La tuberculose bovine est une maladie infectieuse contagieuse causée par Mycobacterium bovis. Cette maladie touche les bovins et de nombreuses espèces de mammifères domestiques et sauvages, ainsi que l’homme. La circulation de la bactérie dans des systèmes multi-hôtes variés favorise l’entretien de la maladie et la contamination des bovins vivant à proximité des animaux sauvages infectés. En marge de la transmission directe de M. bovis par voie respiratoire, la transmission indirecte aux bovins, liée à l’inhalation ou à l’ingestion de matrices environnementales contaminées par un animal infecté excréteur, est suspectée dans plusieurs régions du monde. L’existence de réservoirs environnementaux où le bacille M. bovis est capable de persister, pourrait donc être un facteur important de la réémergence puis du maintien de la maladie dans les systèmes multi-hôtes.En Côte d’Or, département fortement touché par la tuberculose bovine depuis 2004, la transmission indirecte de la bactérie entre la faune sauvage infectée et les bovins est suspectée dans plusieurs élevages. Pour évaluer la présence et la survie de cette bactérie dans l’environnement, nous avons analysé un grand nombre d’échantillons prélevés dans des zones partagées par les bovins et/ou la faune sauvage infectés dans le but de déterminer la distribution environnementale de M. bovis. Pour ce faire, nous avons développé ou modifié des systèmes de détection moléculaire adaptés aux matrices environnementales complexes. Nous avons également évalué l’impact de la température et des propriétés physico-chimiques de deux sols sur la survie de M. bovis, ainsi que le rôle de la mésofaune du sol (lombrics en particulier) dans la dissémination de la bactérie à partir de matière organique contaminée. L’étude environnementale a mis plus particulièrement en évidence la contamination de deux biotopes: les zones humides des pâtures et les sols de terriers de blaireaux. De plus, les études expérimentales ont montré que M. bovis pouvait survivre plusieurs mois dans le sol à 4°C et que les lombrics pouvaient disséminer la bactérie dans le sol, voire jouer un rôle potentiel de vecteur pour les animaux qui les consomment. Ces résultats apportent de nouvelles connaissances sur la persistance et la circulation de M. bovis dans l’environnement en Côte d’Or et permettront de proposer des améliorations aux mesures de biosécurité déjà existantes dans les élevages bovins. / Bovine tuberculosis is an infectious disease caused by Mycobacterium bovis. This disease affects cattle, and many species of domestic and wild mammals, and humans. The circulation of the bacteria in various multi-host systems promotes the maintenance of the disease and the contamination of cattle in the vicinity. Beside direct transmission of the bacteria through the respiratory route, indirect transmission, through inhalation or ingestion of environmental matrices contaminated by an infected animal excretory, is suspected in several countries. Environmental contamination with M. bovis appears to be a crucial factor in the persistence of the infection in multi-host systems.In Côte d'Or, a French department affected by bovine tuberculosis since 2004, the indirect transmission of the bacteria from infected wildlife to cattle is suspected in several cases. To assess this type of transmission of the bacillus, we evaluated the environmental contamination with M. bovis on a large number of samples taken in areas shared by cattle and / or wildlife infected. For this purpose, we developed or modified molecular detection systems adapted for environmental complex matrices. We also assessed the impact of physicochemical properties of both soil and temperature on survival of M. bovis and the role of earthworms in the spread of the bacteria from contaminated organic material. The environmental study showed the contamination of two media in particular: wetlands pastures and soil badger setts. Moreover, experimental studies have shown that M. bovis can survive in soil for several months at 4 ° C and the worms could spread the bacteria in the soil, or even play a potential role for vector animals that consume them. These results will propose improvements to existing biosecurity measures on cattle farms and provide new knowledge about the persistence and circulation of M. bovis in the environment in Côte d'Or.

Pathogenic bacteria such as M.tuberculosis have evolved several mechanisms to aid their intracellular survival and subvert host defenses. One of the contributing factors is thought to be the production and secretion of large amount of cAMP, Mycobacterial genomes encode a large number of adenylyl cyclases distinct in their structure and regulatory mechanisms. The roles of these enzymes in the physiology and pathogenesis of virulent mycobacteria are only now being elucidated. The roles of phosphodiesterases (PDEs), which serve to lower cAMP levels through degradation are, however, relatively unexplored. The Rv0805 gene was previously shown to code for an active phosphodiesterase from Mycobacterium tuberculosis. Bioinformatics analysis revealed that orthologs of Rv0805 were found even in eukaryotes. Biochemical and structural characterization of Rv0805 revealed that it was a class III cAMP phosphodiesterase. Comparative genomics identified a close ortholog of Rv0805 in M. leprae (ML2210). The genome of M. leprae Encodes only 1,604 predicted proteins and possesses the highest number of pseudogenes, 1,116. The retention of a functional PDE, the ortholog of Rv0805, in the minimal genome of M. leprae is indicative of its importance in cellular physiology. Biochemical characterization of proteins from M. leprae and use of heterologous hosts will help understand this human pathogen better, since there are no tools currently available to genetically manipulate this bacterium. Sequence analysis of ML2210 revealed the presence of conserved motifs and residues known to be critical for catalysis and unique to class III phosphodiesterases. ML2210 shares 83% sequence identity with Rv0805 and 24% sequence identity with the phosphodiesterase from E. coli (cpdA). In vitro biochemical characterization of ML2210 using non-nucleotide colorigenic and cyclic nucleotide substrates revealed that it was an enzymatically active phosphodiesterase. Kinetic parameters of ML2210 with respect ot colorigenic substrates revealed that its catalytic properties were similar to that of Rv0805. However, with respect to hydrolysis of 3’, 5’-cAMP, ML2210 was catalytically more efficient than Rv0805, suggesting that in spite of being orthologs, these enzymes have evolved distinct specificities at their active site. A parallel of monoclonal antibodies raised to Rv0805 was also used understand the differences in the biochemical properties of Rv0805 and ML2210 better. It was observed that only one monoclonal antibody was able to recognize ML2210 by ELISA and not by Western blot analysis. These results revealed that conformational differences between ML2210 and Rv0805 exist. Over-expression of ML2210 in M. smegmatis resulted in a modest decrease in intracellular cAMP levels. Despite the absence of a predicted transmembrane region or a membrane-targeting signal, ML2210 localized to the cell envelop fraction upon over expression in M. smegmatis. Moreover, like Rv0805, over-expression of ML2210 also resulted in perturbation of the cell wall of M. smegmatis, arguing for additional cellular roles of this protein. Orthologs of Rv0805 or ML2210 are found only in slow growing mycobacteria suggesting that other cyclic nucleotide phosphodiesterases could regulate cAMP levels in fast growing mycobacteria like M. smegmatis. Since BLAST results did not retrieve an ortholog of Rv0805 or ML2210, COG1409 (COG database) containing Rv0805 was examined for the presence of other mycobacterial phosphodiesterases. Bioinformatics analysis identified Rv2795c as another PDE from M. tuberculosis. Sequence analysis of Rv2795c revealed the presence of all the motifs conserved in the class III PDEs but Rv2795c shared only 22% sequence identity with Rv0805 and 19% sequence identity with CpdA. Importantly, an ortholog of Rv2795c was identified in M. leprae. Interestingly. Rv2795c and its orthologs branched away from Rv0805, making it phylogenetically distinct and hence warranting further characterization. Recombinant, purified MSMEG_2647 (the Rv2795c ortholog from M. smegmatis) was able to hydrolyze cyclic nucleotides and other phosphodiester substrates in vitro. The Km for colorigenic substrates was higher when compared to the Km of ML2210 or Rv0805 for these substrates. However, the kinetic parameters of MSMEG_2647 for cyclic nucleotides were comparable to those of ML2210 or Rv0805. MSMEG_2647 was a metal dependent enzyme and among the panel of metals tested, Mn2+ supported the highest in vitro catalytic activity of MSMEG_2647. Zn2+ inhibited the catalytic activity of MSMEG_2647. In order to gain insight into the catalysis of MSMEG_2647, the end products of cAMP hydrolysis by MSMEG_2647 were analysed using reverse phase HPLC. The assay revealed that the end products of cyclic nucleotide hydrolysis by MSMEG_2647 were different when compared to the end products of hydrolysis of the same substrates by Rv0805 or ML2210. This suggests differences in the architecture of the active site residues of the mycobacterial MPEs. A mutational anlaysis of the active site residues in MSMEG_2647 was carried out to identify residues involved in substrate recognition and metal coordination. Although Rv0805 and MSMEG_2647 shared only a 22% sequence identity, MSMEG_2647 displayed strict conservation in the core MPE motifs. Mutation of the active residues N97 and H98 in Rv0805 had led to an abrogation of its catalytic activity. However, corresponding mutations of N76A and H77A in MSMEG_2647, did not lead to a loss in its catalytic activity. A third mutation known to be important for the catalytic activity of Rv0805 (D19) was incorporated. The corresponding residue at D19 position was mutated to an alanine. The catalytic activity of MSMEG_2647D19AN76AH77A mutant was abrogated, suggesting that while the core MPE motifs are conserved between mycobacterial PDEs, differences in the ensemble of the active site residues contributing to their catalytic activity exist. Thus, at least two biochemically diverse PDE clades are found in mycobacterial species. In order to decipher the function of MSMEG_2647, its expression was monitored during the growth of M. Smegmatis. The promoter of MSMEG_2647 displayed maximum activity during the logarithmic phase of M. smegmatis growth after which its activity declined as M. smegmatis entered the stationary phase. However in contrast to this, the transcript corresponding to msmeg_2647 mRNA was found at both logarithmic and stationary phases. The MSMEG_2647 protein was also detected at both logarithmic and stationary phases of M. smegmatis. These results suggest that additional factors may contribute to the stability of msmeg_2647 mRNA and protein levels. Localization studies of MSMEG_2647 revealed that MSMEG_2647 was present in the cytosol as well as in the cell envelope fractions. Interestingly, over-expression of MSMEG_2647 did not result in a significant increase in PDE activity in various subcellular fractions, suggesting tight regulation on the in vivo activity in various subcellular fractions, suggesting tight regulation on the in vivo activity of MSMEG_2647. In addition, over-expression of MSMEG_2647 in M. smegmatis led to only a modest decrease in cAMP levels in M. smegmatis. These results suggested additional roles of MSMEG_2647 in the biology of mycobacteria. Overexpression of MSMEG_2647 peturbed the integrity of cell wall as assessed by the use of lipophillic indicators of cell growth, crystal violet and malachite green, and a cell wall targeting antibiotic, isoniazid. Analyzing the gene neighborhood of MSMEG_2647 provided an insight into its putative function. It was observed that the stop codon of msmeg_2647 overlapped with the start codon of msmeg_2648 and stop codon of msmeg-2648 overlapped with the start codon of msmeg_2649. RT PCR was carried out at logarhtimic and stationary phases of M. smegmatis growth, which revealed that a polycistronic mRNA was being transcribed. These results confirmed that msmeg_2647, msmeg_2648 and msmeg_2649 were a part of an operon. Interestingly, these three genes as a gene cluster were confined to only those actinobacteria that produced mycolic acids. An operon often encodes products that form multiprotein complexes and operate in a common pathway. Since there were a part of an operon, a GST pull-down approach was employed to test if MSMEG_2647, MSMEG_2648 and MSMEG_2649 could physically interact. It was observed that MSMEG_2647 interacted with MSMEG_2648 and MSMEG_2649. MSMEG_2648 in turn interacted with MSMEG_2649. A role for MSMEG_2647 as a scaffold recruiting MSMEG_2648 and MSMEG_2649 is therefore proposed. In turn, a complex formation with these proteins may regulate the activity of MSMEG_2647. Attempts to generate a knock out of msmeg_2647 in M. smegmatis by homologous recombination were not successful suggesting either the gene was essential or a polar effect on msmeg_2648(an essential gene for the viability of M. smegmatis) may not allow msmeg_2647 to be deleted from the genome of M. smegmatis. In summary, this study has identified and characterized two new phosphodiesterases from mycobacteria, one from the pathogenic mycobacterium, M. leprae and the other, a PDE from M. smegmatis that is conserved in all species of mycobacteria. Several, key biochemical differences were observed using biochemical and biological approaches. It appears that the cellular roles of mycobacterial phsophodiesterases may extend beyond cAMP hydrolysis, with these proteins not only regulating cell wall properties but also acting as scaffolding proteins in the cell.

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.

La tuberculose bovine causée par Mycobacterium bovis (M. bovis) est une zoonose multi-hôtes. Outre les bovins, elle peut être transmise à des populations sauvages variées dont certaines peuvent entretenir l'infection et/ou la retransmettre aux bovins, pouvant empêcher leur assainissement. Les conditions de persistance de M. bovis dans les populations sauvages ont été étudiées dans différentes régions du monde, mettant en lumière des statuts épidémiologiques variables selon les espèces et les contextes géographiques, cynégétiques et zootechniques. Notre objectif était de déterminer le rôle des différentes populations-hôtes sauvages impliquées dans le système multi-hôtes de M. bovis en Côte d'Or où, en plus des bovins, la tuberculose bovine, circule chez le blaireau, le sanglier, le cerf et le renard. Pour cela, nous avons estimé les niveaux d'infection et d'excrétion, les niveaux de densité, et au moyen d'un suivi télémétrique et de dispositif de vidéosurveillance, nous avons estimé les contacts entre les populations sauvages et les bovins. Nos résultats suggèrent que les trois populations ayant le rôle le plus important et aptes à retransmettre l'infection aux bovins sont le blaireau, le sanglier et le cerf, mais d'autres études sont encore nécessaires pour confirmer ces hypothèses et savoir si certaines d'entre elles peuvent entretenir l'infection de façon autonome. Elles pourraient, également, conjointement, constituer une communauté d'hôtes réservoir. Enfin, nos résultats nous ont permis de caractériser le risque de transmission de tuberculose bovine de ces populations sauvages vers les bovins et de proposer des mesures de lutte visant à réduire ce risque / Bovine tuberculosis, caused by Mycobacterium bovis is a multi-host zoonosis. Besides cattle,it can be transmitted to various wild populations and some of them are able to maintain or to spillback the infection to cattle, thus hampering control strategies. The maintenance of M. bovis by wild populations is dependent on species, on geographical configurations, and on hunting and husbandry practices. Our objective was to investigate the role the different wild populations involved in the M. bovis multi-host system of Côte d’Or, where bTB has been reoccuring in cattle since 2002 and has also been found in badgers, wild boar, red deer and foxes. To do so, we have assessed different risk factors. These include infection rate, ability to 10 shed M. bovis, populations densities and level of indirect contact between wild populations and cattle. For the latter factor, we have tracked 11 wild boars and 10 badgers and used remote surveillance in cattle farms. Our results suggest that, in the study site, badgers, wild boar and red deer may be able to spillback the infection to cattle. Nevertheless, further studies are required to confirm these hypotheses and to investigate whether some of these wild populations can act, individually as reservoirs. It might also be the case that, taken jointly, these wild populations could constitute a maintenance community. On the basis of our results, we made recommandations aiming at reducing the risk of spillback transmission

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>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>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.

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