Memoria para optar al Título Profesional de Médico Veterinario / Mycobacterium bovis es el microorganismo zoonótico causante de la tuberculosis bovina, patología infectocontagiosa de distribución mundial responsable de graves problemas económicos y de salud en el sector ganadero. Debido a que varios estudios han establecido la utilidad de las secuencias de inserción IS6110 e IS1081 en la identificación y tipificación de cepas del complejo Mycobacterium tuberculosis, el objetivo de este estudio fue establecer protocolos de PCR (Reacción en Cadena de la Polimerasa) para la amplificación de las secuencias IS6110 e IS1081 y determinar la existencia de diferencias en su capacidad de detectar Mycobacterium bovis en tejidos bovinos. El análisis estadístico de los resultados sugirió que no existen diferencias significativas entre los ensayos de PCR convencional basados en IS1081 e IS6110, lo que contrasta con lo señalado por otros autores, quienes postulan que IS1081 está presente en mayor número de copias en cepas de Mycobacterium bovis, y por tanto, su utilidad en el diagnóstico es mayor. Debido a que la concordancia de ambas pruebas fue baja, se requieren nuevos estudios destinados a determinar la aplicabilidad de ambas secuencias en conjunto, por ejemplo en una prueba de PCR múltiple, ya que este trabajo sugiere que son complementarias para el diagnóstico de tuberculosis bovina / Proyecto FIV 121014019102003

Mycobacterium tuberculosis est la cause d’une des maladies infectieuses les plus fréquentes dans le monde causant la mort de plus de 1,2 millions de personnes chaque année selon l’organisation mondiale de la santé (OMS). Actuellement, la tuberculose à M. tuberculosis émerge chez d’autres espèces comme l’a montré notre revue bibliographique des cas de tuberculose à M. tuberculosis chez les primates non-humains, les éléphants d’Asie, les animaux de ferme, les animaux de compagnie et certains animaux sauvages. Nous avons montré que ces trois espèces survivent dans le sol pendant au moins 12 mois et restent pathogènes dans un modèle souris. Egalement nous avons montré que le sol infecté par M. tuberculosis est une source potentielle de contamination pour les animaux. Nous avons ensuite développé un protocole de culture rapide de M. tuberculosis, incluant un nouveau milieu de culture solide, des conditions optimisées d’incubation, et la détection des microcolonies par autofluorescence. Notre travail de thèse a permis de mettre en place des techniques et protocoles qui révolutionnent la culture et l’isolement de M. tuberculosis en réduisant les délais de culture et des antibiogrammes, un point déterminant pour la lutte contre la tuberculose notamment dans les pays à ressources limitées et les pays à forte émergence de souches de M. tuberculosis de plus en plus résistantes. Ces protocoles sont en cours de transfert pour la routine de laboratoire. / Mycobacterium tuberculosis is the cause of one of the most common infectious diseases in the world killing more than 1.2 million people each year according to the World Health Organization (WHO). Currently, M. tuberculosis tuberculosis emerges in other species like non-human primates, Asian elephants, farm animals and some wild animals. We have shown that three species of M. tuberculosis complex survive in the soil for at least 12 months and are pathogenic in a mouse model and M. tuberculosis-infected soil is a potential source of infection for animals. We then developed a protocol for rapid culture of M. tuberculosis, including a new solid culture medium, optimized conditions of incubation, and detection of microcolonies by autofluorescence. Our thesis has helped develop techniques and protocols that are revolutionizing the culture and isolation of M. tuberculosis by reducing delays culture and susceptibility testing, a crucial point for the fight against tuberculosis, especially in countries limited resources and countries with strong emergence of M. tuberculosis strains more resistant. These protocols are being transferred to the routine laboratory.

L’agent pathogène responsable de la tuberculose, Mycobacterium tuberculosis, utilise principalement les lipides comme source d’énergie lors du processus infectieux. Cela suggère que les enzymes lipolytiques (ELs) sont des facteurs d’une importance cruciale, qui jouent un rôle essentiel pour la survie et la persistance du bacille au sein des granulomes tuberculeux. Dans ce contexte, nous nous sommes intéressés au rôle physiologique de la protéine LipY (Rv3097c), une TAG-hydrolase apparentée à la lipase Hormono-sensible humaine. En utilisant un modèle expérimental de « foamy » macrophages, nous avons démontré que cette protéine sécrétée par le système de sécrétion ESX-5 était responsable de l’hydrolyse des triacylglycerols (TAG) de l’hôte dans la lumière phagosomale. Dans une seconde étude, nous avons développé un modèle expérimental robuste et réversible basé sur la biodisponibilité en azote et en carbone, deux facteurs essentiels qui gouvernent la formation et l’hydrolyse de TAG sous la forme d’inclusions lipidiques intracytoplasmiques (ILI) chez les mycobactéries. La simplicité d’utilisation de ce modèle s’avère être un excellent système biologique permettant d’étudier les propriétés phénotypiques des bacilles riches en ILI. L’ensemble des résultats obtenus au cours de ces travaux de thèse confirme que certaines ELs sont directement impliquées dans la virulence et/ou la survie des mycobactéries pathogènes. De plus, une très grande partie de ce travail ouvre de nouvelles perspectives sur la caractérisation et l’inhibition d’acteurs protéiques essentiels au métabolisme des lipides in vivo, offrant ainsi de nouvelles opportunités pour lutter contre M. tuberculosis. / Mycobacterium tuberculosis, the pathogenic agent responsible for tuberculosis (TB), mainly uses lipids as a source of energy during the infectious process. This suggests that lipolytic enzymes (LEs) are crucial metabolic agents that play a critical role in the survival and persistence of the bacillus within TB granulomas. In this context, we investigated the physiological role of the LipY protein (Rv3097c), a TAG-hydrolase related to the human Hormono-sensitive lipase. Using an experimental model of "foamy" macrophages, we demonstrated that this protein which is secreted by the ESX-5 secretion system, was responsible for the hydrolysis of host-derived triacylglycerols (TAGs) within the phagosomal compartment. In a second study, we developed a robust and reversible experimental model based on the bioavailability of nitrogen and carbon, two essential factors that govern the formation and hydrolysis of TAGs in the form of intracytoplasmic lipid inclusions (ILI) in mycobacteria. The simplicity of this model proves to be an excellent biological system for studying the phenotypic properties of ILI-rich bacilli. All the results obtained during this thesis confirm that some LEs are directly involved in the virulence and/or survival processes of pathogenic mycobacteria. In addition, we truly believe that this work opens up new perspectives on the characterization and inhibition of protein, that are essential actors for lipid metabolism in vivo, thus offering new opportunities to better control M. tuberculosis.

Thesis presented in fulfilment of the requirements governing the degree of Philosophy in the School of Medicine, University of the Witwatersrand. Johannesburg / Sonicates derived from Mycobacterium tuberculosis suppressed lymphocytes proliferation. Pulsing of monocytes with mycobacterial sonicates resulted in the release of high molecular weight lipids. Both these lipids and those prepared by column fractionation of mycobacterial sonicates suppressed lymphocyte blastogenesis.This effect was due to the activation and not the proliferation of CD8+ lymphocytes by the lipid containing mycobacterial fractions of Mr>200kDa that could be obtained in vitro by column fractions. / IT2018

A research report Submitted to the faculty of Health Sciences, University of Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Masters of Science in Medicine (Pharmaceutical Affairs). / Pathogenic mycobacterial infection was in recent decades a health concern so well controlled that eradication seemed imminent. However, it is once again reaching epidemic proportions following the increasing prevalence of AIDS. One important means of curbing this resurgence, is a robust method that has the capability of identifying to a species level speciating disease causing mycobacteria in a matter of days. Several new methodologies are now available that enable dramatic reductions in turn-around times. In this study High Performance Liquid Chromatography was investigated to determine how this system compared with the current mycobacterial system of methodologies adopted in South African laboratories. Four species of pathogenic mycobacteria, with a high prevalence in South Africa, were tested in a sample size of 80. Samples were subjected to HPLC, Gene Probes and Biochemical testing. HPLC was the most capable of identifying the mycobacteria to species level displaying a sensitivity to the organisms of 96.25 %. Gene probes and Biochemical testing had sensitivity values of 82.5 % and 80 % respectively. HPLC was also more cost efficient and displayed a wider range of identification. It is therefore suggested that HPLC replace Gene probes and Biochemical testing for purposes of MOTT identification in the comprehensive mycobacterial identification system. The result is a time saving of at least 3 weeks and a cost reduction of approximately 30 %. The large initial capital investment required for the implementation of the HPLC system is justified by the long term cost saving as well as the additional utility derived from early identification. As a consequence treatment is not empiric but rather tailored to the organism infecting the patient, hence preventing multiple drug resistance developing and ultimately saving a life through rational drug use. / WHSLYP2016

MSc., Faculty of Science, University of the Witwatersrand, 2011 / The increasing emergence of drug-resistant pathogens poses a major threat to public health. Although influenced by multiple factors, resistance is often associated with mutations in drug target-encoding or associated genes. The potential fitness cost of such resistance mutations is, in turn, a key determinant of the spread of drug-resistant strains. Rifampicin (RIF) is a frontline anti-tuberculosis agent that targets the rpoB-encoded β-subunit of the DNA-dependent RNA polymerase (RNAP). RIF resistance (RIFR) maps primarily to mutations in rpoB that might be expected to affect transcription and so the ability of the organism to cause disease. Accordingly, numerous studies have assessed the impact of RIFR on key fitness indicators in pathogens including Mycobacterium tuberculosis (MTB). In contrast, the specific consequences of RIFR for bacterial physiology remain poorly understood. Notably, previous studies of the effects of RIFR-associated rpoB mutations on mycobacterial physiology have been conducted using strains generated by RIF exposure, without accounting for the potential impact of second-site mutations that may compensate for fitness costs or contribute to drug resistance. In this study, site-directed mutagenesis and allelic exchange were employed to generate a panel of M. smegmatis (MSM) strains containing clinically-relevant RIFR-associated point mutations. Importantly, this methodology enables the introduction of rpoB mutations into defined strain backgrounds in the complete absence of RIF. Using this approach, we constructed “RIF naive” MSM rpoB mutant strains carrying either an S531L or H526Y mutation. The resulting mutants were 100-fold less susceptible to RIF than the isogenic, parental strain. Notably, the inclusion of selected efflux inhibitors in susceptibility assays had little impact on mutant susceptibility to RIF. In contrast, restoration of the wild-type allele returned the observed susceptibility to parental levels, thereby providing strong evidence of the sufficiency of a single rpoB mutation for clinical RIFR in mycobacteria. Competitive growth assays utilizing the S531L mutant and the parental strain exposed a growth defect for the S531L mutant. However, discriminating between wild-type and mutant rpoB strains proved a significant technical challenge, again highlighting the difficulties associated with inferring in vivo fitness from in vitro assays conducted under a limited number of different conditions. In summary, our results suggest the benefit of a deeper exploration of the physiological and fitness implications of RIFR-associated mutations. In addition, in coupling a system which enables an evaluation of the physiological consequences of drug resistance-associated mutations with evolutionary analyses, we provide preliminary evidence of the benefits of a multipronged approach to elucidating the physiological implications of drug resistance in MTB.

A Pirazinamida (Z), droga de primeira linha usada no tratamento da tuberculose, necessita ser hidrolisada pela enzima bacteriana pirazinamidase (PZase) para que o seu metabólito ativo, o ácido pirazinóico (POA), possa agir. O principal mecanismo molecular de resistência a esta droga envolve mutações no gene pncA, que codifica a PZase. Com base nestas informações e tendo em vista a ausência de estudos acerca de resistência à Z em isolados clínicos de M. tuberculosis em nosso país, o presente trabalho propôs caracterizar as mutações envolvendo o gene pncA, bem como relacioná-las com os resultados do teste de atividade da enzima PZase e da concentração inibitória mínima (CIM) de Z. A caracterização molecular dos isolados foi realizada por \"Spoligotyping\", sendo que, todos os isolados testados foram confirmados como pertencentes à espécie M. tuberculosis. A CIM foi realizada por três metodologias: técnica em microplaca utilizando o Alamar Azul como revelador (MABA), método de microdiluição em caldo (BMM), e método das proporções em Lowenstein-Jensen. Os resultados obtidos dão conta de uma boa associação entre as metodologias, e a determinação da CIM pelo método MABA mostrou-se uma nova e segura opção a ser utilizada para Z. A maioria dos isolados clínicos de M. tuberculosis resistentes à Z (88%), apresentaram também atividade de PZase negativa, bem como mutações no gene pncA. Algumas exceções foram encontradas, já que 12% dos isolados clínicos resistentes não apresentaram mutações no gene pncA e tiveram atividade da PZase positiva, sugerindo a existência de outro mecanismo envolvido com resistência à Z. Das 22 mutações encontradas no gene pncA, 9 estão sendo descritas apenas neste estudo. Registrou-se também a presença de 5 isolados clínicos apresentando fenótipo de monorresistência à Z. / Pyrazinamide (Z), a first-line antituberculous drug, is a prodrug that must be activated by bacterial pyrazinamidase (PZase) to the active form pyrazinoic acid, which kills M. tuberculosis. Many studies have shown that mutation in the gene encoding PZase (pncA) is the major mechanism of Z-resistance in M. tuberculosis. Based on this information and taking into consideration the absence of studies concerning Z-resistance in Brazilian M. tuberculosis strains, this study was aimed at characterizing pncA mutations and investigating its correlation with Z-resistance and PZase activity. The molecular characterization carried out by Spoligotyping revealed that all tested strains belong to M. tuberculosis species. The minimal inhibitory concentration (MIC) of Z was determined by three methods: microplate Alamar Blue assay (MABA), broth microdilution method (BMM) and method of proportions on Lowenstein-Jensen medium. The results showed a good association between the 3 methods, and MABA for MIC determination signalized a new and safe option to be used for Z. Most of Z-resistant strains (88%) presented pncA mutations as well as loss of PZase activity. Some exceptions were found since 12% of Z-resistant strains presented neither pncA mutations nor loss of PZase activity, what suggests the existence of another Z-resistance mechanism. Nine of 22 mutations found in pncA gene were described only in this study. During the course of this investigation were identified 5 Z-monoresistant M. tuberculosis strains.

by Au Lai Yin, Cathy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 50-66). / Chapter I. --- ABSTRACT --- p.i / Chapter II. --- ACKNOWLEDGMENTS --- p.iii / Chapter III. --- TABLE OF CONTENTS --- p.iv / Chapter IV. --- LIST OF TABLES --- p.viii / Chapter V. --- LIST OF FIGURES --- p.x / Chapter VI. --- INTRODUCTION --- p.1 / Chapter VII. --- LITERATURE REVIEW --- p.3 / Chapter A. --- Mycobacterial tuberculosis Infections --- p.3 / Chapter B. --- Diagnostic Criteria forM .tuberculosis Infections --- p.3 / Chapter C. --- Mycobacteriological Laboratory Investigations for M. tuberculosis --- p.4 / Chapter 1. --- Conventional methods --- p.4 / Chapter 2. --- Rapid methods --- p.4 / Chapter D. --- Polymerase chain reaction (PCR) - the Principle --- p.5 / Chapter E. --- Application of PCR for Detection of M. tuberculosis --- p.6 / Chapter 1. --- Choice of target sequences --- p.6 / Chapter 2. --- Choice of method for the detection & identification of PCR-amplified product --- p.7 / Chapter 3. --- Studies on pure cultures --- p.9 / Chapter a. --- Detection limit - target DNA --- p.9 / Chapter b. --- Detection limit - Colony forming units --- p.9 / Chapter c. --- Detection limit - Number of cells --- p.10 / Chapter 4. --- Studies on clinical specimens --- p.10 / Chapter 5. --- Problems --- p.12 / Chapter a. --- Availability of target DNA --- p.13 / Chapter (i) --- Cell breakage efficiency --- p.13 / Chapter (ii) --- Target sequence --- p.14 / Chapter b. --- Inhibitory factors for Taq polymerase --- p.14 / Chapter c. --- Contamination --- p.15 / Chapter VIII. --- MATERIALS AND METHODS --- p.16 / Chapter A. --- Bacterial Strains and Strain Maintenance --- p.16 / Chapter 1. --- Reference Strains --- p.16 / Chapter 2. --- Clinical isolates --- p.16 / Chapter B. --- Growth media and culture conditions --- p.17 / Chapter C. --- Restriction Fragment Length Polymorphism (RFLP) --- p.17 / Chapter 1. --- Extraction of chromosomal DNA from M. tuberculosis --- p.18 / Chapter 2. --- Digestion of chromosomal DNA by PVU II --- p.19 / Chapter 3. --- Separation of digested DNA fragment by electrophoresis --- p.19 / Chapter 4. --- Southern Blotting --- p.19 / Chapter 5. --- Preparation of DNA probes by Polymerase Chain Reaction --- p.20 / Chapter 6. --- Hybridization --- p.21 / Chapter 7. --- Detection --- p.21 / Chapter D. --- Assessment of number of organisms --- p.22 / Chapter 1. --- Viable cell count --- p.22 / Chapter 2. --- Direct cell count --- p.22 / Chapter E. --- Assessment of the presence of IS6110/986 in M. tuberculosis isolates --- p.23 / Chapter F. --- Human leukaemic monocytic cell line (THP-1) --- p.23 / Chapter 1. --- Growth media and maintenance --- p.23 / Chapter 2. --- Culture Conditions --- p.24 / Chapter 3. --- Uptake of M. tuberculosis --- p.24 / Chapter G. --- Cell breakage and DNA extraction methodologies --- p.25 / Chapter H. --- Polymerase chain reaction (PCR) methodologies --- p.28 / Chapter 1. --- Primer and probe --- p.28 / Chapter 2. --- PCR conditions --- p.28 / Chapter 3. --- Detection --- p.29 / Chapter I. --- Patients and Clinical specimens --- p.30 / Chapter 1. --- Patients recruitment --- p.30 / Chapter 2. --- Clinical specimens --- p.30 / Chapter IX. --- RESULTS --- p.32 / Chapter A. --- "Development or Selection of a ""Standardized"" PCR Protocol for the Detection of M. tuberculosis Using Pure Cultures In Vitro" --- p.32 / Chapter 1. --- Selection of organisms for verification of the PCR protocol --- p.32 / Chapter 2. --- Optimization of the PCR conditions --- p.32 / Chapter 3. --- Detection limit of target DNA using the PCR procedure --- p.33 / Chapter B. --- Initial Screening of Six Different Cell Breakage Procedures Using Pure Cultures of M. tuberculosis Isolates TB19 &22a Based on Detection Limits of Colony Forming Units and Number of Cells --- p.34 / Chapter C. --- Comparison of Method 1 and Method 2 Based on Detection Limits of Colony Forming Units and Number of Cells Using Pure Cultures of the Eight Clinical Isolates of M. tuberculosis with variable copies of IS6110/986 --- p.34 / Chapter D. --- Detection of M. tuberculosis Isolates Within Macrophages --- p.35 / Chapter 1. --- Uptake of M. tuberculosis cells by THP-1 --- p.35 / Chapter 2. --- Comparison of the Six Different Cell Breakage Procedures Using Pure Cultures of M. tuberculosis Isolates TB19 & 22a Phagocytized by Activated THP-1 Macrophages --- p.35 / Chapter 3. --- Comparison of Method 1 and Method 2 Using Pure Cultures of the Eight Clinical Isolates of M. tuberculosis Phagocytized by Activated THP-1 Macrophages --- p.36 / Chapter E. --- Analysis of Clinical Specimens Using Method 1 & 2 with the Optimized PCR Protocol --- p.36 / Chapter 1. --- Bronchial Aspirate & Bronchoaveolar Lavage Fluid --- p.36 / Chapter 2. --- Pleural Fluid --- p.37 / Chapter 3. --- Tissue --- p.37 / Chapter 4. --- Sputum --- p.38 / Chapter 5. --- Cerebrospinal Fluid --- p.38 / Chapter X. --- DISCUSSION --- p.39 / Chapter A. --- Selection of IS6110/986 for DNA amplification --- p.39 / Chapter B. --- Optimization of PCR conditions reflected by detection limit of target DNA --- p.40 / Chapter C. --- Selection of cell breakage methods based on detection limits of CFU and/or number of mycobacterial cells --- p.41 / Chapter D. --- Application of Methods 1 & 2 and the optimized PCR protocol for clinical specimens --- p.43 / Chapter 1. --- Bronchial aspirates and bronchoaveolar lavage fluids --- p.43 / Chapter 2. --- Pleural fluids --- p.44 / Chapter 3. --- Tissues --- p.45 / Chapter 4. --- Sputa --- p.46 / Chapter 5. --- Cerebrospinal fluids --- p.46 / Chapter XI. --- CONCLUSION --- p.48 / Chapter XII. --- LITERATURE CITED --- p.50 / Chapter XIII --- TABLES --- p.67 / Chapter XIV. --- FIGURES --- p.85

Orientador: Ana Marisa Fusco Almeida / Coorientador: Cleslei Fernando Zanelli / Banca: Carlos Peleschi Taborda / Banca: Cristiano Gallina Moreira / Resumo: Paracoccidiodomicose (PCM) é uma micose sistêmica endêmica na América Latina, de alta prevalência no Brasil, que tem como agente etiológico os fungos dimórficos do gênero Paracoccidioides, P. brasiliensis e P. lutzii. No processo de infecção as adesinas, substâncias sintetizadas por estes fungos que se ligam a matriz extracelular, são importantes fatores de virulência para o estabelecimento da infecção. A proteína de 30kDa, pertencente a família de proteínas 14-3-3, se destaca no processo adesivo deste fungo. As proteínas 14-3-3 são uma família de proteínas presentes em todas as células eucariotas e possuem múltiplas funções. Em P. brasiliensis, a proteína 14-3-3Pb, liga-se a laminina, principal componente da matriz extracelular das células hospedeiras, e durante a infecção sua expressão é aumentada. Com o intuito de compreender melhor a função desta proteína o objetivo deste estudo foi realizar a análise funcional da 14-3-3Pb através da obtenção de um homólogo funcional em S. cerevisiae e análise do anticorpo monoclonal anti-14-3-3Pb. S. cerevisiae foi escolhida como modelo de estudo por ser uma levedura amplamente utilizada na pesquisa genética, ao contrário de P. brasiliensis, e por possuir duas isoformas de 14-3-3, Bmh1p e Bmh2p, com alta identidade com 14-3-3Pb Para obtenção do homólogo funcional, foi realizada a transformação do vetor pYES-14-3-3Pb em S. cerevisiae. A partir da obtenção dos transformantes a avaliação da complementação foi realizada e foi observado uma complementação parcial das proteínas Bmh1p e Bmh2p por 14-3-3Pb. Ainda foram realizados teste de sensibilidade aos antifúngicos e a derivados semissintéticos do ácido gálico, onde foi possível observar um aumento da sensibilidade das linhagens com nocaute para os genes BMH1 e BMH2, S. cerevisiae Δbmh1 e Δbmh2, quando comparado à linhagem selvagem. A análise do anticorpo monoclonal foi realizada através de ensaio... / Abstract: Paracoccidioidomycosis (PCM) a systemic mycosis endemic in Latin America, with high prevalence in Brazil, has as etiological agent dimorphic fungi Paracoccidioides spp., P. brasiliensis e P. lutzii. During the infection process the adhesins, substances synthetized by fungi that interacts with host's extracellular matrix (ECM), are important virulence factors for the establishment of infection. A 30kDa protein that belongs to 14-3-3 proteins family stands out in the adhesion process of this fungus. The 14-3-3 proteins are present in all eukaryotic cells and play multiple functions. In P. brasiliensis, the 14-3-3Pb protein binds to lamin, the major component of ECM from host cells, and during the infection the expression is increased. In order to have a better understanding of 14-3-3Pb functions the aim of this study is to perform a functional analysis of this protein through achieving a functional homologous in S. cerevisiae and to analyze the monoclonal antibody anti-14-3-3Pb during infection. S. cerevisiae was chosen as model because it's widely use in genetic research, unlike P. brasiliensis, and has two isoforms of 14-3-3 proteins, Bmh1p and Bmh2p, with high identity with 14-3-3Pb. The functional homologous were obtained by yeast transformation of pYES-14-3-3Pb vector. Complementation assay was performed with obtained transformants, and it was observed that 14-3-3Pb partially complements Bmh1p and Bmh2p, with higher complementation of Bmh2p. Also susceptibility assays were performed with antifungal drugs and semi-synthetic compounds derived from gallic acid where it was observed that S. cerevisiae wild type was less sensitive to antifungals than the knockout strains, S. cerevisiae Δbmh1 and Δbmh2. Monoclonal antibody anti14-3-3Pb was evaluated alone and in association with substances with antifungal activities through inhibition adhesion assay, where it was observed that the anti-14-3-3Pb alone is able to inhibit P. brasiliensis ... / Mestre

One third of the world's population is infected with Mycobacterium tuberculosis, the bacterium that causes TB. During an infection, bacteria often survive host immune system attacks, which include oxidative stress conditions for bacteria growing inside macrophages. This makes treatment difficult and time-consuming. We hypothesize bacteria can adapt to environmental conditions by changing their mRNA maturation and degradation profiles. Using a model system, Mycobacteruim smegmatis, we focus on how mRNA expression is affected by oxidative stress. After construction and sequencing of RNA expression libraries, preliminary analysis showed that after three hours of H2O2 exposure most upregulated genes were related to DNA repair, while downregulated genes included transport proteins. After six hours of exposure, upregulated genes were similar to three hours and downregulated genes included tRNAs. 5' end mapping libraries were also constructed to access differential cleavage site abundance under oxidative stress conditions. We also investigated the roles RNase J may have in stress response and mRNA processing in Mycobacteria. RNase J and RNase E are thought to be the major RNases in bacteria. While most bacteria only have one of them, mycobacteria encode both in their genome, with RNase J being non-essential. We constructed a set of 4 strains (WT, RNase J overexpression, RNase J deletion, and complemented RNase J deletion) and tested their drug resistance and stress tolerance. Results suggests that RNase J deletion and overexpression alter drug sensitivity. Stress tolerance assays showed that WT is more tolerant to oxidative stress, followed by RNase J deletion strain and overexpression and complemented RNase J deletion strains, with the last two showing no growth when cultured with H2O2. Analysis of the expression profile of these strains was performed to help understand if gene expression differences are responsible for the phenotypes observed. For the complemented RNase J deletion, one operon had almost all its genes upregulated. This operon encodes a hydrogenase (Hyd3), suggesting that redox balance in the strain is perturbed.