La tuberculose demeure un problème majeur de santé publique. Mycobacterium tuberculosis infecte un tiers de la population mondiale et environ 3 millions de décès, des suites de l’infection, sont recensés chaque année dans le monde. La mise au point d’une stratégie vaccinale efficace constitue dès lors la solution idéale pour tenter d’éradiquer la bactérie. Le système immunitaire humain répond à l’infection par l’induction d’une réponse cellulaire, caractérisée essentiellement par la sécrétion de médiateurs pro-inflammatoires, comme l’interféron-gamma (IFN-?). A la fois les lymphocytes T CD4+ et T CD8+ produisent cette cytokine, mais les seconds sont également doués de propriétés cytotoxiques, entraînant la mort de la cellule infectée et du bacille. L’évaluation du potentiel de nouveaux candidats vaccins implique dès lors la caractérisation exhaustive de la réponse immunitaire induite. <p>La « heparin-binding hemagglutinin (HBHA) » est une protéine de 28-kDa, sécrétée et exprimée à la surface de M. tuberculosis et de M. bovis BCG. Montrant une affinité importante pour les gycoconjugués sulfatés, elle favorise la dissémination hématogène du bacille de Koch. <p><p>Nos résultats démontrent que la HBHA stimule l’immunité à médiation cellulaire humaine avec, toutefois, des différences selon que le sujet infecté souffre ou non de tuberculose active. En effet, les cellules mononuclées circulantes, de la majorité des individus infectés mais non-malades, secrètent de l’IFN-? en réponse à la HBHA, alors qu’une minorité de sujets malades produit de faibles quantités d’IFN-? après stimulation in vitro avec l’antigène. Lors de l’infection naturelle par le bacille de Koch, la HBHA devient dès lors une cible pour le système immunitaire, et plus particulièrement au sein des sujets, généralement considérés, comme protégés.<p>L’analyse de la réponse cellulaire, spécifique à l’adhésine, démontre que les lymphocytes T CD4+, mais également T CD8+, des sujets infectés mais non-malades, produisent de l’IFN-? L’antigène est effectivement présenté aux lymphocytes T grâce aux glycoprotéines du complexe majeur d’histocompatibilité de classe I et de classe II. Le phénotypage des cellules productrices d’IFN-? témoigne également la participation des cellules « natural killer (NK) » dans la réponse immunitaire contre la HBHA. En l’absence des lymphocytes T restreints à l’antigène, les cellules NK se montrent toutefois incapables de secréter de l’IFN-? au contact de la HBHA. Les interactions entre les lymphocytes T, spécifiques à l’antigène, déterminent également la production de cytokines. Alors que la déplétion des cellules T CD8+ diminue légèrement la production d’IFN-? l’absence des lymphocytes T CD4+ abolit toute sécrétion résiduelle d’IFN-? lors de la stimulation avec la HBHA. Par contre, les lymphocytes T CD8+, pré-stimulés avec l’antigène en présence de cellules T CD4+, répondent secondairement à la présentation de la HBHA par des macrophages. Ce résultat suggère une coopération entre ces deux sous-populations cellulaires, afin de produire de l’IFN-? à l’encontre de la HBHA. Grâce à un contact cellulaire, les lymphocytes T CD4+ spécifiques à la HBHA soutiennent effectivement l’activation des cellules T CD8+.<p>Outre la production de cytokines, la participation des lymphocytes T CD8+ à la lutte contre M. tuberculosis, se traduit également par leurs fonctions cytotoxique et bactéricide. La caractérisation des cellules T CD8+, spécifiques à la HBHA, s’est dès lors poursuivie par l’évaluation de leur potentiel cytolytique. Après expansion clonale, les lymphocytes T CD8+ induisent la mort des macrophages présentant la HBHA. Le mécanisme cytotoxique engage la libération du contenu des granules cytoplasmiques, comme le montre l’augmentation de la synthèse de perforine et de granzyme A, lorsque les cellules T CD8+ sont stimulées avec la HBHA. Privés de ces médiateurs solubles, les lymphocytes T CD8+, spécifiques à la HBHA sont alors incapables de lyser les cellules cibles. En définitive, l’activité microbicide constitue actuellement le meilleur corrélat de protection. La culture de macrophages infectés par M. bovis BCG, en présence de cellules T CD8+ spécifiques à la HBHA, limite partiellement la croissance de la bactérie phagocytée, soulignant le pouvoir anti-mycobactérien de l’immunité cellulaire induite par la HBHA, chez le sujet infecté mais non-malade.<p>D’autre part, l’analyse biochimique, menée à l’Institut Pasteur de Lille, démontre que la HBHA subit une modification post-traductionnelle, lors de sa synthèse. Il s’agit d’une méthylation des multiples résidus lysine, qui composent son extrémité C-terminale. La comparaison des formes native méthylée et recombinante non-méthylée de la HBHA démontre que la méthylation détermine l’immunogénicité et le pouvoir protecteur de la HBHA. En effet, contrairement à la HBHA native, la forme recombinante stimule faiblement la production d’IFN-? chez les individus infectés mais non-malades, et ne protège pas la souris contre l’infection par le bacille de Koch. La sécrétion d’IFN-? est, par ailleurs, partiellement restaurée lorsque la HBHA est artificiellement méthylée in vitro. Les splénocytes murins se comportent également différemment, selon qu’ils ont été immunisés avec la forme méthylée ou non. Alors que la HBHA recombinante est immunogène chez la souris et chez l’homme, l’immunité cellulaire murine induite demeure impassible face à l’infection des phagocytes par les mycobactéries, ce qui se traduit par l’absence de protection.<p><p>En conclusion, la HBHA se compose d’épitopes protecteurs, qui dépendent de la présence des groupements méthyls, associés à son domaine C-terminal. Il s’agit, à notre connaissance, de la première mise en évidence de l’implication de la méthylation dans la réponse d’immunité cellulaire à l’encontre d’une protéine. De plus, l’immunité adaptative spécifique à la HBHA, chez le sujet infecté mais non-malade, se caractérise par les trois principaux corrélats de protection, actuellement décrits chez l’homme. Le potentiel vaccinal de cette adhésine mycobactérienne est donc bien réel. / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished

Le facteur bactérien Rho est une ARN hélicase toroïdale utilisant l’énergie issue de l’hydrolyse d’ATP pour transloquer le long de brins ARN et dissocier les obstacles moléculaires se trouvant sur son chemin. La fonction majeure de Rho est de provoquer la terminaison de la transcription. Les nombreuses études du facteur Rho d’E. coli (EcRho) ont montré que l’enzyme se fixe aux régions nues des transcrits naissants au niveau de sites Rut (Rho utilization) et utilise son activité de translocase ATPase-dépendante pour rattraper l’ARN polymérase et provoquer la dissociation du complexe d’élongation de la transcription. EcRho est impliquée dans 20 à 50 % des évènements de terminaison de la transcription chez E. coli et contribue à la régulation globale ainsi qu’à la protection du génome bactérien via divers mécanismes complexes. Le spectre d’activité restreint de la Bicyclomycine (BCM) – un antibiotique inhibant la fonction ATPase de Rho – suggère que Rho, bien qu’essentielle chez la plupart des espèces Gram- comme E. coli, est superflue chez la plupart des espèces Gram+. Pour vérifier cette hypothèse et mieux comprendre l’importance de Rho au sein de la diversité bactérienne, nous avons évalué la distribution et la conservation phylogénétique de Rho en utilisant les banques publiques de données génomiques et protéomiques. Nous avons observé que ~92% des espèces analysées (représentant la plupart des phyla bactérien) possèdent Rho (ou un gène rho) et que la présence de ce dernier semble être corrélée avec la complexité du génome, du programme de régulation et de l’écosystème de la bactérie. Cette complexité est illustrée par notre découverte que la protéine régulatrice CsrA contrôle la terminaison Rho-dépendante dans la partie 5’ UTR de l’opéron pgaABCD dont l’expression est critique pour la formation de biofilms par E. coli. En parallèle de ce travail, nous avons testé la proposition récente que le facteur Rho de Mycobacterium tuberculosis (MtbRho) opère par un mécanisme atypique indépendant de son activité ATPase, ce qui le rendrait insensible à la BCM. Nos résultats réfutent cette hypothèse et démontrent sans équivoque que MtbRho utilise un mécanisme similaire à celui de EcRho, pouvant être inhibé par la BCM (mais nécessitant des doses particulièrement élevées). L’ensemble de ces travaux apporte de nouvelles informations illuminant le rôle, le mécanisme et l’importance de Rho au sein de la biodiversité et conforte Rho comme cible prometteuse pour le développement de nouveaux antibiotiques. / The bacterial Rho factor is a ring-shaped, hexameric RNA helicase which uses the energy derived from ATP hydrolysis to translocate along RNA strands, disrupting molecular roadblocks in its way. A major function of Rho is to induce termination of transcription. Extensive studies of the prototypical Rho factor from E. coli (EcRho) indicate that the enzyme binds naked regions of nascent RNA transcripts at the level of Rut (Rho utilization) sites and, then, uses its ATPase-dependent translocase activity to catch up with RNA polymerase and to trigger dissociation of the transcription elongation complex. EcRho is implicated in 20 to 50 % of all transcription termination events in E. coli and contributes to the global regulation and protection of the bacterial genome through various sophisticated mechanisms. The limited spectrum of activity of the antibiotic Bicyclomycin (BCM) – an inhibitor of Rho’s ATPase – suggests that Rho, while critical in many Gram- species such as E. coli, is dispensable in most Gram+ species. To verify this assumption and better understand the importance of Rho across the biodiversity, we assessed the phylogenetic distribution and conservation of Rho using public ‘omics’ databases. We found Rho (or a rho ORF) in ~92% of analyzed species (representing most bacterial phyla), its presence seemingly related to the complexity of the bacterial genome, regulatory program, and ecosystem. An illustration of this complexity is our discovery that regulatory protein CsrA mediates Rho-dependent termination in the 5’UTR of the pgaABCD operon whose expression is critical for biofilm formation by E. coli. In parallel, we tested the recent proposal that the Rho factor of Mycobacterium tuberculosis (MtbRho) operates by an atypical, ATPase-independent mechanism that would make it immune to BCM. Our results rule out this possibility and unambiguously show that MtbRho uses a mechanism similar to that of EcRho, one that can be inhibited by BCM (albeit at unusually high concentrations). Overall, this work provides key, new information to better understand the role, mechanism, and importance of Rho across the bacterial diversity and endorses Rho as a promising target for the development of new antibiotics.

Introduction Nucleotide excision repair (NER) is a highly conserved pathway involved in repair of a wide variety of structurally unrelated DNA lesions. One of the well characterized NER systems is from E. coli which involves UvrABC nucleases. NER consists of two related sub-pathways: global genomic repair (GGR), which removes lesions from the overall genome, and transcription coupled repair (TCR), which removes lesions from the transcribed strand of active genes. Bulky DNA lesions such as cyclobutane pyrimidine photodimers (CPD) induced by UV irradiation block RNA polymerase (RNAP) during transcription. In bacteria, a gene product of mfd called transcription repair coupling factor (TRCF) or Mfd is required for TCR. Bacterial Mfd interacts with the stalled RNAP, displaces it from the DNA and recruits NER proteins at the site of damage. Mfd, thus contributes to the faster repair of the transcribed strand compared to the non-transcribed strand for similar kind of lesions. Intracellular pathogens like M. tuberculosis are constantly exposed to a variety of stress conditions inside the host, mainly due to host defense systems and antibiotic treatments. It is therefore, extremely important for bacteria to have DNA damage repair and reversal mechanisms that can efficiently counteract these effects. However, very little is known about DNA repair systems in M. tuberculosis compared to other bacteria. Sequencing of M. tuberculosis genome revealed the presence of NER associated genes including a putative mfd. Additionally, due to the high GC content of genome as well as the DNA damage prone host environment, the transcription in M. tuberculosis may encounter the problems, which are not apparent in other bacteria. Therefore, the gene like mfd may play very important role in physiology of M. tuberculosis. In the present study, we describe the biochemical and functional characterization of Mfd from M. tuberculosis (MtbMfd) and discuss its unusual properties. Biochemical characterization of MtbMfd Genome analysis of M. tuberculosis as well as the sequence alignment studies revealed that MtbMfd is 1234 amino acids long multifunctional protein having various domains specialized for different functions. Cloning of Mtbmfd was carried out by reconstructing the full length gene from three PCR amplified fragments using genomic DNA as a template. Complementation study using Mtbmfd suggested that the gene of interest complements E. coli counterpart and increases survival of UV irradiated cells. To further characterize the function of Mtbmfd, a road block reporter assay was performed, which indicates that the MtbMfd interacts with stalled E. coli RNAP and displaces it from the site of transcription resulting in low reporter gene activity. The MtbMfd protein was expressed and purified by using various chromatographic techniques, and confirmed by mass spectrometry. In addition to full length protein, a number of truncated MtbMfd constructs were generated and purified to homogeneity. Mfd is a motor protein and requires ATP hydrolysis in order to translocate along DNA. The signature motifs of superfamily 2 helicases / ATPases are present at the C-terminal of Mfd along with translocase motif which is highly homologous to motif present in RecG helicase. To analyze the kinetics of ATP hydrolysis of MtbMfd and its truncated proteins, ATPase reactions were carried out using γ32P-ATP as a tracer. Wild-type MtbMfd exhibited ATPase activity, which was stimulated ~1.5 fold in presence of dsDNA. The mutant MtbMfd (D778A), which harbors mutation in one of the key residues of Walker B motif of the ATPase domain showed negligible ATPase activity indicating the importance of residue D778 for ATP hydrolysis. While the C-terminal domain (CTD) comprising amino acids 600 to 1234 showed elevated ATPase activity, the N-terminal domain (NTD) containing the first 500 amino acid residues was able to bind ATP but deficient in hydrolysis. Deletion of 184 amino acids from the C-terminal end of MtbMfd (MfdΔC) increased the ATPase activity by ~10-fold compared to full-length MtbMfd. The translocase activity of MtbMfd was measured by an oligonucleotide displacement assay and it was found that full length MtbMfd and CTD have a very weak translocase activity whereas, MfdΔC exhibited efficient translocation along DNA in ATP dependent manner. These results provide a direct correlation between translocase and ATPase activity of MtbMfd, and suggest possibly an auto-regulatory function for the extreme C-terminus of MtbMfd. Oligomeric status of MtbMfd was determined using various techniques including gel filtration chromatography and it was found that MtbMfd exists as monomer and hexamer in solution. The monomer showed increased ATPase activity and susceptibility to proteases compared to the hexameric form. MfdΔC, on the other hand, was predominantly monomer in solution implicating importance of the extreme C-terminal region in oligomerization of protein. Taken together, the biochemical evidence suggests that monomeric MtbMfd is an active form and oligomerization provides stability to the protein. One important finding of the present study is the binding of ATP to NTD of MtbMfd. All Mfd NTDs resemble UvrB and possesses the degenerate ATPase motifs. Indeed, on the basis of sequence and structural similarities, it has been suggested that Mfds have evolved from UvrB incorporating an additional translocase activity. UvrB has a cryptic ATPase activity while the NTD of Mfd may have lost the activity as it possesses degenerate Walker motifs. In contrast, NTD of MtbMfd binds ATP but is hydrolysis deficient. A closer comparison of the amino acid sequences in the Walker A motif reveal that conserved K 45 of UvrB has been replaced by R in case of NTD of MtbMfd. It has been shown previously that mutation of K 45 to A, D and R led to a loss of ATPase activity of UvrB. Thus, MtbMfd seems to be a natural mutant of UvrB. Since NTD harbors an intact UvrA interacting domain, when it is expressed it may sequester the cellular pool of UvrA leading to dominant negative phenotype. When UV survival assays were carried out, cells expressing NTD showed hyper-sensitivity to UV light – a typical characteristic of NER deficiency. In addition, in vitro NER assay clearly suggested that NTD sequesters pool of UvrA inside the cell and blocks both GGR and TCR which further affects the mutation frequency of bacterial cells. Influence of MtbMfd on elongation state of RNAP The movement of RNAP along the template during transcription elongation is not uniform and is interrupted due to various factors. To overcome transcription elongation interruptions, a number of proteins viz. Mfd, Gre and Nus act on RNAP and modify its activity. RNAP displacement and transcript release experiments showed that MtbMfd influenced the elongating RNAP by more than one way. MtbMfd displaced stalled RNAP, which was blocked by NTP starvation on T7A1 promoter based template in a concentration and time-dependent manner. RNAP displacement activity of MtbMfd was shown to depend on ATP or dATP hydrolysis. On the other hand nucleotides like ADP, GTP, CTP and ATPγS did not support the RNAP displacement activity. However, in presence of ATPγS, MtbMfd was able to bind stalled complex but unable to displace RNAP suggesting that ATP or dATP hydrolysis is important for MtbMfd function. On the other hand, MtbMfd did not affect initiating RNAP when σ factor was still bound suggesting that upstream DNA is necessary for Mfd function. To assay RNA or transcript release activity of MtbMfd after transcription complex disruption, immobilized transcription complex assay was carried out. Immobilized stalled complex was generated by UTP and CTP starvation on biotinylated T7A1 promoter based template which can be affixed to temporary pellet in presence of streptavidin beads. It was found that MtbMfd released RNA into a supernatant fraction in a concentration-dependent manner suggesting that MtbMfd releases transcript after ternary complex disruption. MtbMfd released transcript in an energy-dependent manner and both ATP and dATP supported the activity, which allows the complete separation of RNA release from RNA synthesis inside the cell. An ATPase mutant of MtbMfd (MfdD778A) failed to release transcript, which further supported that ATP hydrolysis is important for MtbMfd function. Since both Mfds and RNAPs are evolutionary conserved proteins, to analyze the effect of MtbMfd on other bacterial RNAPs, displacement and release assays were carried out. Stalled complexes were generated using EcoRNAP (E. coli), MsRNAP (M. smegmatis) and MtbRNAP (M. tuberculosis) on T7A1 promoter based template. It was observed that MtbMfd was able to displace all the three RNAPs from stalled elongation complex as well as released transcript with varying efficiency. MtbMfd showed optimal displacement and release activity in presence of mycobacterial RNAPs. Transcription elongation complexes adopt various conformations and exist as different isomerized states during elongation. In an active elongation complex the 3'-OH polymerizing end of transcript aligns with an active centre of the RNAP. However, one of the most common and intrinsic properties of RNAP is backtracking or reverse translocation, which leads to misalignment of 3'-OH polymerizing end from an active centre of the polymerase. It is of interest to know if backtracking affects MtbMfd function. It is likely that complexes blocked by lesions inside the cell might tend to backtrack, and different translocational isomers possibly have different sensitivities to MtbMfd action which may illuminate the overall mechanism of MtbMfd. Backtracking of RNAP was induced on +20 and +39 stalled complexes and the effect of MtbMfd was analyzed in presence of NTPs in the reaction. It was found that arrested or backtracked complexes were restored to the forward position by the activity of MtbMfd in presence of NTP resulting into productive elongation. These results suggest that arrested RNAP again resumes transcription if conditions are favorable; otherwise, MtbMfd further assists RNAP to dissociate which leads to release of transcript. Anti-backtracking activity of MtbMfd might have important function in cellular metabolism and it has been speculated that Mfd could play more general role during transcription apart from repair. To explore the role of MtbMfd as a transcription factor and effect of MtbMtb on transcription processes in the mycobacteria, a variety of T7A1 promoter based templates were generated. These templates were derived from genes of M. tuberculosis and E. coli having varying GC content (39-81 %). The rationale behind this experiment is that the high GC content of mycobacteria and the template derived from mycobacterial genes may pose as sequence dependent structural constraints and hence block the RNAP during transcription. By anti-backtracking activity of MtbMfd these paused complexes may get relieved, leading to efficient transcription by RNAP which may lead to the formation of more full length transcript. To analyze the effect of MtbMfd, purified templates of different GC content were incubated with RNAP and MtbMfd to carry out in vitro transcription. Although, in case of multiple rounds of transcription, multiple pauses were observed even in presence of MtbMfd. However, in presence MtbMfd around 1.5 - 2 fold increased full-length transcripts were observed suggesting that MtbMfd assisted RNAP during elongation to overcome sequence dependent pause. To avoid multiple pauses that are likely to occur due to the initiation of multiple round of transcription, and trailing effect of RNAP itself, single round of transcriptions were carried out in presence of heparin. Sequence specific pauses were observed with increasing GC percentage in template suggesting that indeed high GC content contributes to transcription pause. At the same time, MtbMfd in the reaction increased the amount of full length transcript by 1.5 - 2.0 fold probably by pushing paused RNAP forward to resume elongation. Taken together, this study investigates the biochemical properties of MtbMfd and its mechanism of action. In addition, it explores the importance of the coupling of transcription to repair in M. tuberculosis as well as the overall proof reading mechanism of transcription elongation in the GC rich genome of mycobacteria.

L'une des particularités de Mycobacterium tuberculosis, agent pathogène de la tuberculose, est sa capacité à accumuler des lipides dans son cytoplasme, ce qui favorise son entrée en dormance. Le séquençage du génome de M. tuberculosis a permis d'identifier certains gènes codant pour des enzymes lipolytiques, parmi ceux-ci : le gène codant pour la protéine Rv3097c aussi appelée LipY (composée d'un domaine PE et d'un domaine lipase relié par un Linker). Dans la première partie de ce travail de thèse, nous avons procédé à la caractérisation biochimique de LipY, mais aussi de ses formes mutantes LipY(∆PE), LipY(∆149) et LipY(∆170), et à l'étude d'inhibition des membres de la famille Lip apparentés à la lipase hormono-sensible humaine (Lip-HSL). Nous avons pu déterminer les propriétés cinétiques de l'activité lipase de LipY et de ses différentes formes mutantes.Dans la seconde partie, nous nous sommes intéressés à la compréhension du rôle des différents domaines de LipY, et du linker dans l'hydrolyse des lipides au cours de l'infection en utilisant des macrophages spumeux (macrophage riche en lipides) infectés au préalable par des souches de M. bovis BCG recombinantes. Ces résultats et les hypothèses posées durant ce travail de thèse, pourraient être appuyés par l'obtention de la structure tridimensionnelle de LipY. Pour cela, nous avons initié et procédé à la cristallogenèse de LipY. La poursuite des études d'optimisation des cristaux obtenus pourrait permettre d'aller plus en profondeur dans l'élucidation du rôle et du mécanisme d'action de LipY. / Mycobacterium tuberculosis is a pathogenic agent, responsible of the tuberculosis, which can store lipids into the cytoplasm. This accumulation allows the bacteria to enter in the dormancy phase. The sequencing of M. tuberculosis genome, allows to identify some genes coding for lipolytic enzymes, among which a gene coding for Rv3097c protein, also called LipY. (possessing PE domain linkto a lipase domain). During my PhD thesis, we first biochemically characterized LipY and its mutant forms LipY(ΔPE); LipY(Δ149) and Lip(YΔ170) and studied the inhibition of Lip family members related to the human hormone-sensible lipase (Lip-HSL). We determined the kinetic properties for the lipase activity of LipY and its mutants. In the second part, based on these previous results, we studied the role of these different domains and the linker on the hydrolysis of lipids during the infection phase, in infected foamy macrophages (lipids rich). For these studies, we used foamy macrophages infected by recombinants strains of M. bovis BCG (LipY(wt) and its mutants.These results and hypothesis can be confirmed and supported by resolving the tridimensional structure of LipY. Crystallogenesis tests allowed us to have some crystals of LipY(wt), which after optimization would allow us to have a better understanding of the role and action mechanism of LipY.

Orientador: Marcelo de Carvalho Ramos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T12:21:17Z (GMT). No. of bitstreams: 1 Basso_AudreyJordao_M.pdf: 998979 bytes, checksum: 6b29af1ec17385aea2f4b4f550bb349e (MD5) Previous issue date: 2006 / Resumo: A técnica do estudo do polimorfismo de fragmentos de restrição, com a pesquisa da seqüência de inserção IS6110 (IS6110-RFLP), é o método de genotipagem mais empregado mundialmente para a caracterização de isolados de M. tuberculosis. Ela pode ser empregada para o estudo de surtos, epidemias ou para estudos de genética populacional. Em Moçambique, onde a tuberculose tem uma elevada prevalência, não há informação suficiente sobre os padrões genotípicos obtidos com a IS6110-RFLP de cepas locais de M. tuberculosis. A descrição dos padrões obtidos com essa metodologia pode ser útil localmente para propósitos epidemiológicos ou, internacionalmente, para descrever o relacionamento de cepas isoladas em Moçambique com outras áreas do mundo. Neste estudo, uma coleção de 158 isolados de M. tuberculosis, identificados com o emprego da análise de fragmentos de restrição após a amplificação de trecho do gene hsp65 (hsp65-PRA), recuperados de pacientes infectados pelo HIV com tuberculose pulmonar e que residiam em Maputo, Moçambique, foram genotipados. O número de seqüências IS6110 obtido variou de 1 to 18, com 21.5% dos isolados exibindo menos de seis cópias. Um total de 10 ¿clusters¿ foram caracterizados, um com três isolados e os demais com dois cada. Os isolados que exibiram menos de seis seqüências não foram incluídos na análise, dado o baixo poder discriminatório do método. Baseado no coeficiente de similaridade, 85% dos isolados tinham mais do que 65% de homologia. Esses dados mostram que, isolados de M. tuberculosis obtidos em Moçambique, África, podem ser analisados, para fins epidemiológicos com o auxílio dessa técnica de genotipagem. Entretanto, um considerável número de isolados exibiu um número pequeno de cópias da seqüência IS6110 e um segundo marcador genético, como a espoligotipagem, deve ser utilizado / Abstract: IS6110 RFLP has been the most widely used genetic subtyping method for M. tuberculosis strains, to characterize disease outbreaks or for evolutionary genetics studies. In Mozambique, where tuberculosis exhibits a high prevalence, there is not enough information about IS6110-RFLP patterns of local M. tuberculosis strains. The description of the fingerprinting patterns obtained with this methodology can be useful locally for epidemiological purposes, and internationally to investigate the relatedness of strains isolated in Mozambique to other areas of the world. In this study, a collection of 158 isolates of M. tuberculosis strains, as identified by using hsp65-PRA, recovered from HIV-infected patients with pulmonary tuberculosis residing in Maputo, Mozambique, was genotyped. The number of IS6110 copies ranged from 1 to 18, with 21.5% of strains exhibiting less than six copies. A total of 10 clusters were found, one consisting of three strains and all the others of two strains. Isolates showing less than six bands were not included in the cluster analyses due to low discriminatory power of the analysis. Based on similarity coefficients 85% of strains had more than 65% homology. This data show that M. tuberculosis strains obtained in Mozambique, Africa can be analyzed for epidemiological purposes with the use of this genotyping technique. However, a considerable number of strains exhibited a low number of IS6110 copies, and a second genetic marker as spoligotyping has to be used. / Mestrado / Clinica Medica / Mestre em Clinica Medica

La tuberculose est une maladie causant encore aujourd'hui plus d'un million de mort chaque année. De nouvelles solutions vaccinales sont nécessaires pour enrayer cette épidémie. Les sulfoglycolipides, trouvés chez Mycobacterium tuberculosis, se sont révélés capables d'activer le système immunitaire et pourraient ainsi représenter une solution thérapeutique intéressante dans la création d'un nouveau vaccin. Dans ce cadre, nous avons souhaités élaborer de nouveaux analogues de sulfoglycolipides. Pour cela, nous avons employé une méthode de protection régiosélective par catalyse tandem au chlorure de fer(III) hexahydrate précédemment développée au laboratoire pour préparer les cœurs glycosidiques des différents mimes. La méthode d'alkylation asymétrique développée par Myers a également été utilisée pour la préparation des acides polydéoxypropionates portés par les différents analogues. / Tuberculosis is still responsible for more than one million deaths each year. New therapeutic solutions are needed to fight this disease. Sulfoglycolipids, found in Mycobacterium tuberculosis's cell wall, seem to be able to activate immune system and could represent an interesting therapeutic solution for the development of a new vaccine. In this context, we wished to elaborate new sulfoglycolipid analogues. For the synthesis of the glycoside moieties of these analogues, we have used a tandem regioselective protection catalyzed by iron(III) chloride, previously developed in our laboratory. Myers's asymmetric alkylation has also been used for the synthesis of polydéoxypropionate chains.

La tuberculose (TB), maladie pulmonaire causée par le Mycobacterium tuberculosis (Mtb), reste la première cause de mortalité par un agent infectieux. La TB est responsable de près de 1,7 million de morts et de 10,4 millions de nouveaux cas par an dans le monde. L’émergence et la propagation de souches bactériennes multi-résistantes aux antibiotiques (MDR) représentent une menace majeure grandissante et reflètent l’efficacité partielle des thérapies actuelles. Le traitement des patients atteints de TB-MDR est constitué actuellement de combinaisons d’antibiotiques, souvent toxiques, administrés pendant une longue durée, avec une efficacité limitée. Il existe donc un besoin urgent de développer de nouveaux traitements antituberculeux. L’immunothérapie, dont l’objectif est d’améliorer la réponse immunitaire de l’hôte contre le Mtb, représente une approche complémentaire intéressante dans le but de diminuer la durée et d’augmenter l’efficacité des traitements actuels de la TB-MDR. Le premier objectif de cette thèse a été de caractériser un nouveau vaccin thérapeutique, basé sur le virus de la vaccine modifié d’Ankara (Modified Vaccinia virus Ankara, MVA), le MVATG18598, qui exprime dix antigènes représentatifs des trois phases de l’infection par Mtb. En utilisant différentes lignées de souris, nous avons montré que la vaccination par MVATG18598 entraîne l'induction de réponses spécifiques cellulaires et humorales. Les cellules T produisent plusieurs cytokines de type Th1 et présentent une activité cytolytique. Dans des modèles murins d’efficacité, le MVATG18598, associé à un traitement antibiotique, réduit significativement la charge bactérienne dans les animaux infectés ainsi que le taux de rechute de la maladie après l’arrêt du traitement. Le deuxième objectif de cette thèse a été d'analyser l'impact des antituberculeux sur l'immunogénicité du vaccin MVATG18598. Nous avons montré que les antibiotiques de première ligne, et principalement l’isoniazide, diminuent la réponse immunitaire Th1 induite par le MVATG18598. De plus, nous avons démontré que la réponse humorale induite par le candidat vaccin est modifiée et s’oriente vers une augmentation du rapport IgG1/IgG2a en présence d’antibiotiques. En conclusion, nous montrons qu'un vaccin immunothérapeutique, tel que le MVATG18598, a la capacité de contribuer au contrôle de la tuberculose en augmentant l'efficacité des traitements antituberculeux. De plus, nos résultats indiquent que les antibiotiques modulent la réponse immunitaire induite par le vaccin, données devant être prises en compte lors du développement des futures stratégies immunothérapeutiques / Tuberculosis (TB), a lung disease caused by Mycobacterium tuberculosis (Mtb), remains the leading cause of death worldwide from an infectious disease. TB is responsible for an estimated 1,7 million of deaths and 10,4 million new cases annually. The emergence and spreading of multidrug resistance (MDR) Mtb strains represent a major global threat and reflect limitation of current treatments. Patients with MDR-TB are currently treated with multiple drug regimens, often toxic, given for long durations, with a limited efficacy. Therefore, developing novel TB therapies is urgently needed. Immunotherapy aiming at triggering specific immune response against Mtb represents an attractive approach to shorten the duration and increase the efficacy of current MDR-TB treatment. The first aim of this PhD project was to characterize a novel therapeutic vaccine, based on the Modified Vaccinia virus Ankara (MVA), MVATG18598, expressing ten antigens representative of the three phases of Mtb infection. Using different strains of mouse, we showed that MVATG18598 vaccination is able to trigger Mtb antigens-specific humoral and cellular responses. Both CD4 and CD8 T cells display the capacity to produce multiple Th1-cytokines together with cytolytic activity. In post-exposure mouse models, MVATG18598 combined with an antibiotic regimen decreases significantly the bacterial burden in lungs of infected mice as well as the disease relapse rate after treatment completion. The second aim of this project was to analyze the impact of TB antibiotics on the immunogenicity of the MVATG18598 vaccine. We showed that first-line antibiotic regimen, mostly isoniazid, decreases antigen-specific Th1 immune response triggered by MVATG18598 vaccination in mice. Moreover, we demonstrated that Mtb-specific antibody response induced by the vaccine candidate is modified and shifted towards an increase of IgG1/IgG2a ratio in presence of drugs. Altogether, these results illustrate that immunotherapeutic vaccine such as MVATG18598 has the capacity to contribute to the control of TB by improving efficiency of anti-TB drugs treatment. In addition, our results indicate that antibiotics are able to modulate vaccine-induced immune response, a feature to consider for the future development of immunotherapies

Tuberculosis is a fatal airborne disease caused by bacteria from the Mycobacterium tuberculosis complex (MTBC). The incidence of contracting tuberculosis is estimated to be around 10.6 million cases each year. Increased drug resistance among mycobacteria has led to the need to develop new treatments. The study's purpose was to determine the antimycobacterial ability of drug complexes and how toxic these complexes are against human cells. Drug complexes from "phage derived endolysins" and "A pyrazine amide-4 aminoquinoline hybrids" were studied to possibly be included as a treatment against tuberculosis in the future. The minimum inhibitory concentrations (MIC) of the drug complexes were analyzed by the method Resazurin microtiter assay (REMA), where the results were assessed visually. The toxicity of the drug complexes was studied by growing THP1-Blue™ NF-κB cells, which then were exposed to the drug complexes. The results could then be obtained by absorbance measurement with spectrophotometry. One-way ANOVA showed a non-significant value, as the P-value was 0.44 (P&gt;0.05). However, more supplementary studies need to be carried out to obtain a significant result. All performed concentrations of the drug complexes were assessed as non-toxic against human THP1-Blue™ NF-κB cells. / Tuberkulos är en dödlig luftburen sjukdom som orsakas av bakterier från Mycobacterium tuberculosis complex (MTBC). Den årliga incidensen i världen för insjuknande i tuberkulos beräknas vara 10.6 miljoner. Ökad läkemedelsresistens bland mycobakterier har lett till att nya behandlingar behöver utvecklas. Syftet med studien var att studera två olika läkemedelskomplex antimycobakteriella förmåga samt hur toxiska dessa komplex är mot humana celler. Läkemedelskomplex från "phage derived endolysins" och "A pyrazine amide-4 aminoquinoline hybrids" studerades för att eventuellt kunna ingå som behandling mot tuberkulos framöver. Läkemedelskomplexens minsta inhibitoriska koncentration (MIC) analyserades genom metoden Resazurin microtiter assay (REMA) där resultaten bedömdes visuellt. Läkemedelskomplexens toxicitet studerades genom odling av THP1-Blue™ NF-κB celler som sedan exponerades för läkemedelskomplexen och resultaten  kunde sedan fås genom absorbansmätning med spektrofotometri. One-way ANOVA påvisade ett icke-signifikant värde,  då p-värdet blev 0,44 (P&gt;0,05). Dock behövs fler kompletterande studier genomföras för att studera detta ytterligare. Alla studerade koncentrationer av läkemedelskomplexen bedömdes vara icke-toxiska mot humana THP1-Blue™ NF-κB celler.

M. Tech. (Department of Health Sciences, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Tuberculosis (TB) remains one of the most difficult infectious diseases to control in the world today. The disease spreads easily in overcrowded, badly ventilated places and among people who are undernourished. Trends in the incidence of TB together with the development of multi-drug (MDR-TB) and extensively drug resistant (XDR-TB) strains of TB raises the need to intensify the search for more efficient drugs to combat this disease. Herbal remedies used in traditional medicine provide an interesting and largely unexplored source for the discovery of potentially new drugs for infections such as TB. The aim of the study was to evaluate the in vitro antimycobacterial activity of synthesized compounds from medicinal plants against Mycobacterium tuberculosis (M. tuberculosis). About 40 synthesized compounds isolated from South African medicinal plants were screened against H37RV using microplate alamar blue assay (MABA). Identified active compounds were screened against resistant strains of M. tuberculosis (MDR, XDR and pre-XDR) and sensitive clinical isolates of TB. Cytotoxicity and synergistic drug combination studies were done on active compounds to validate their toxicity and synergy levels. Cytotoxicity was done by sulforhodamine assay (SRB) against the C2C12 cell line. Only six compounds showed activity against M. tuberculosis with minimum inhibitory concentration (MIC) below 10μg/ml. The results obtained indicated that the cytotoxicity effects of the three compounds on C2C12 cells demonstrated marginal toxicity except for MVB 282/61215 which showed a high toxicity at the lowest concentration of 0.156μg/ml with over 100% viable cells at the highest concentration (5μg/ml). MVB 282/61271 had the highest percentage cell viability (65%) at the lowest concentration. Only two compounds had a higher potency evoking a bigger response at low concentrations with treated cells still viable after 3 days of incubation with the compound which was comparable with the treatment of isoniazid (INH). Synergistic activity of the six compounds was less in INH combination as compared to the rifampicin’s (RIF) combination. The results demonstrated that the synergistic interaction between the compounds and RIF could the antituberculosis acitivity. In conclusion the synergistic effects with RIF translate to lower dosing requirements of the compounds and the potential to combat multidrug resistant TB. In deed there is no doubt that natural products, with their range of interesting chemical structures and powerful antimycobacterial effects are certain to remain important participants in the development of new generations of antimycobacterial drugs.

Tuberculosis (TB) is an infectious and fatal disease that ranks as the second leading killer worldwide. It is caused by Mycobacterium tuberculosis (Mtb) which is an obligate intracellular parasite that colonizes the alveolar macrophages of the immune system. The major health concern associated with TB is its co-infection with HIV and the development of strains with multi-drug resistance. The elimination of TB has been hindered due to the lack of understanding of the survival strategies used by this pathogen. Thus, research towards discovering new effective antibacterial drugs is necessary and a group of Mtb kinase enzymes were targeted in this study because these enzymes are crucial for metabolism, pathogenesis and, hence, the survival of Mtb. Kinases are a group of structurally distinct and diverse proteins that catalyze the transfer of the phosphate group from high energy donor molecules such as ATP (or GTP) to a substrate. The phosphorylation of proteins modifies the activity of specific proteins which is subsequently used to control complex cellular processes within Mtb. The starting point of this research targeted eight specific Mtb kinases namely; Nucleoside diphosphokinase, Homoserine kinase, Acetate kinase, Glycerol kinase, Thiamine monophosphate kinase, Ribokinase, Aspartokinase and Shikimate kinase. The aim of this project was to subclone the gene sequences for these eight recombinant Mtb kinases and express them in Escherichia coli, to purify the proteins and determine their activity. In the effort to find new lead compounds, the final stage of this study focused on the basic screening of the TB kinases against an extract prepared from Pelargonium sidoides, a medicinal plant, to identify any inhibitory effects. Although this traditional medicinal plant has been broadly researched and extensively used to treat TB, there is still a lack of understanding of this plant’s scientific curative effect. Various molecular and biochemical methods were used to achieve the aims of this project. The putative gene sequence was obtained from the annotated genome of H37Rv, deposited at NCBI as NC_000962.2. The genes encoding the kinases were successfully PCR-amplified from genomic DNA, cloned into an expression vector in-frame with a C- or N-terminal 6-histidine-tag and expressed in E. coli BL21 (DE3). The purification of the protein was complex, but various different methods and techniques were explored to obtain sufficient amounts of protein. The functional characterization of the kinases involved an HPLC enzyme assay that showed that the recombinant kinases were active. These enzymes were then screened against the potential inhibitory compounds in P. sidoides using enzyme assays to generate dose-response curves. This allowed an effective comparison not only of the Mtb kinases’ activity under normal conditions but also the kinases’ activity in the presence of a potential inhibitor. Overall, the inhibition of the enzymes required the presence of higher concentrations of the P. sidoides extract. However, the SK enzyme results presented a significantly higher inhibition and the lowest IC50 value, in comparison to the other kinases, which makes this kinase an attractive potential drug target against TB. In summation, cloning and purification of SK was successful, resulting in a concentration of 2030 μg/ml of purified enzyme and its activity analysis demonstrated enzyme functionality. This activity was reduced to zero in the presence of 1 x 102 mg/ml dilution of P. sidoides plant extract. This research conducted has extended the quality of information available in this field of study. These interesting results, proposing and identifying SK as a suitable potential target can be a starting point to significantly contribute and progress in this field of research, with the eventual goal of developing a drug to combat this fatal disease. / Life Sciences / M. Sc. (Life Sciences)