Gotowa bibliografia na temat „Mycobacterium tuberculosis σ Factors”

ABSTRACT Mycobacterium tuberculosis and Mycobacterium bovis are responsible for infections that cause a substantial amount of death, suffering, and loss around the world. Still, relatively little is known about the mechanisms of gene expression in these bacteria. Here, we used genome-wide location assays to identify direct target genes for mycobacterial σ factors. Chromatin immunoprecipitation assays were performed with M. bovis BCG for Myc-tagged proteins expressed using an anhydrotetracycline-inducible promoter, and enriched DNA fragments were hybridized to a microarray representing intergenic regions from the M. tuberculosis H37Rv genome. Several putative target genes were validated by quantitative PCR. The corresponding transcriptional start sites were identified for σF, σC, and σK, and consensus promoter sequences are proposed. Our conclusions were supported by the results of in vitro transcription assays. We also examined the role of each holoenzyme in the expression of σ factor genes. Our results revealed that many σ factors are expressed from autoregulated promoters.

Promoter binding specificity is one of the important characteristics of transcription by Mycobacterium tuberculosis (Mtb) sigma (σ) factors, which remains unexplored due to limited structural evidence.

σ factors are transcriptional regulatory proteins that bind to the RNA polymerase and dictate gene expression. The extracytoplasmic function (ECF) σ factors govern the environment dependent regulation of transcription. ECF σ factors have two domains σ2 and σ4 that recognize the -10 and -35 promoter elements. However, unlike the primary σ factor σA, the ECF σ factors lack σ3, a region that helps in the recognition of the extended -10 element and σ1.1, a domain involved in the autoinhibition of σA in the absence of core RNA polymerase. Mycobacterium tuberculosis σC is an ECF σ factor that is essential for the pathogenesis and virulence of M. tuberculosis in the mouse and guinea pig models of infection. However, unlike other ECF σ factors, σC does not appear to have a regulatory anti-σ factor located in the same operon. We also note 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 σC4 domains of σC were determined. These promoter recognition domains are structurally similar to the corresponding domains of 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 interdomain interaction. Interaction 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.

Mycobacterium tuberculosishas multiple σ factors which enable the bacterium to reprogram its transcriptional machinery under diverse environmental conditions. σJ, an extracytoplasmic function σ factor, is upregulated in late stationary phase cultures and during human macrophage infection. σJgoverns the cellular response to hydrogen peroxide-mediated oxidative stress. σJdiffers from other canonical σ factors owing to the presence of a SnoaL_2 domain at the C-terminus. σJcrystals belonged to the tetragonal space groupI422, with unit-cell parametersa=b= 133.85,c= 75.08 Å. Diffraction data were collected to 2.16 Å resolution on the BM14 beamline at the European Synchrotron Radiation Facility (ESRF).

ABSTRACT The numerous sigma (σ) factors present in Mycobacterium tuberculosis are indicative of the adaptability of this pathogen to different environmental conditions. In this report, we describe the M. tuberculosis σB regulon and the phenotypes of an M. tuberculosis sigB mutant strain exposed to cell envelope stress, oxidative stress, and hypoxia. The sigB mutant was especially defective in survival under hypoxic conditions in vitro, but it was not attenuated for growth in THP-1 cells or during mouse and guinea pig infection.

Abstract Bacterial RNA polymerase (RNAP) forms distinct holoenzymes with extra-cytoplasmic function (ECF) σ factors to initiate specific gene expression programs. In this study, we report a cryo-EM structure at 4.0 Å of Escherichia coli transcription initiation complex comprising σE—the most-studied bacterial ECF σ factor (Ec σE-RPo), and a crystal structure at 3.1 Å of Mycobacterium tuberculosis transcription initiation complex with a chimeric σH/E (Mtb σH/E-RPo). The structure of Ec σE-RPo reveals key interactions essential for assembly of E. coli σE-RNAP holoenzyme and for promoter recognition and unwinding by E. coli σE. Moreover, both structures show that the non-conserved linkers (σ2/σ4 linker) of the two ECF σ factors are inserted into the active-center cleft and exit through the RNA-exit channel. We performed secondary-structure prediction of 27,670 ECF σ factors and find that their non-conserved linkers probably reach into and exit from RNAP active-center cleft in a similar manner. Further biochemical results suggest that such σ2/σ4 linker plays an important role in RPo formation, abortive production and promoter escape during ECF σ factors-mediated transcription initiation.

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.

The monograph is devoted to an important medical and social problem - improving the effectiveness of treatment of tuberculosis patients with surgical interventions. Evaluation of the effectiveness of surgical treatment in the long-term period made it possible to establish the main controllable risk factors for reactivation of the tuberculosis process in the postoperative period. The influence of the timing of surgical treatment on the long-term results of surgical treatment and the formation of drug resistance of Mycobacterium tuberculosis has been proved. An innovative approach of preoperative planning with the help of 3D computer modeling through the Autopilot program for planning surgical interventions on the lungs is presented. A personalized approach is proposed when choosing surgical treatment and improving the tactics of preoperative preparation. It is intended for doctors — thoracic surgeons, phthisiologists.

This chapter covers the key facts about tuberculosis (TB), then goes on to describe the epidemiology and pathophysiology of the disease. Risk factors, and signs and symptoms, and investigations are all covered. The treatment phase and standard drug therapies are shown, and directly observed therapy for the patient with an increased risk of poor adherence is described. Mono-resistant, multidrug-resistant, and extensively drug-resistant types are included in this chapter, as well as contact tracing in line with NICE clinical guidelines. Mycobacterium Tuberculosis (MTB) is part of a family of mycobacterium which includes Mycobacterium bovis. M. bovis is uncommon in humans, although it frequently affects cattle and badgers. MTB can affect any organ in the body; this chapter will concentrate on the diagnosis, treatment, and prevention of pulmonary MTB in adults in the UK.

Tuberculosis is caused by organisms of the Mycobacterium tuberculosis complex, including M. tuberculosis (the most important), M. bovis, and M. africanum. It has been present since antiquity and is the leading infectious cause of death ahead of HIV infection. An estimated 2 billion people worldwide carry latent infection, when M. tuberculosis persists within cells and granulomas, with the potential to reactivate to cause disease decades later. Tubercle bacilli are transmitted between people by aerosols generated when an infectious person coughs. Proximity to an infectious person determines the risk of infection. Host immunity and factors affecting it—most importantly HIV infection but also diabetes, cigarette smoking, and alcohol and drug abuse—determine the risk of active disease following infection.

This chapter investigates how the epidemiology of tuberculosis (TB) has changed in recent years due to shifting demographic trends. Although the incidence of TB in the United States has followed a generally downward trend in recent decades, the HIV/AIDS epidemic and an increase in homelessness, poverty, and drug abuse continue to remain the major risk factors for infection by Mycobacterium tuberculosis, the bacteria causing TB. Influxes of refugees and immigrants from areas of the globe endemic to tuberculosis contribute to the growing incidence of TB. Although M. tuberculosis is thought of primarily as a disease of the lungs, the bacteria can attack any organ in the body, most commonly the kidney, brain, and spine. This chapter details the testing, evaluation, surveillance, and treatment of patients with TB.

The principal diseases caused by mycobacteria are tuberculosis and leprosy. Descriptions of both diseases can be found in pre-Christian Greek literature and DNA from both has been recovered from ancient skeletons. Leprosy is caused by Mycobacterium leprae and Mycobacterium lepromatosis. Leprosy still exists in many parts of the world but for unknown reasons, it has died out in Europe. Tuberculosis is the best studied mycobacterial disease and is caused by members of the Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, M. africanum, and M. canetti). Genome analysis has shown that mycobacteria show a large variation in size and that the smallest genomes are found in the pathogenic species (genomic reduction). Genome analysis has not provided an explanation for the inability of Mycobacterium leprae to grow in the laboratory. Genomic analysis has revealed some of the key virulence factors of Mycobacterium tuberculosis and these include the type VII secretion system (known as ESX), the PE and PPE family of proteins, and mycolic acid. The latter helps to protect the bacterium from phagocytosis. Effective protection against tuberculosis is provided by vaccination with either Mycobacterium microti or the BCG strain of Mycobacterium tuberculosis. Both have a deletion of a genomic region encoding three cell wall proteins. Treatment of tuberculosis requires months of antibiotic therapy and multi-drug resistance is common. The use of DNA sequencing can greatly reduce the time to determine antibiotic sensitivity.