Rozprawy doktorskie na temat „M. tuberculosis Infection”

A central feature of Mycobacterium tuberculosis pathogenesis is the ability to survive within macrophages and colonize hostile environments that are acidic and rich in cholesterol and fatty acids. The genetic variability among clinical isolates may have dramatic consequences on the outcome of infections. Many in vitro and in vivo studies have demonstrated strain-dependent variation in key aspects of virulence such as stress survival, transmission, pathology, and lethality. This variability in MTB clinical isolates went neglected for decades; however, recently the scientific community recognized it causes important consequences on the progression of infection. As reduced host response is supposed to be the key to enabling MTB persistence and transmissibility, the immunological state of the host is also important to consider when studying the relative virulence of clinical MTB strains. In this context, the polarization states of macrophages have a huge impact on their function. It affects how macrophages can react to external signals, changing gene expression, membrane composition, receptor exposure, and cytokines production. In our work, we focused our attention on MTB diversity and its role in pathogenesis and drug resistance acquisition. We proposed a comprehensive approach for better understanding MTB virulence taking into consideration both human (phenotypic) and MTB (genetic) variability. We selected well-characterized strains belonging to specific MTB lineages and adopt the THP-I-derived macrophage infection model, but considering host variability by deriving M1 or M2 polarized macrophages. Confocal live microscopy was used to study at a single-cell level different macrophage pathways during the infection. These included phagolysosomal acidification, autophagic flux, and apoptosis. All are mechanisms that MTB hijacks in order to survive within the host. Cytokine profiles were measured in response to the different MTB lineages, along with the survival of both the macrophages and the bacteria at different time points post-infection. In M1 macrophages, whereas less virulent/ancient are more efficient in blocking the phagosomal acidification, more virulent/modern strains are likely better at tolerating the acidic environment. However, the number of responsive macrophages remained relatively low. In M2a macrophages, whereas less virulent/ancient strains are less efficient in blocking the phagolysosomal acidification, more virulent/modern strains are blocking phagolysosomal acidification. The number of responsive macrophages was found to vary depending upon the features of the infecting strains, with the most virulent ones inducing the lowest percentages of acidifying cells. The analysis of the autophagic flux showed less heterogeneity among both the bacterial strains and the macrophage phenotypes considered. Indeed, the induction of autophagy was found negligible in our observations. In M1 macrophages, bystander non-infected cells of more virulent/ modern showed increased apoptosis (40%). Stratification of the data by mycobacterial burden showed similar apoptotic levels irrespective of the mycobacterial load, despite a trend displaying slightly higher apoptotic levels in bystander non-infected cells or in cells infected with a lower number of mycobacteria could be noted. In contrast, none of the categories induced apoptosis in M2 macrophages at the time point considered. In M1 cells, the infection with less virulent/ancient lineages reduced the production of pro-inflammatory IL1-β. Likewise, IL-18 levels were lower in M1 cells infected with less virulent/ancient lineages. The chemokine GROα showed a similar pattern between MTB categories considered. In M2 cells we observed the modulation of anti-inflammatory IL-10 when comparing clinical to laboratory strains. Most of the statistically significant differences found were observed within 24h for both polarization types. Our findings on macrophage survival corroborate the results obtained at the single-cell level, with more virulent/modern lineages causing increased cell death. Interestingly, these data seem to contrast the finding that such strains showed lower macrophage entry in colony-forming units evaluation studies. Similarly, there was not a direct correlation between pro-inflammatory cytokine production and macrophage killing. Nevertheless, more virulent/modern strains showed increased intramacrophagic replicative capacity compared to less virulent/ancient lineages. We also studied the genetic variability of the MTB strains during drug resistance acquisition. Among newly investigated mechanisms, the role of smallRNAs in the development of drug resistance has been considered in other bacteria but remains unexplored in MTB. We characterized the smallRNA ncRv0842c, cis-encoded to the Rv0842 gene which codes for a putative efflux pump reported to be involved in rifampicin resistance (RIF-R) development. Accordingly, the present study characterized the role of ncRv0842c during RIF challenge and validated the role of a lineage-specific synonymous mutation on the coding region of Rv0842 affecting the promoter region of the cis-encoded smallRNA. In order to understand the role of the smallRNA in modulating the efflux pump, we characterized its expression during challenge experiments using sub-inhibitory concentrations of rifampicin. We then generated overexpressing mutants from MTB-selected ancient and modern strains. qPCR showed basal downregulation of ncRv0842 in ancient lineages compared to modern lineages. This result confirmed the hypothesis that the synonymous mutation in Rv0842 (L45L) specific for the ancient lineages, and abrogating the -10 promoter region of the antisense smallRNA is severely affecting the expression of the smallRNA. Under RIF-induced stress, we observed upregulation of Rv0842, and downregulation of the smallRNA only in H37Rv (modern lineage) whereas in L5 (ancient lineage) the smallRNA expression was not affected. MABA assay performed on modern lineage mutants overexpressing ncRv0842c showed a 1-dilution reduction of the MIC in comparison with their respective control (mock) while the ancient lineage strains did not show any MIC shift despite the overexpression of the smallRNA. Our analysis showed that the unraveling of smallRNA may provide new insights on MTB lineage-specific adaptation to drug-related stress and uncover the role of silent mutations in determining different phenotypes in MTB. Our study can contribute to better understanding lineage-specific pathogenicity and highlights the importance of strain- and lineage-specific rational design and development of effective diagnostic tools and vaccines.

There are so many people who I would like to thank. If it takes a village to raise a child, it certainly takes a city to train a scientist. Firstly, I would like to thank my supervisors, A/Prof Reto Guler, Prof Frank Brombacher and Dr Mumin Ozturk for allowing me to further my studies and allowing me to work on several interesting projects. Specifically, I would like to take this opportunity to thank A/Prof Reto Guler for his insightful patient advice, training and ever willingness to talk about my work. His brilliant example has made me a better scientist. Further, I would also like to thank Dr Mumin Ozturk for his constant, patient mentorship, help, advice and friendship. His influence, guidance and example have affected me more than he'll ever know. Lastly, but certainly not least, I would like Professor Bavesh Kana and for all his advice and support. I would also like to express my gratitude to my labmates from the Brombacher group. All the conversations, laughs, celebrations and commiserations have made this journey undeniably easier. In particular, I would like to thank Shelby-Sara Jones for her constant willingness to help with lab work whilst chatting about everything under the sun. To my friends and family, there are no words to express my unending gratitude. Without their love and support along the way, I would never have gotten to this stage in my life. To my parents and sister, I would like to say a huge thank you for their constant support and love during my academic career so far. You guys have been wonderful. A huge thank you to Daniela de Almeida and the French's for their support and love from afar– you guys have been great. I would like to say a special thank you to Dustin Fischer who has always been there for a beer and good old-fashioned rant. I can only hope that my friendship and advice have been even the smallest bit as helpful to him as he has been to me during our long trek through academia. To the Cunniffes, thank you for all your support down this long road and truly making me feel like one of the family. Last but by no means least, I would like to thank my partner, Teagan Cunniffe, whose effortless grace, wit, humour, friendship and constant love have been the single greatest gifts I have ever received. I look forward to our adventures to come. Thank you for being there every step of the way and keeping me sane - This dissertation is dedicated to you.

Mycobacterium tuberculosis, the aetiological agent of tuberculosis, is an intracellular pathogen commonly infecting macrophages, and has also been shown to infect dendritic cells (DC). As DC are particularly effective antigen-presenting cells, it is likely that they play a principle role in initiating anti-mycobacterial T cell responses. This work investigates the activation of DC in response to M. tuberculosis infection using murine bone marrow-derived DCs, generated in the presence of granulocyte- macrophage colony-stimulating factor (GM-CSF). It was found that both unsorted DC populations and those sorted on CD11c+, were capable of supporting the survival and replication of wild type M. tuberculosis (H37Rv) in a manner similar to that observed in macrophages. Mycobacterial infection was found to be sufficient to activate the DC populations, particularly CD11c+ DC, to acquire a mature phenotype, as measured by cytokine production and expression of costimulatory and antigen presentation molecules on the cell surface. Further study showed that mycobacteria-infected DC could prime protective immunity in an experimental model of murine tuberculosis. This was carried out using a lysine auxotroph of M. tuberculosis. Infected DC were used to vaccinate syngeneic or allogeneic mice. Protection against challenge with wild type M. tuberculosis was observed in both cases, suggesting that recipient antigen-presenting cells crosspresented mycobacterial antigen from donor DC to induce a protective immune response. A similar protective response was observed on using a xenogeneic model, in which infected murine DC were used to vaccinate guinea pigs. Both CD4+ and CD8+ T cells harvested from spleens of vaccinated mice, showed specific production of interferon-y in response to mycobacterial antigen, indicating that crosspresentation by recipient antigen-presenting cells results in the effective priming of mycobacteria-specific T cells in vivo.

Tuberculosis (TB) remains the main cause of death in people living with HIV (PLHIV). Indeed, PLHIV have a 20-30% greater risk of developing TB compared to HIV-uninfected subjects and have lower TB treatment success rates. In 2014, among the 9.6 million incident cases of TB reported worldwide, 12% occurred in PLHIV and 0.4 million deaths from HIV-associated TB were recorded.Mycobacterium tuberculosis is the main etiological agent for TB. For a majority of individuals, the immune response upon infection by M. tuberculosis is sufficient to prevent the development of disease, but insufficient to clear the bacteria. This leads to the persistence of viable M. tuberculosis in diverse cells with no resulting clinical manifestations, an entity known as latent tuberculosis infection (LTBI). The resulting reservoir of M. tuberculosis is vast, and an estimated one third of the world population is concerned. For subjects with LTBI, the life-time risk of reactivation and progression to TB lies between 5 and 10%. However, if co-infected with HIV, the risk is much greater and reaches 10% per year. According to a Cochrane review in 2010, the screening and treatment of LTBI in PLHIV reduces this risk by 30-60%. This prevention strategy is therefore widely recommended. However, the implementation of LTBI screening and treatment into standard HIV-care has been limited. In this work, three different approaches have been used to understand and address this issue, focusing on a low TB-incidence and high-income setting.The first approach was to assess the implementation of LTBI screening in HIV-care across Belgium and identify its barriers as perceived by the caregivers on the field. Raising awareness to this issue was an indirect objective of the study. A multi-choice questionnaire was sent to 55 physicians working in a Belgian AIDS reference center or satellite clinic. A response rate of 62% was obtained. Only 20% of participants performed LTBI screening on all their patients and notable variations in the screening methods used were observed. A large majority of participants were in favor of targeting LTBI screening to HIV-infected patients at highest risk of TB rather than a systematic screening of all PLHIV. These results have been communicated to the Belgian LTBI working group, currently updating the national LTBI screening guidelines. Indeed, targeting screening to those at highest risk of TB is an attractive strategy in low-TB incidence countries and is already recommended in the United Kingdom. However, to date, no score assessing the risk of TB in PLHIV has been validated. Among the barriers to LTBI screening identified by the participants of this first study, the most frequently reported were lack of sensitivity of screening tools, risk associated to polypharmacy and toxicity of treatment. Improving the sensitivity of LTBI screening was the cornerstone of the second approach. The available screening tools for LTBI are the tuberculin skin test (TST) and two Interferon-gamma release assays (IGRAs): the QuantiFERON-TB Gold-IT (QFT-GIT) and the T-SPOT.TB®. All three lack sensitivity in PLHIV. Various strategies to discover superior LTBI screening tools are therefore being explored, including the development of IGRAs in response to alternative M. tuberculosis antigens to those used in the QFT-GIT or T-SPOT.TB®. A potential candidate is the native Heparin-Binding Haemagglutin (nHBHA), a methylated M. tuberculosis protein regarded as a latency-associated antigen. An in-house IGRA based on nHBHA (nHBHA-IGRA) has been shown to be a promising LTBI screening tool both in immunocompetent adults and in hemodialysed patients. The contribution of this nHBHA-IGRA to the detection of M. tuberculosis in PLHIV was therefore investigated. Treatment-naïve HIV-infected subjects were recruited from 4 Brussels-based hospitals. Subjects underwent screening for latent TB using the nHBHA-IGRA in parallel to the classical method consisting of medical history, chest X-ray, TST and QFT-GIT. Prospective clinical and biological follow-up ensued, with repeated testing with nHBHA-IGRA. Among 48 candidates enrolled for screening, 9 were diagnosed with LTBI by combining the TST and QFT-GIT results (3 TST+/QFT-GIT+, 1 TST+/QFT-GIT- and 5 TST-/QFT-GIT+). All 3 TST+/QFT-GIT+ patients, the TST+/QFT-GIT- patient as well an additional 3 subjects screened positive with the nHBHA-IGRA. These 3 additional patients had known M. tuberculosis exposure risks compatible with LTBI. During follow-up (median 14 months) no case of TB was reported and nHBHA-IGRA results remained globally constant. Multiplex analysis confirmed IFN- as the best read-out for the assay. From this study, we concluded that the nHBHA-IGRA appears complementary to the QFT-GIT for the screening of LTBI in PLHIV and the combination of the two tests may increase the sensitivity of screening. A large-scale study is however necessary to determine whether combining nHBHA-IGRA and QFT-GIT offers sufficient sensitivity to dismiss TST, as suggested by our results. In the same study, a group of HIV-infected adults with clinical suspicion of active TB were also recruited and tested with nHBHA-IGRA. Contrary to results in HIV-uninfected subjects, the nHBHA-IGRA could not discriminate between LTBI and active TB in PLHIV. This is an important caveat as HIV-infected subjects may present subclinical TB.A different angle was used for the third approach to the problem of LTBI in PLHIV. Systemic immune activation (SIA) is one of the principal driving forces in the natural course of HIV-infection. Despite long-term viral suppression by combination antiretroviral treatment (cART), a low-level SIA persists and is associated with an early-onset of age-associated disorders such as cardiovascular disease, dementia and osteoporosis. Causes of SIA in PLHIV are multiple and certain chronic infections appear to be implicated. A recent study in South Africa found that LTBI in PLHIV was associated with an increase in circulating activated CD8+ T-cells. If LTBI should contribute to the persistence of SIA, its screening and treatment could have an additional benefit on the clinical outcome of PLHIV. To investigate this theory, the expression of T-cell activation markers (CD38 and HLADR) as well as the level of plasmatic markers of immune activation (IL-6, sCD14, D-Dimers) were compared between subjects presenting active TB, subjects with LTBI and M. tuberculosis-free persons, with and without HIV-infection. In accordance with previous studies, active TB was associated with higher levels of SIA biomarkers in both HIV-infected and -uninfected groups. Among the HIV-uninfected subjects, no significant difference in biomarker level was found between those presenting LTBI and those with no evidence of M. tuberculosis. The effect of LTBI on activation biomarkers in the HIV-infected groups remained inconclusive because of the small number of individuals in the HIV+/LTBI group. Further investigation is therefore warranted. Interestingly, it was found that plasmatic markers may have a greater sensitivity for the detection of M. tuberculosis-associated SIA than the T-cell activation markers, an important result for future studies.Overall, LTBI in PLHIV is a challenging topic, in particular because of the lack of a gold-standard for the diagnosis of LTBI. Despite suboptimal tools, the evident clinical impact of LTBI screening and treatment in PLHIV on TB incidence justifies its implementation in standard HIV-care. In low TB-incidence countries, who, when and how to screen for LTBI in PLHIV remains unclear. This work offers an overview on the subject with particular focus on possible measures for improvement in the field.
Doctorat en Sciences médicales (Médecine)
info:eu-repo/semantics/nonPublished

Oligodeoxynucleotides, containing unmethylated CpG dinucleotides within specific sequence contexts in bacterial or viral DNA, are detected as a danger signal by the vertebrate immune system. CpG motifs are sequences with a potent antymycobacterial activity and we tested a panel of eight synthetic CpG ODNs for their capacity to reduce Mycobacterium tuberculosis growth analyzing their effects in vitro in human macrophages. CpG2 ODN is able significantly to reduce pathogen growth while CpG3 ODN determines an enhancement in replication than control macrophages. In term of molecular and cellular mechanisms of defence in the host-pathogen interaction we found a specificity of CpG ODN sequence in intracellular pH decrease but not in Reactive Oxygen Species production, both influenced by CpG. Moreover we analyzed environment of macrophages, stimulated with these two CpG ODNs, before and after infection showing a correlation between mycobacterial growth and IFN- transcription following 1 hour of CpG treatment, and a down-regulation in IFN-mRNA upon pathogen contact. Finally we described a pathway of cytokines, among them SerpinE1 never associated before in M. tuberculosis infection involved in macrophages migration. In conclusion CpG ODNs could be used as vaccine adjuvants confirming their capacities to improve protection against M. tuberculosis involving mechanisms of defence.

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Sabe-se que existem inúmeros trabalhos envolvendo a modulação da resposta imune ao Mycobacterium. No entanto, o número de indivíduos apresentando tuberculose é cada vez maior. A resposta imune ao Mycobacterium é desencadeada principalmente por linfócitos Th1, com a produção de IFN-γ. As parasitoses intestinais também representam um importante problema médico-sanitário, tendo em vista o grande número de pessoas acometidas e as inúmeras alterações orgânicas que podem provocar no hospedeiro. Essas infecções helmínticas induzem preferencialmente uma resposta Th2 com a produção de IL-4, IL-5 e IL-13. Este trabalho avaliou a regulação da resposta imune no pulmão de camundongos co-infectados ou não por S. venezuelensis (SV) e/ou Mycobacterium bovis-BCG (MB), em dois pontos das duas infecções, denominados como ponto 1 (4° e 7° dia pós-imunização [dpi]) e ponto 2 (7° e 10° dpi) por MB e SV, respectivamente. Os animais foram infectados com 700 larvas de SV pela via subcutânea e, após 3 dias, com 1x106 UFC de MB cepa selvagem pela via intravenosa. Realizou-se a quantificação do número de ovos e vermes, a dosagem de citocinas (IFN-γ, IL-4 e IL-10) e quimiocinas (CCL2 e CCL5), o envolvimento de MPO e EPO, a detecção da infecção pelo MB por PCR, a avaliação histopatológica e a expressão de moléculas coestimulat órias/imunomodulatórias (CD80, CD86, CD28, CTLA-4 e CD25) em células ou tecidos do pulmão dos animais infectados e/ou co-infectados. Os resultados mostraram que a presença do MB favoreceu para o aumento do número de ovos e vermes do SV observados nos animais nos dias 4° e 7° (ponto 1) e 7° e 10° (ponto 2) após a infecção por MB e SV, respectivamente, nos animais co-infectados (COIN). A reação de PCR foi efetiva em detectar a presença do MB no pulmão dos animais. Foi observado um aumento de IFN-γ e uma diminuição de IL-4 e EPO no pulmão dos animais do grupo COIN, além de aumento na expressão da molécula co-estimulatória CD80 no ponto 1 e uma diminuição no ponto 2. Houve uma alta produção de IL-10 no pulmão dos animais dos grupos MB e COIN, sendo que a histopatologia neste sítio mostrou formação de granulomas com grande influxo de neutrófilos, macrófagos e células epitelóides na periferia nos pulmões dos animais do grupo MB e um granuloma bem mais avançado, com centro necrótico nos animais do grupo COIN. Baseado nesses resultados, conclui-se que o MB modula a infecção pelo SV, fazendo com que os animais fiquem mais suscetíveis à infecção helmíntica. Por outro lado, o SV modula a infecção pelo MB, fazendo com haja uma modificação na formação de granuloma no pulmão dos animais do grupo COIN no ponto 1 da infecção pelo MB, que poderia ser justificada pela diminuição de IL-4 nos animais do grupo COIN.
A rising number of people have been contracting tuberculosis around the world even though a multitude of reports involving a modulation of the immune response to Mycobacterium have been published. The response to Mycobacterium is mainly mediated by Th1 lymphocytes through IFN-gamma production. Parasitic diseases account for a large proportion of human morbidity and mortality, considering the number of people affected by them and several pathologies associated to parasitic infection. Helminthic infections drive towards Th2 response which leads to IL-4, IL5 and IL-13 production. The present study evaluated the immune response of coinfected animals or not with Strongyloides venezuelensis (SV) and Mycobacterium bovis-BCG (MB) on pulmonary cells collected from BALB/c mice at time points 1 (4th and 7th days post-immunization [dpi] by MB and SV, respectively) and 2 (7th and 10th dpi by MB and SV, respectively). Animals were infected with 700 SV larvae subcutaneously, and 3 days after, 1x106 CFU of wild MB strain intravenously. The number of worms and eggs was counted as well as cytokine (IFN-gamma, IL-4 and IL-10) and chemokine (CCL2 and CCL5) assessments, and the MPO and EPO levels determination on pulmonary tissue from infected and/or coinfected animals. In addition, PCR for MB detection, the histopathology and the expression of costimulatory molecules such as CD80, CD86, CD28, CTLA-4 and CD25 on pulmonary tissue were also assessed. The results pointed that MB led to increase SV parasite burden in coinfected mice (COIN) at both time points analyzed. The PCR technique detected effectively MB. Moreover, elevated IFN-gamma and reduced IL-4 and EPO levels were detected on pulmonary tissue in the COIN group. In regard to CD80 molecule, there was an increased expression at time point 1 and diminished expression at time point 2. Also, higher amounts of IL-10 were found on pulmonary tissue in MB and COIN groups. The histopathological analysis revealed pulmonary granulomas with a number of neutrophils, macrophages and epithelial cells-like in the MB group as well as granulomas in an advanced stage with caseous necrosis in the COIN group. Based on these findings, it may be concluded that MB modulated the immune response to SV, leading coinfected animals to be more susceptible to helminthic infection. On the other hand, SV modulated the MB infection by modifying the characteristics of the pulmonary granulomas in the COIN group at time point 1 probably due the reduced IL-4 production in this group.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
A interleucina-6 (IL-6) à uma importante citocina que exerce papel fundamental na imunopatogÃnese de diversas doenÃas infecciosas. O objetivo deste estudo foi investigar o nÃvel de produÃÃo sistÃmica de IL-6 e aferir o papel funcional do polimorfismo -174 G>C do gene dessa citocina em indivÃduos diagnosticados como portadores de infecÃÃo latente pelo Mycobacterium tuberculosis (ILTB). Para controle, foram utilizados dois grupos de comparaÃÃo: um deles composto por portadores de tuberculose pulmonar ativa (TB) e o outro formado por indivÃduos saudÃveis, doadores de sangue. O grupo ILTB foi composto por 15 indivÃduos, selecionados dentre os contactantes de portadores de TB pulmonar ativa, atendidos no Hospital SÃo Josà de DoenÃas Infecciosas e no Centro de SaÃde da FamÃlia AnastÃcio MagalhÃes. O grupo TB foi formado por 38 pacientes com TB pulmonar ativa, procedentes do Hospital de Messejana, Hospital de Maracanaà e Hospital Geral Dr. CÃsar Cals. O grupo de indivÃduos saudÃveis contava com 63 doadores voluntÃrios de sangue do Centro de Hematologia e Hemoterapia do CearÃ. A dosagem sÃrica de IL-6 foi realizada por meio de um ensaio imunoenzimÃtico (ELISA), com kit especÃfico fornecido pela Invitrogen Corporation. Para purificaÃÃo do DNA, foi utilizado o kit GFX Genomic Blood DNA Purification, da GE Healthcare. O polimorfismo -174GC do gene da IL â 6 foi tipificado pela tÃcnica de reaÃÃo em cadeia da polimerase (PCR), utilizando-se iniciadores de sequÃncia especÃfica (PCR-SSP) (One-Lambda). As medianas de concentraÃÃes sÃricas de IL-6 para os grupos ILTB, TB e saudÃveis foram de, respectivamente, 1,7 pg/mL, 4,3 pg/mL e 0,5 pg/mL (p < 0,0001). Nos trÃs grupos estudados, o genÃtipo encontrado com maior frequÃncia foi o G/G [ILTB = (80%); TB = (58,9%); saudÃveis = (62,8%)]. Em conclusÃo, podemos inferir que a IL-6 deve desempenhar um papel importante na manutenÃÃo do estado de latÃncia, haja vista que sua concentraÃÃo, nos indivÃduos com ILTB, foi 3,4 vezes maior que no grupo saudÃvel. Ademais, constatamos que, na populaÃÃo estudada, o polimorfismo -174GC nÃo se mostrou funcional no Ãmbito da infecÃÃo latente pelo Mycobacterium tuberculosis.
Interleukin-6 (IL-6) is an important cytokine involved in the pathogenesis of multiple infectious diseases. The aim of this study was to investigate the levels of IL-6 production and to correlate to the -174G>C polymorphism at the IL-6 gene in latent infection with M. tuberculosis (ILTB). As controls, two groups were used. One of them with active pulmonary tuberculosis (TB) patients and the other with healthy blood donors. ILTB group was composed by 15 individuals, selected among active pulmonary TB contacts seen at the Hospital SÃo Josà de DoenÃas Infecciosas and the Centro de SaÃde da FamÃlia AnastÃcio MagalhÃes. TB group had 38 patients with active pulmonary disease seen at the Hospital de Messejana, Hospital de Maracanaà and the Hospital Geral Dr. CÃsar Cals. The third group was composed by 63 healthy blood donors from the Centro de Hematologia e Hemoterapia do CearÃ. Serum levels of IL-6 were measured by an ELISA using specific kits from Invitrogen Corporation. For DNA purification a GFX Genomic Blood DNA Purification kit (GE Healthcare) was used. The -174GC polymorphism was analyzed by a SSP-PCR method using One-Lambda kits. Median values of serum levels of IL-6 from ILTB, TB and healthy groups were, respectively, 1.7 pg/mL, 4.3 pg/mL and 0.5 pg/mL (p < 0.0001). For the three studied group, the most frequent genotype found was the G/G (ILTB = 80%; TB = 58.9%; saudÃveis = 62.8%). In conclusion, it is possible to consider that IL-6 should play an important role in the maintenance of latent infection state as its concentrations were 3.4 fold higher in ILTB group than that of healthy controls. Moreover, the -174GC polymorpism was not functional in the ILTB group.

碩士
國立臺灣大學
獸醫學研究所
89
The 4 major pathogenic mycobacteria, M. tuberculosis, M. bovis, M. avium subsp. avium, and M. avium subsp. paratuberculosis, have been identified in Taiwan for many years, remaining important for public health and animal production. The characteristics of the 4 mycobacteria are very similar and it is difficult to differentiate them clinically and histopathologically. The conventional diagnosis for animal mycobacterial infection include PPD, bacterial culture, and acid-fast staining etc. However, these methods have innate defects that may sometimes cause perplexity in the diagnosis. The molecular biotechnologies have been widely used in disease diagnosis. The polymerase chain reaction (PCR) has become a new tool for a more rapid and accurate diagnosis of tuberculosis and paratuberculosis. The objective of the present study was to establish a multiplex-PCR for the diagnosis and differentiation of the 4 major pathogenic mycobacteria simultaneously. The primers and DNA probes were selected and designed according to other’s publication and gene bank comparison, respectively. A two-step multiplex-PCR using 5 pairs of primer was established with standard bacteria. In the first step, 3 pairs of primers were included; primer GroEL amplified a 383 bp nucleotide fragment encoding for the GroEL gene of Mycobacterium spp; primer PT1-2 amplified a 396 bp nucleotide fragment encoding for the mtp40 gene of M. tuberculosis; primer JB21-22 amplified a 495 bp nucleotide fragment of M. bovis. In the second step, 2 pairs of primer were included; primer IS1245 amplified a 427 bp nucleotide fragment encoding for the IS1245 gene of M. avium subsp. avium; primer IS900 amplified a 229 bp nucleotide fragment encoding for the IS900 gene of M. avium subsp. paratuberculosis. Formalin-fixed and paraffin-embedded (FP) tissues from various animal species and samples of blood, milk, nasal discharge, and feces from PPD positive and negative cows were then tested by this method. When the result of multiplex-PCR was obscure, southern blotting was used for further confirmation. For the FP tissues, the 1 M. tuberculosis and 7 M. avium subsp. paratuberculosis cases diagnosed by bacterial culture and/or histopathological examination were all positive by the multiplex-PCR. There were only 4/7 cases of M. bovis-infected PPD-positive cows were positive due to insufficient tissues in the blocks or lack of acid-fast bacteria in the tissue. For other 28 tuberculosis-speculated cases by pathology or PPD test, 8/9 cases and 1/9 case were multiplex-PCR negative due to lack of enough tissues and possibly DNA breakdown, respectively; the remaining 12/19 and 5/19 cases were M. bovis and Mycobacterium spp. positive by multiplex-PCR, respectively; and 2/19 cases (F91-129 and F90238) were multiplex-PCR negative although 1 was acid-fast positive and both had sufficient tissues in the blocks. Tissues from 5 cases suspected having M. avium subsp. avium infection pathologically were all negative by multiplex-PCR. It was possible that primer IS1245 is not specific for all the serotypes of M. avium subsp. avium; additionally, M. avium subsp. intracellulare is very similar to M. avium subsp. avium clinically and histopathologically and primer IS1245 can not detect M. avium subsp. intracellulare. The feces and nasal discharge from PPD positive and negative cows were all negative by multiplex-PCR. For the blood and milk, 34/57 blood samples were M. bovis positive by multiplex-PCR within which 26 were PPD-positive; there were also 7 PPD-positive cases were multiplex-PCR negative. Four out of 13 milk samples were M. bovis positive by multiplex-PCR; blood samples from the 4 cases were M, bovis positive and 3 of them were PPD-positive as well. Besides, one milk case was PPD-positive but both blood and milk were multiplex-PCR negative. It is believed that PPD test combined with blood multiplex-PCR will improve the detection of mycobacterial infection more effectively.

archives@tulane.edu
In order to develop better therapeutics and treatment strategies for tuberculosis (TB) infection, it is imperative to understand interactions that occur in the host in response to the bacilli that contribute to disease progression. Modeling of TB in simple animal models such as mice and zebrafish is often incomplete as there are evolutionary differences, as well as structural issues, that reduce the faithfulness to human TB infection. The non-human primate model of TB infection provides the added benefit of providing a long-established model of SIV infection that recapitulates HIV infection in humans and has been expanded to model TB/HIV co-infection. Here, we have sought to identify correlates of protection in TB and TB/SIV co-infection using rhesus macaques. In the first experiment, we used two strains of Mycobacterium tuberculosis (Mtb), CDC1551 and Erdman, to investigate strain-specific mechanisms of virulence. As increased virulence of Mtb Erdman was associated with excess inflammatory responses, we sought to evaluate a host-directed therapeutic in a lethal challenge model of Mtb CDC1551 infection. We found that use of a type I interferon antagonist significantly improved host survival in the absence of antibiotic treatment and survival was associated with the presence of increased levels of granzyme B producing T cells. A major producer of granzyme B, mucosal-associated invariant T (MAIT) cells was investigated in Mtb/SIV co-infection, but was found to not contribute to protection in TB or TB/SIV. In order to further expand our model of Mtb/SIV co-infection, we co-infected latent Mtb-infected rhesus macaques with a non-pathogenic strain of SIV, SIVmac239ΔGY, and administered a CD4-depleting antibody, CD4R1, in place of SIVmac239 co-infection. Using SIVΔGY, we found that virulent viral replication was necessary for TB reactivation. Using both SIVΔGY and antibody-mediated CD4+ T cell depletion, we found that immune responses are disregulated in Mtb/SIV reactivators in a divergent manner, illustrating the presence of SIV-dependent factors that contribute to TB/SIV reactivation. Overall, these results indicate that immune mechanisms, especially those of inflammation, are significant in determining host outcomes. Developing ways to better control inflammation are necessary to supplement antibiotic treatment and cure TB.
1
Allison Bucsan

M. tuberculosis (Mtb) senses and responds to changes in its environment primar-ily through two-component signalling systems (TCSs). Each TCS contains a trans-membrane histidine kinase (HK ) protein and a cytoplasmic response regulator (RR) protein. HK detects a stimulus and gets phosphorylated. It then binds and transfers the phosphoryl group to the RR of the same TCS. Activated RR then triggers gene ex-pression, including upregulation of the HK and RR involved, eliciting responses that are essential for the bacterium to adapt. Though di erent TCSs detect distinct stimuli, the binding regions of the HK s and RRs share signi cant similarity. This raises the possibil-ity of crosstalk, where HK s dissipate signals to RRs that do not belong to the same TCS. Studies have argued that such dissipation of signals impairs the fitness of the organism, as it decreases the output levels as well as triggers unwanted responses. In contrast, a recent experimental study has discovered that TCSs of Mtb share extensive crosstalk, violating the widely accepted specificity paradigm. In this study, we have attempted to unravel the evolutionary underpinnings of this extensive crosstalk observed in Mtb. We hypothesised that such crosstalk may be advantageous in programmed environments, where there are well-defined sequences of stimuli. In such situations, crosstalk can up-regulate HK s and RRs of non-cognate TCSs. This up-regulation primes the latter TCSs for upcoming signals, increasing their sensitivity. We constructed a mechanistic model of the functioning of TCSs and a fitness variable to qualitatively measure the response of a TCS to a signal, to test the hypothesis. We performed population genetics simulations of the evolution of phenotypes of different crosstalk patterns. We found that in a random environment, the phenotype without any crosstalk is selected over time, which is in agreement with prevalent arguments in favour of specificity of TCSs. But when the environment is programmed, the phenotype with a crosstalk pattern mirroring the pattern of stimuli dominates the population. Finally, we found evidence for the evolutionary preference to preserve crosstalk in gene sequences of HK s and RRs encoded in Mtb. We found that the binding domains of HK s and RRs, which were predicted to share crosstalk, are under greater pressure to be similar than those domains which do not crosstalk. Our study thus provides a plausible explanation of the unexpected presence of crosstalk in Mtb. Since these cross-interactions aid the pathogen to adapt in the host, inhibitors of such interactions are likely to have therapeutic potential.

M. tuberculosis (Mtb) senses and responds to changes in its environment primar-ily through two-component signalling systems (TCSs). Each TCS contains a trans-membrane histidine kinase (HK ) protein and a cytoplasmic response regulator (RR) protein. HK detects a stimulus and gets phosphorylated. It then binds and transfers the phosphoryl group to the RR of the same TCS. Activated RR then triggers gene ex-pression, including upregulation of the HK and RR involved, eliciting responses that are essential for the bacterium to adapt. Though di erent TCSs detect distinct stimuli, the binding regions of the HK s and RRs share signi cant similarity. This raises the possibil-ity of crosstalk, where HK s dissipate signals to RRs that do not belong to the same TCS. Studies have argued that such dissipation of signals impairs the fitness of the organism, as it decreases the output levels as well as triggers unwanted responses. In contrast, a recent experimental study has discovered that TCSs of Mtb share extensive crosstalk, violating the widely accepted specificity paradigm. In this study, we have attempted to unravel the evolutionary underpinnings of this extensive crosstalk observed in Mtb. We hypothesised that such crosstalk may be advantageous in programmed environments, where there are well-defined sequences of stimuli. In such situations, crosstalk can up-regulate HK s and RRs of non-cognate TCSs. This up-regulation primes the latter TCSs for upcoming signals, increasing their sensitivity. We constructed a mechanistic model of the functioning of TCSs and a fitness variable to qualitatively measure the response of a TCS to a signal, to test the hypothesis. We performed population genetics simulations of the evolution of phenotypes of different crosstalk patterns. We found that in a random environment, the phenotype without any crosstalk is selected over time, which is in agreement with prevalent arguments in favour of specificity of TCSs. But when the environment is programmed, the phenotype with a crosstalk pattern mirroring the pattern of stimuli dominates the population. Finally, we found evidence for the evolutionary preference to preserve crosstalk in gene sequences of HK s and RRs encoded in Mtb. We found that the binding domains of HK s and RRs, which were predicted to share crosstalk, are under greater pressure to be similar than those domains which do not crosstalk. Our study thus provides a plausible explanation of the unexpected presence of crosstalk in Mtb. Since these cross-interactions aid the pathogen to adapt in the host, inhibitors of such interactions are likely to have therapeutic potential.

Tuberculosis (TB) is a deadly disease responsible for the death of approximately 1.5 million people each year, with the highest being from developing nations. Tuberculosis affects mostly the lungs, and other parts of the body like nerves, bones and liver. Mycobacterium tuberculosis (Mtb) is the causative agent of TB in humans. The onset of infection is via the deposition of aerosol droplets containing the pathogen, M. tuberculosis, onto the lung alveolar surfaces. About one third of the world’s population asymptomatically harbors latent M. tuberculosis bacterium with a constant risk of disease activation. Due to the emergence of drug-resistant strains and the evolution through multi-drug resistance (MDR) to extensive drug resistance (XDR), the fight against TB has become extremely challenging. Standard treatment for TB comprises four first-line antimicrobials: isoniazid, rifampicin, pyrazinamide and ethambutol. However, resistance to all of these drugs has been observed in several MDR strains of Mtb. Despite the recent advances in target identification and drug discovery, there is a relentless need for novel inhibitors against vital pathways of Mtb. The novel drug-development regimens endorse strategies wherein the pre-approved drugs for other ailments could be re-purposed, thereby cutting down the cost and time associated with the process of drug discovery. Also, the target selection strategy requires to aim at the key enzymes from the essential biosynthetic pathways, keeping an eye on their underlying dissimilarities when compared to human host. The challenges in finding a suitable target for anti-Mtb drug discovery is it’s ever evolving stride and the conserved nature of the essential proteins. Many novel small molecule inhibitors of Mtb are undermined, during the course of studies, by cross reactivity with homologs proteins in the host. Traditionally, the replication machinery has been at the heart of drug discovery and the processes associated with logarithmic growth phase are vastly exploited for drug targeting. However, targeting these vital cellular components may result in some serious non-specific effects to the host. On the other hand, the intricate network of metabolic pathways provides novel avenues for specific targeting of pathogens, precisely for three main reasons: 1. There is an acute shortage of cellular nutrients due to the constant competition between the pathogen and the host, throughout the course of infection. 2. Infectious cycles often lead to the disruption of metabolic pathways, again leading to nutrient scarcity. 3. Survival of the pathogen within the hostile niche and under oxygen starvation conditions further potentiate the demands of crucial metabolites (amino acids, nitrogen bases, carbohydrates etc.) that are used as the building blocks for cellular machinery. 4. Metabolic pathways have evolved with time, to provide the much-required specificity for exclusive targeting of the pathogen, thereby limiting the cross-reactivity with the host pathways. In order to persist and efficiently replicate in host cells, intracellular pathogens must adapt their metabolism to the available nutrients and physical conditions (mainly pH, oxygen availability and osmotic pressure) in the host. Among the major metabolic, amino acid metabolism holds great importance; as they not only serve to meet the nutritional needs of the pathogen but also play a key role in the strategies employed during pathogenesis. Although the host and the pathogen compete for many metabolites, three amino acids, Arginine, Asparagine and Tryptophan seems to be a focus of this competition because the availability of these amino acids or their derivatives influence both pathogen behavior and the immune response. Arginine constitutes a major proportion of the total proteins in the cell and arginine and its precursor ornithine are used for the biosynthesis of the most common polyamines, putrescine and spermidine. These molecules are required for optimal growth of the organism and are involved in several physiological processes. Apart from being a critical amino acid for the synthesis of cellular proteins, arginine can also be used as a nitrogen source, under conditions of nitrogen starvation, hence crucial for pathogenesis. The glutamate and glutamine are the key metabolites in the central nitrogen metabolism; both serve as endogenous nitrogen acceptor as well as nitrogen donor. However, reports demonstrate that Mtb utilizes arginine and asparagine as the key sources of nitrogen during infection in mice models of tuberculosis. Therefore, our study focuses on the process of Arginine biosynthesis in M. tuberculosis, wherein it is essential for the survival and pathogenesis. Since the arginine metabolism is essential for both the host and the pathogen, and competition for arginine may shift the balance, and thus determine the outcome of the infection. The enzymes involved in this pathway will be a promising target for anti-TB drug development. Despite the acknowledged significance of Arginine biosynthesis in the pathogens like M. tuberculosis, inhibitors to target this pathway remain to be discovered. Moreover, inhibitors of this pathway may provide novel insights to the significance of arginine biosynthesis in Mtb associated stress responses and persistence. Ornithine acetyltransferase (MtArgJ), one of the crucial enzymes during the biosynthesis of arginine in Mtb, is essential for its survival and pathogenesis. MtArgJ lacks a homolog in human genome, thereby being a good target against Mtb. We hypothesize that a targeted inhibitor against this key player of mycobacterial metabolism has the potential to combat the Mtb survival and pathogenesis. In the present thesis, we have characterized the potential of MtArgJ from M. tuberculosis as a valuable target for drug design against tuberculosis. Most importantly, the approach is to specifically target a novel allosteric site identified in this study, on the MtArgJ surface. Since we are not using the age-old approach of substrate analog as an inhibitor, we hereby further minimize or even eliminate the chances of cross-reactivity with the host cellular proteins. In the later parts, we have identified an allosteric inhibitor of MtArgJ, that significantly reduces the survival of pathogenic Mtb through the pre-clinical models of tuberculosis. Chapter 1 of this thesis gives a detailed account of the history of Tuberculosis, and its pathogenesis. The chapter further elaborates on the metabolic pathways of Mycobacterium tuberculosis, with special emphasis on the arginine biosynthesis pathway. The drug discovery regime and therapeutic challenges associated with the disease are discussed in the later parts of the chapter. All the information discussed in this chapter serves a preface for the work done throughout the thesis, and outlinesthe objectives for rest of the chapters. Chapter 2 describes the characterization and kinetic analysis of MtArgH, the last enzyme from the arginine biosynthetic pathway in M. tuberculosis. This chapter demonstrates the importance of a c-terminal cysteine residue (Cys441) in the catalysis and thermal stability of the enzyme. We further propose the existence of a product mediated feed-back inhibition of MtArgH, wherein fumarate, one of the product of MtArgH, gradually modifies the Cys441 through succination. Chapter 3 to 5 discuss about the work carried out on the enzyme Ornithine acetyltransferase (MtArgJ), a crucial enzyme for arginine biosynthesis in M. tuberculosis. We have identified a selective allosteric inhibitor against this key player of mycobacterial metabolism, employing the below-mentioned strategy. First step was to characterize the target, followed by a structure based in-silico screen. The best hits were subjected to in-vitro validation, leading to the in-vivo testing of the potential molecule, in the pre-clinical model of tuberculosis. Target characterization In-silico screen In-vitro validation Pre-clinical testing Chapter 3 starts with the characterization of the MtArgJ, wherein we identified a novel hydrophobic pocket present on the enzyme surface. We further characterized the potential of this pocket in allosterically modulating the active site. This was then followed by a structure based in-silico screen with a library of FDA approved drugs, specifically targeting this novel allosteric pocket on MtArgJ. Chapter 4 deals with the in vitro validation of the identified compounds from in-silico screen. We here identified two lead molecules, Pranlukast (PRK) and Sorafenib (SRB), to have significant affinity for the allosteric site on MtArgJ, leading to the inhibition of its enzymatic activity. We further propose the key residues involved in this interaction, thereby suggesting a potential molecular mechanism of inhibition. Chapter 5 leads us to the in-vitro and in-vivo characterization of these compounds as a potent anti-tubercular agent. We first demonstrate its efficacy in deducing the survival of the pathogenic strains of Mtb in-vitro and in the macrophage models of infection. We also tested the efficacy of these compounds in combination with the standard of care TB therapy drugs, and found PRK to work efficiently in such combinations. Finally, we evidence the potency of PRK in compromising the survival and pathogenesis of Mtb in mice models of tuberculosis infection. PRK is presently being used as a drug against chronic asthma, therefore its human safety is already assured. This will facilitate its induction into the direct clinical trials against tuberculosis. Taken together, the work done in this thesis demonstrates a novel metabolic inhibitor of Mtb pathogenesis, through the pre-clinical models of infection with the potential for development of advanced combinatorial therapy against tuberculosis.

碩士
嘉南藥理科技大學
生物科技系暨研究所
98
Abstract Mycobacterium avium complex (MAC) belongs to a group of slow growing mycobacteria. Many studies showed that chronic pulmonary disease is most commonly manifestated by a non-tuberculous mycobacteria (NTM) infection. MAC, M. kansasii, and M. abscessus, in order of frequency, are the most common pathogens of NTM lung disease. We found the patients with NTM lung disease were more than those with pulmonary tuberculosis between July, 2007 and December, 2008 and MAC was the most common pathogen of NTM lung disease. A case-control was conducted study and 30 patients with a positive sputum culture for MAC as the case group and 30 patients with a positive sputum culture for M. tuberculosis as the control group were enrolled. The two groups were matched with sex, residential areas, and patient services (outpatients or inpatients). Several variables as predictors of risk factors were used to evaluate acquiring NTM lung disease, including age, height, weight﹐BMI﹐DM, smoking, upper lung fields﹐lower lung fields, and pulmonary cavities. We also compared the gene sequence by sequencing to identify the Rifampicin resistance gene of MAC. Involvement of lower lung fields were more common in MAC lung disease (McNemar test, p &lt;0.05), while the pulmonary cavities were more common in pulmonary tuberculosis (Logistic regression, p &lt;0.05). Finally, EMBOSS were used for the database of M.avium AF060366 and sequence analysis. Three strains with a point mutation (Glycine→Aspartic acid) in the nucleotide sequence 433 was noted. Twenty-six strains (87%) presented the phenotype of Rifampicin resistance, while 4 strains were sensitive to Rifampicin (13%). However, genotypes of Rifampicin resistance were noted for only 11% of the strains. It implies that a new criteria for clinical susceptibility test should be established to provide a more convenient, cheap and accurate method for the in-vitro susceptibility test.

Bovine Tuberculosis is a chronic debilitating disease of cattle and other animals with a worldwide distribution and transmitted mainly through the inhalation of aerosols. The aim of this study was to determine the prevalence of BTB in the cattle population of selected dip tanks in Swaziland. Furthermore, the zoonotic risk to farmers whose cattle are infected with BTB was assessed by means of a questionnaire survey. Abattoir surveillance identified 16 dip tanks of study where at least 10 % of the cattle were tested for BTB using the comparative intra-dermal skin. In five of these dip tanks, the same cattle were tested for BTB using the IFN-γ Test. Eight BTB skin test positive animals were slaughtered and a detailed post mortem examination was conducted and samples collected for the isolation of M. bovis. Concurrent with BTB testing, a questionnaire survey was conducted to determine risk factors for humans. The prevalence of BTB was found to be 6.75 % in the study population and 20 % of BTB positive animals were diagnosed by both the CIST and IFN-γ, indicating a correlation for the test positive animals in the two tests. M. bovis was isolated from seven of the eight animals slaughtered. Farmers’ knowledge of BTB as a cattle disease and serious zoonosis is insufficient and inadequate while consumption practices of products of bovine origin exposes them to the risk of infection by M. bovis. There is a need to investigate the extent of M. bovis infections in the human population.
Dissertation (MSc)--University of Pretoria, 2013.
gm2014
Veterinary Tropical Diseases
unrestricted