacteria. In this regard, evaluation of the host-pathogen interactions during Mtb infection in terms of key the signaling pathway would provide us a critical insight into details of immune responses and its regulation. The present study addresses the role of WNT/ β-CATENIN pathway-dependent factors in modulating the host immune responses during Mtb infection. WNT/ β-CATENIN pathway-dependent G9a (histone methyltransferase) and Sirtuin6 (SIRT6; histone deacetylase) regulate cholesterol metabolism/homeostasis and mycobacterial survival (Chapter 3); AMPK-WNT/ β-CATENIN pathway-dependent COP1, an E3 ubiquitin ligase, skews the macrophage towards M2 phenotype hence aiding mycobacterial survival (Chapter 4) and finally the ability of mycobacterial surface antigen Ac2PIM in selectively suppressing NOD2-induced immunomodulators COX2, SOCS3 and MMP9 (Chapter 5). Formation of highly structured aggregate of immune cells, called granulomas, is a hallmark of mycobacterial infection. One of the characteristic cells constituting the granulomas is the lipid-laden foamy macrophages (FM). These FMs serve as a source of nutrients as well as immune-modulators that aid in the survival of the pathogen. Amongst other lipids, cholesterol and cholesterol esters are significantly accumulated in the FMs. Further, it has been reported that cholesterol is one of the major carbon source for mycobacteria and helps in its persistence within the host. However, the molecular mechanisms that regulate intracellular cholesterol accumulation during the course of infection are not clear. Here, we analyzed the role of two histone modifiers G9a (histone methyltransferase; H3K9me1) and SIRT6 (histone deacetylase; H3K9Ac), which have been implicated in regulating immune responses. Mtb infection augments the levels of G9a and SIRT6 in macrophages as well as in the lungs of infected mice. Further, genes involved in cholesterol biosynthesis, uptake and efflux are differentially regulated upon mycobacterial infection. G9a was found to regulate genes involved in uptake (Lrp2) and biosynthesis (Aacs, Hmgcs1, Mvd, Dhcr24) of cholesterol by recruiting at the respective promoters and bringing about H3K9me1leading to their enhanced expression. On the contrary, cholesterol efflux genes (Abca1, Abcag1) promoters were found to be enriched with SIRT6 with loss of H3K9Ac upon infection indicating their repression. Corroborating these observation mouse aerosol infection model of TB was utilized to ascertain the role of G9a and SIRT6 in mycobacterial survival wherein mice treated with G9a inhibitor or Sirt6-/+ mice showed significant decrease in mycobacterial burden in organs such as spleen and lung. Immunofluorescence and transcript level analyses of the target genes in infected and uninfected lungs further substantiated our in vitro findings. Also, G9a and SIRT6 were found to be under the tight regulation of Mtb activated WNT/ β-CATENIN pathway. Thus our study unveiled the role of WNT/ β-CATENIN dependent G9a and SIRT6 in regulating cholesterol levels and consequently pathogen survival during mycobacterial infection. The host induces the production of pro-inflammatory cytokines to counter any bacterial infection, including that by mycobacteria. However, mycobacteria skews the immune cells towards eliciting anti-inflammatory cytokine response, which prove more conducive for their survival. In this context, we investigated the role of AMPK, a crucial energy-sensing molecule that has been reported to play critical role in polarizing macrophage towards M2 phenotype and an E3 ubiquitin ligase COP1 in modulating immune responses during mycobacterial infection. AMPK is activated upon mycobacterial infection in macrophages through the action of upstream kinase PKCζand LKB1. Activated AMPK cross-talk with WNT/ β-CATENIN signaling that leads to the expression of COP1, an E3 ubiquitin ligase. Chromatin immunoprecipitation analysis confirms the recruitment of β-CATENIN at the promoter of COP1. Further, knockdown studies show that AMPK and COP1 are important for the expression of M2 cytokines/chemokines like IL-10, IL-4, CCL-4, CCL-17 and ARGINASE-1 that could aid the survival of mycobacteria in the host. Thus, our study highlights the activation of PKCζ-LKB1-AMPK-WNT/ β-CATENIN-COP1 pathway upon mycobacterial infection and postulates the essential role of the axis in modulating cytokine levels, thereby aiding in mycobacterial survival. Specific and coordinated regulation of innate immune receptor-driven signaling networks often determines the net outcome of the immune responses. Here, we investigated the cross-regulation of toll-like receptor (TLR)2 and nucleotide-binding oligomerization domain (NOD)2 pathways mediated by Ac2PIM, a tetra-acylated form of mycobacterial cell wall component and muramyl dipeptide (MDP), a peptidoglycan derivative respectively. While Ac2PIM treatment of macrophages compromised their ability to induce NOD2-dependent immunomodulators like cyclooxygenase (COX)-2, suppressor of cytokine signaling (SOCS)-3 and matrix metalloproteinase (MMP)-9, no change in the NOD2-responsive NO, TNF-α, VEGF-A and IL-12 levels was observed. Further, genome-wide microRNA expression profiling identified Ac2PIM-responsive miR-150 and miR-143 to target NOD2 signaling adaptors, RIP2 and TAK1 respectively. Interestingly, Ac2PIM was found to activate the SRC-FAK-PYK2-CREB cascade via TLR2 to recruit CBP/P300 at the promoters of miR-150 and miR-143 and epigenetically induce their expression. Loss-of-function studies utilizing specific miRNA inhibitors establish that Ac2PIM, via the miRNAs, abrogate NOD2-induced PI3K-PKCδ-MAPK pathway to suppress β-CATENIN-mediated expression of COX-2, SOCS-3 and MMP-9. Our investigation has thus underscored the negative regulatory role of Ac2PIM-TLR2 signaling on NOD2 pathway, which could broaden our understanding of cross-regulation between two PRRs upon activation. Altogether, we have established an implication of novel molecular players in the pathogenesis of TB. We have found that an intricately woven network of signaling pathways and epigenetic factors fine tune the execution of mycobacterial survival strategies such as accumulation of cholesterol and swerving of the inflammatory milieu. Further, we gained insights into the extensive implication of cross-talk between PRRs in coining the outcome of conditions where they are simultaneously stimulated. Such detailed investigations would confer a holistic perspective of host-pathogen interactions and would bear potential in effective disease control by aiding the search for efficacious therapeutics.

Submitted in fulfillment of the requirements for the degree of Master’s of Technology: Radiography, Durban University of Technology, Durban, South Africa, 2015.
Introduction Tuberculosis remains a leading cause of death, second to the Human Immunodeficiency Virus. The risk of latent tuberculosis infection and active tuberculosis disease is a known occupational hazard. In South Africa, a high tuberculosis burden country, the potential of Mycobacterium tuberculosis transmission to health care workers is high. This includes diagnostic radiographers and other radiology staff working in radiology departments. Purpose of the Study This study aimed to investigate the association of demographic and occupational factors with latent tuberculosis infection in radiology staff in public sector hospitals of the eThekwini Health District. Methodology This cross-sectional study was conducted from 26 February 2013 to 07 June 2013. Quantitative methods were used to test for associations of demographic and occupational factors with latent tuberculosis infection in participants. A sample size of 181 participants for an estimated population of 340 radiology staff was recommended at the proposal stage. The study consisted of two phases; the questionnaire survey (phase one) and the administration of a two-step tuberculin skin test (phase two). Data was obtained with regard to demographics, occupational history, social behaviours, medical history; and family and home histories. Demographic and occupational associations with latent tuberculosis infection were made in relation to the size of the first tuberculin skin test induration. Frequency distributions were developed to describe data categories. Pearson’s and Spearman rho’ correlation coefficients were used to test for correlations between the independent variables. The chi-square test was used to determine associations between the categorical independent variables and the dependent variable. Bivariate analyses were performed using these tests. The multivariate analysis was performed using logistic and linear regression on the dependent variable. Results A total of 182 questionnaires were returned from approximately 280 radiology staff. At the outset, all doctors working in the radiology department had to be excluded due to numerous failed attempts to enlist their participation. Fifty-three (29.12 percent) participants were excluded from phase one of the study and a further thirteen participants were excluded from phase two. The total sample was 116 participants. Of the 116 participants, 86.2 percent tested positive for latent tuberculosis infection at the first step of the two-step testing method used. One (0.86 percent) participant went on to convert at the second step, testing positive at this level. Demographic associations with latent tuberculosis infection included age (older) as an associated factor. A significant demographic association with latent tuberculosis infection was the use of alcohol (p-value 0.033 on the multivariate analysis). Occupational associations with latent tuberculosis infection included longer durations of employment. The annual income (higher income earners) displayed significant associations with latent tuberculosis infection (p-value 0.048 on the multivariate analysis). It is necessary in this study to note that participants include support personnel (lower income earners) making up 37.8 percent of the study, diagnostic radiographers making up 48.3 percent; and radiography managers/assistant managers (highest income earners) making up 13.8 percent of the study. Conclusion and recommendations The risk of transmission of Mycobacterium Tuberculosis to health care workers is a known occupational hazard. This study has described the prevalence of latent tuberculosis infection in radiology staff, at district and regional hospitals within the eThekwini Health District. With 23.62 percent of all participants already having active TB disease and 86.2 percent of the tested group displaying positive results for latent tuberculosis infection, using the tuberculin skin tests, the need for tuberculosis screening is essential. The findings of this study will be used as a health improvement mechanism for stakeholders, having identified potential gaps in medical screening in healthcare in Kwa-Zulu Natal. This study makes recommendations for the early detection of active tuberculosis infection and the monitoring of health care workers that are latently infected, thus assisting in reducing the rate of conversion of latent tuberculosis infection to active tuberculosis disease in radiology staff. This reduces long-term exorbitant costs related to health care associated infections, such as tuberculosis. It also reduces rates of transmission and cross infection to both co-workers and already immunocompromised patients, helping to curb the overall epidemic in South Africa.