All organisms must sense and respond to their environment in order to survive. The processes that allow a living cell to sense changes in its environment, and respond appropriately are collectively referred to as ‘signal transduction’. Cyclic AMP is a ubiquitously used second messenger molecule that plays diverse roles from hormone signalling in mammalian cells to catabolite repression in enteric bacteria. In several bacterial pathogens such as Pseudomonas aeruginosa, cAMP has also been found to mediate pathogenesis, usually by regulating the production of several virulence factors aiding in colonisation of the host. Cyclic AMP signalling has been suggested to regulate the virulence of the obligate intracellular Mycobacterium tuberculosis. Mycobacteria, including M. tuberculosis, code for a large number of adenylyl cyclases, enzymes that synthesise cAMP. Of the 16 putative adenylyl cyclases encoded by M. tuberculosis H37Rv, 10 have received extensive biochemical attention. A knockout of one of these cyclases, Rv0386, resulted in compromised virulence of M. tuberculosis. Ten proteins predicted to bind cAMP and mediate its cellular roles have also been identified in M. tuberculosis. Among these are the cAMP-regulated transcription factor, CRPMt, and cAMP-regulated protein acetyl transferase, KATmt. Comparatively little information is available, however, regarding the roles of cAMP-degrading machinery in mycobacteria. Two phosphodiesterases, with modest activity against cAMP in vitro, have been identified from M. tuberculosis, and are encoded by the Rv0805 and Rv2795c loci. Of these, Rv2795c has orthologs in all sequenced mycobacterial genomes. However, Rv0805-like proteins are coded only by slow growing mycobacteria such as the M. tuberculosis-complex, M. marinum and M. leprae, several of which are human or animal pathogens. Rv0805 belongs to the metallophosphoesterase superfamily of proteins, consisting of metal-dependent phosphoesterases with substrates ranging from large polymers like nucleic acids to small molecules like cAMP and glycerophospholipids. Like other metallophosphoesterases, Rv0805 displays promiscuous substrate utilisation and efficient hydrolysis of 2’3’-cAMP in vitro. Rv0805 also hydrolyses 3’5’-cAMP in vitro. Overexpression of Rv0805 is reported to lead to reduction in intracellular cAMP levels in M. smegmatis and M. tuberculosis, suggesting that it is capable of hydrolysing cAMP in the bacterial cell as well. The structure of Rv0805 revealed a sandwich-like α/β fold, typical of metallophosphoesterases, along with a relatively flexible C-terminal domain of unknown function. Despite extensive biochemical and structural information on Rv0805 however, its roles in mycobacteria remain unknown. In this study, the cellular roles of Rv0805 are explored and using information from biochemical and structural analyses, novel activities and interactions of Rv0805 have been identified. Rv0805, when expressed in M. smegmatis, led to a reduction in intracellular cAMP, as previously reported. However, the extent of reduction was modest (~30 %) and limited to the exponential phase of growth when both Rv0805 and intracellular cAMP are at their highest levels. Overexpression of Rv0805 also resulted in hypersensitivity to cell wall perturbants like crystal violet and sodium dodecyl sulphate (SDS) indicative of a change in the properties of the cell envelope of M. smegmatis. Importantly, these effects were independent of cAMP-hydrolysis by Rv0805, as overexpression of catalytically inactive Rv0805N97A also elicited similar changes. Unexpectedly, Rv0805 was localised to the cell envelope, both in M. tuberculosis as well as in M. smegmatis. The ability of Rv0805 to localise to the cell envelope was dependent on it C-terminus, as truncation of Rv0805 in this region (Rv0805Δ10, Rv0805Δ20 and Rv0805Δ40) resulted in progressively greater enrichment in the cytosol of M. smegmatis. Overexpression of Rv0805Δ40, which was localised almost completely to the cytosol, did not result in hypersensitivity to SDS, suggesting that cell envelope localisation, rather than cAMP-hydrolysis, was crucial for the cell envelope modifying roles of Rv0805. A possible mechanism behind the cell envelope-related effects of Rv0805 overexpression was the ability of the protein to interact with the cell wall of mycobacteria in a C-terminus-dependent manner. Purified Rv0805, but not Rv0805Δ40, could associate with crude mycobacterial cell wall as well as purified cell wall core polymer (mycolyl-arabinogalactan-peptidoglycan complex) in vitro. In addition to the C-terminus, the architecture of the active site was also crucial for this interaction as mutations in the active site that compromised metal-binding also resulted in poor interaction with the cell wall. Most significant among these residues was His207, which when mutated to Ala almost completely abrogated interaction with the cell wall in vitro. Further, Rv0805H207A was unable to localise to the cell envelope when expressed in M. smegmatis, even in the presence of the C-terminus, highlighting the importance of this residue in maintaining the structural integrity of Rv0805, and demonstrating that the structure of the C-terminus, rather than its sequence alone, played a role in cell envelope localisation and interaction. In order to verify that the observed sensitivity of Rv0805-overexpressing M. smegmatis to cell wall perturbants was due to a change in cell envelope properties atomic force microscopy was employed. Two distinct modes of operation were used to analyse surface and bulk properties of the mycobacterial cell envelope. These were tapping mode phase imaging, and contact mode force spectroscopy. Using tapping mode phase imaging, it was found that the cell surface of M. smegmatis was inherently heterogeneous in its mechanical properties. Further, contact mode force-spectroscopy revealed that the cell envelope of M. smegmatis in cross-section had at least three layers of varying stiffness. Typically, a middle layer of high stiffness was observed, sandwiched between two lower stiffness layers. This organisation is reminiscent of the current model of the mycobacterial cell envelope, possessing a central polysaccharide rich layer and outer and inner lipid rich layers. Treatment of wild type M. smegmatis with cell wall-perturbing antibiotics isoniazid and ethambutol resulted in markedly altered phase images, as well as significantly lower stiffness of the bacterial cell envelopes, validating that the methodology employed could indeed be used to assess cell wall perturbation in mycobacteria. Further, M. smegmatis harbouring deletions in cell envelope biosynthesis related genes, MSMEG_4722 and aftC, showed significantly lower cell wall stiffness than wild type M. smegmatis, providing evidence that genetic perturbation of the cell wall of mycobacteria could also be studied using atomic force microscopy. While phase imaging revealed similar surface properties of Rv0805-overexpressing and control M. smegmatis, force spectroscopy revealed significantly lower cell envelope stiffness, particularly of the middle layer, of the former. Cell envelope stiffness was, however, unaffected by expression of Rv0805Δ40 in M. smegmatis, providing direct evidence for C-terminus-dependent cell envelope perturbation upon Rv0805 overexpression. Additionally, overexpression of Rv0805N97A, but not Rv0805H207A led to reduced stiffness of the cell envelope of M. smegmatis, demonstrating that the cell wall remodelling activity of Rv0805 was independent of cAMP-hydrolysis, but dependent on cellular localisation and cell wall interaction. Like in M. smegmatis, overexpression of Rv0805 also led to lower cAMP levels in M. tuberculosis. Using a microarray-based transcriptomics approach, pathways affected by Rv0805 overexpression were identified. Rv0805 overexpression elicited a transcriptional response, leading to the down-regulation of a number of virulence associated genes such as whiB7, eis, prpC and prpD. Importantly, Rv0805-overexpression associated gene expression changes did not include genes regulated by CRPMt, the primary cAMP-regulated transcription factor in M. tuberculosis. Further, Rv0805N97A overexpression in M. tuberculosis led to similar changes in gene expression as overexpression of the wild type protein. These observations reiterated that, at least upon overexpression, the effects of Rv0805 were largely independent of cAMP-hydrolysis. Using overexpression in M. smegmatis and M. tuberculosis, cAMP-hydrolysis independent roles of Rv0805 in mycobacteria were identified. To further validate these observations, a knockout strain of the Rv0805 gene was generated in M. bovis BCG, a well-established model to study M. tuberculosis. Curiously, deletion of Rv0805 did not lead to a change in intracellular cAMP levels, demonstrating that cAMP-hydrolysis by Rv0805 may not contribute to the modulation of mycobacterial cAMP levels under standard laboratory growth conditions. Rv0805 deletion led to altered colony morphology and possible reduction in cell wall thickness, reaffirming the roles of this phosphodiesterase in regulating cell envelope physiology of mycobacteria. Additionally, Rv0805 deletion also resulted in compromised growth of M. bovis BCG in fatty acid-deficient media, implicating Rv0805 as a possible regulator of carbon metabolism. In summary, this thesis explores novel links between Rv0805 and the mycobacterial cell wall and elucidates the critical importance of the C-terminus domain of this metallophosphodiesterase in modulating its cellular localisation to, and interaction with, the mycobacterial cell envelope. En route to understanding the effects of Rv0805 overexpression on the cell wall of M. smegmatis, an atomic force microscopy-based methodology to assess perturbation of the cell envelope of mycobacteria was also developed. Finally, using a combination of biochemical and genetic analyses, cellular roles of Rv0805, independent of cAMP-hydrolysis, were identified in slow-growing mycobacteria. This study therefore provides direct evidence against the sole role of this mycobacterial phosphodiesterase as a regulator of intracellular cAMP levels, and opens up new avenues to understanding the cellular functions of Rv0805 and indeed other members of the metallophosphoesterase superfamily.

The current, grim world-TB scenario, with TB being the single largest infectious disease killer, warrants a more effective approach to tackle the deadly pathogen, Mycobacterium tuberculosis. The deadly synergy of this pathogen with HIV and the emergence of drugresistant strains of the organism present a challenge for disease treatment (Russell et al., 2010). Thus, there is a pressing need for newer drugs with faster killing-kinetics which can claim both the actively-multiplying and latent forms of this pathogen causing the oldest known disease to man. This thesis entitled “Mechanistic and Regulatory Aspects of the Mycobacterium tuberculosis Dephosphocoenzyme A Kinase” describes one such potential drug target, which holds promise in future drug development, in detail. The development of efficacious antimycobacterials now requires previously unexplored pathways of the pathogen and cofactor biosynthesis pathways present a good starting point. Therefore, the mycobacterial Coenzyme A (CoA) biosynthesis was chosen for investigation, with the last enzyme of this pathway, dephosphocoenzyme A kinase (CoaE) which was shown to be essential for M. tuberculosis survival, as the focus of the present study (Sassetti et al., 2003). This thesis presents a detailed biochemical and biophysical characterization of the enzymatic mechanism of mycobacterial CoaE, highlighting several hitherto-unknown, unique features of the enzyme. Mutagenic studies described herein have helped identify the critical residues of the kinase involved in substrate recognition, binding and catalysis. Further, a role has been assigned to the UPF0157 domain of unknown function found in the mycobacterial CoaE as well as in several organisms throughout the living kingdom. Detailed insights into the regulatory characteristics of this enzyme from this work further our current understanding of the regulation of the universal CoA biosynthetic pathway and call for the attribution of a greater role to the last enzyme in pathway regulation than has been previously accredited. The thesis begins with a survey of the current literature available on tuberculosis and where we stand today in our fight against this dreaded pathogen. Chapter 1 details the characteristic features of the causative organism M. tuberculosis, briefly describing its unique genome and the cellular envelope which the organism puts forward as a tough shield to its biology. This is followed by a brief description of the infection cycle in the host, the pathogen-host interplay in the lung macrophages, the deadly alliance of the disease with HIV and our current drug arsenal against tuberculosis. Further, emphasizing on the need for newer, faster-acting anti-mycobacterials, Chapter 1 presents the rationale for choosing the mycobacterial coenzyme A biosynthetic pathway as an effective target for newer drugs. A detailed description of our current understanding of the five steps constituting the pathway follows, including a comparison of all the five enzymatic steps between the human host and the pathogen. This chapter also sets the objectives of the thesis, describing the choice of the last enzyme of the mycobacterial CoA biosynthesis, dephosphocoenzyme A kinase, for detailed investigation. As described in Chapter 1, the mycobacterial CoaE is vastly different from its human counterpart in terms of its domain organization and regulatory features and is therefore a good target for future drug development. In this thesis, Rv1631, the probable mycobacterial dephosphocoenzyme A kinase annotated in the Tuberculist database (http://genolist.pasteur.fr/TubercuList), has been unequivocally established as the last enzyme of the tubercular CoA biosynthesis through several independent assays detailed in Chapter 2. The gene was cloned from the mycobacterial genomic DNA, expressed in E. coli and the corresponding recombinant protein purified via a single-step affinity purification method. The mechanistic details of the enzymatic reaction phosphorylating dephosphocoenzyme A (DCoA) to the ubiquitous cofactor, Coenzyme A, have been described in this chapter which presents a detailed biochemical and biophysical characterization of the mycobacterial enzyme, highlighting its novel features as well as unknown properties of this class of enzymes belonging to the Nucleoside Tri-Phosphate (NTP) hydrolase superfamily. The kinetics of the reaction have been biochemically elucidated via four separate assays and the energetics of the enzyme-substrate and enzymeproduct interactions have been detailed by isothermal titration Calorimetry (ITC). Further details on the phosphate donor specificity of the kinase and the order of substrate binding to the enzyme provide a complete picture of the enzymatic mechanism of the mycobacterial dephosphocoenzyme A kinase. Following on the leads generated in Chapter 2 on the unexpected strong binding of CTP to the enzyme but its inability to serve as a phosphate donor to CoaE, enzymatic assays described in Chapter 3 helped in the identification of a hitherto unknown, novel regulator of the last enzyme of CoA biosynthesis, the cellular metabolite CTP. This chapter outlines the remarkable interplay between the regulator, CTP and the leading substrate, dephosphocoenzyme A, possibly employed by the cell to modulate enzymatic activity. The interesting twist to the regulatory mechanisms of CoaE added by the involvement of various oligomeric forms of the enzyme and the influence of the regulator and the leading substrate on the dynamic equilibrium between the trimer and the monomer is further detailed. This reequilibration of the oligomeric states of the enzyme effected by the ligands and its role in activity regulation is further substantiated by the fact that CoaE oligomerization is not cysteine-mediated. Further, the effects of the cellular metabolites on the enzyme have been corroborated by limited proteolysis, CD and fluorescence studies which helped elucidate the conformational changes effected by CTP and DCoA on the enzyme. Thus, the third chapter discusses the novel regulatory features employed by the pathogen to regulate metabolite flow through a critical biosynthetic pathway. Results presented in this chapter highlight the fact that greater importance should be attributed to the last step of CoA biosynthesis in the overall pathway regulation mechanisms than has been previously accorded. The availability of only three crystal structures for a critical enzyme like dephosphocoenzyme A kinase (those from Escherichia. coli, Haemophilus influenzae and Thermus thermophilus) is indeed surprising (Obmolova et al., 2001; O’Toole et al., 2003; Seto et al., 2005). In search of a structural basis for the dynamic regulatory interplay between the leading substrate, DCoA and the regulator, CTP, a computational approach was adopted. Interestingly, the mycobacterial enzyme, unlike its other counterparts from the prokaryotic kingdom, is a bi-domain protein of which the C-terminal domain has no assigned function. Thus both the N- and C-terminal domains were independently modeled, stitched together and energy minimized to generate a three-dimensional picture of the mycobacterial dephosphocoenzyme A kinase, as described in Chapter 4. Ligand-docking analyses and a comprehensive analysis of the interactions of each ligand with the enzyme, in terms of the residues interacted with and the strength of the interaction, presented in this chapter provide interesting insights into the CTP-mediated regulation of CoaE providing a final confirmation of the enzymatic inhibition effected by CTP. These homology modeling and ligand-docking studies reveal that CTP binds the enzyme at the site overlapping with that occupied by the leading substrate, thereby potentially obscuring the active site and preventing catalysis. Further, very close structural homology of the modeled full-length enzyme to uridylmonophosphate/cytidylmonophosphate kinases, deoxycytidine kinases and cytidylate kinases from several different sources, with RMSD values in the range of 2.8-3 Å further lend credence to the strong binding of CTP detailed in Chapter 2 and the regulation of enzymatic activity described in Chapter 3. Computational analyses on the mycobacterial CoaE detailed in this chapter further threw up some interesting features of dephosphocoenzyme A kinases, such as the universal DXD motif in these enzymes, which appears to play a crucial role in catalysis as has been assessed in the next chapter. It is interesting to note that the P-loop-containing nucleoside monophosphate kinases (NMPK), with which the dephosphocoenzyme A kinases share significant homology, have three catalytic domains, the nucleotide-binding domain, the acceptor substrate-binding domain and the lid domain. Computational analyses detailed in Chapter 4 including the structural and sequential homology studies, helped in the delineation of the three domains in the mycobacterial enzyme as well as highly conserved residues potentially involved in crucial roles for substrate binding and catalysis. Therefore important residues from all three domains of the mycobacterial CoaE were chosen for mutagenesis to study their contributions to catalysis. Conservative and non-conservative replacements of these residues detailed in Chapter 5 helped in the identification of crucial residues involved in phosphate donor, ATP binding (Lys14 and Arg140); leading substrate, DCoA binding (Leu113); stabilization of the phosphoryl transfer reaction (Asp32 and Arg140) and catalysis (Asp32). Thus, the results reported here present a first attempt to identify the previously unknown functional roles of highly conserved residues in dephosphocoenzyme A kinases. Chapter 5 also delineates the dependence of this kinase on the divalent cation, magnesium, for catalysis, describing a comparison of the kinetic activity by the wild type and the mutants, in the presence and absence of Mg2+. Therefore, this chapter presents a thorough molecular dissection of the roles played by crucial amino acids of the protein and the results herein can serve as a good starting point for targeted drug development approaches. As described above, another unusual characteristic of the mycobacterial CoaE is the fact that it carries a domain of unknown function, UPF0157, C-terminal to the N-terminal dephosphocoenzyme A kinase domain. The function of this unique C-terminal domain carried by the mycobacterial CoaE has been explored in Chapter 6. The failure of the Nterminal domain (NTD) to be expressed and purified in the soluble fraction in the absence of a domain at its C-terminus (either the mycobacterial CoaE CTD or GST from the pETGEXCT vector) pointed out a possible chaperonic activity for the CTD. A universal chaperonic activity by this domain in the cell was ruled out by carrying out established chaperone assays with insulin, abrin and -crystallin. In order to delineate the CTD sequence involved in the NTD-specific chaperoning activity, deletion mutagenesis helped establish the residues 35-50 (KIACGHKALRVDHIG) of the CTD in the N-terminal domain-specific assistance in folding. Chapter 6 further details the several other potential roles of the mycobacterial CTD probed, including the 4’-phosphopantethienyl transfer, SAM-dependent methyltransferase activity, activation of the NTD via phospholipids among others. Thus the results presented in this chapter are a first attempt at investigating the role of this domain found in several unique architectures in several species across the living kingdom. Chapter 7 is an attempt to stitch together and summarize the results presented in all the preceding chapters, giving an overview of our present understanding of the mycobacterial CoaE and its novel features.

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

<p>While the many completely sequenced genomes of bacterial pathogens contain all the determinants of the host-pathogen interaction, and also every possible drug target and recombinant vaccine candidate, computational tools for selecting suitable candidates for further experimental analyses are limited to date. The overall objective of my PhD project was to attempt to design reusable systems that employ the two most important features of bacterial evolution, horizontal gene transfer and adaptive mutation, for the identification of potentially novel virulence-associated factors and possible drug targets. In this dissertation, I report the development of two novel technologies that uncover novel virulence-associated factors and mechanisms employed by bacterial pathogens to effectively inhabit the host niche. More importantly, I illustrate that these technologies may present a reliable starting point for the development of screens for novel drug targets and vaccine candidates, significantly reducing the time for the development of novel therapeutic strategies. Our initial analyses of proteins predicted from the preliminary genomic sequences released by the Sanger Center indicated that a significant number appeared to be more similar to eukaryotic proteins than to their bacterial orthologs. In order determine whether acquisition of genetic material from eukaryotes has played a role in the evolution of pathogenic bacteria, we developed a system that detects genes in a bacterial genome that have been acquired by interkingdom horizontal gene transfer.. Initially, 19 eukaryotic genes were identified in the genome of Mycobacterium tuberculosis of which 2 were later found in the genome of Pseudomonas aeruginosa, along with two novel eukaryotic genes.</p> <p>Surprisingly, six of the M. tuberculosis genes and all four eukaryotic genes in P. aeruginosa may be involved in modulating the host immune response through altering the steroid balance and the production of pro-inflammatory lipids. We also compared the genome of the H37Rv M. tuberculosis strain to that of the CDC- 1551 strain that was sequenced by TIGR and found that the organisms were virtually identical with respect to their gene content, and hypothesized that the differences in virulence may be due to evolved differences in shared genes, rather than the absence/presence of unique genes. Using this observation as rationale, we developed a system that compares the orthologous gene complements of two strains of a bacterial species and mines for genes that have undergone adaptive evolution as a means to identify possibly novel virulence &ndash / associated genes. By applying this system to the genome sequences of two strains of Helicobacter pylori and Neisseria meningitidis, we identified 41 and 44 genes that are under positive selection in these organisms, respectively. As approximately 50% of the genes encode known or potential virulence factors, the remaining genes may also be implicated in virulence or pathoadaptation. Furthermore, 21 H. pylori genes, none of which are classic virulence factors or associated with a pathogenicity island, were tested for a role in colonization by gene knockout experiments. Of these, 61% were found to be either essential, or involved in effective stomach colonization in a mouse infection model. A significant amount of strong circumstantial and empirical evidence is thus presented that finding genes under positive selection is a reliable method of identifying novel virulence-associated genes and promising leads for drug targets.</p>

We aimed to characterize well-know PIDs, novel genetic defects and immunopathological mechanisms in Brazilian patients with susceptibility to mycobacterial diseases. The patients developed different mycobacterial diseases and M. tuberculosis was the most frequent species. Molecular and genetic analysis revealed mutations in different genes: RAG1 (P1), CD40LG (P2, P3, P4), NEMO (P5), NCF1 (P6), TLR2 (P7) IL-12Rb2 (P8), IL-12Rb1 (P9), TLR10 (P10), DKC1(P11), SOCS-1(P12) and IRAK2 (P13). Finally, MDMs from patients phagocytose normally but were unable to appropriately control intracellular M. tuberculosis growth in comparison to MDMs from healthy subjects. We concluded that the Brazilian patients have heterogeneous mutations previously associated with susceptibility to mycobacterial diseases and novel genetic variations were identified suggesting novel PIDs. In addition, the inability of MDMs to control the intracellular growth of M. tuberculosis indicates this contributes to patients´ susceptibility to mycobacterial infections. / Objetivamos identificar novos defeitos genéticos e mecanismos imunopatológicos em pacientes brasileiros com suscetibilidade a infecções por micobactérias. Os pacientes foram investigados se portadores de imunodeficiencias previamente caracterizadas tais como SCID, deficiência de CD40L, MSMD, defeitos na sinalização via TLRs e CGD. A análise genética foi realizada por sequenciamento Sanger e \'\'whole exome sequencing\'\' para identificar possíveis novas imunodeficiências primárias. Além disso a função dos macrófagos dos pacientes foi avaliada. Infecções por diferentes espécies de micobactérias foram apresentadas pelos pacientes, sendo M. tuberculosis a espécie mais frequentemente identificada. Mutações em diferentes genes foram encontradas: RAG1 (P1), CD40LG (P2, P3, P4), NEMO (P5), NCF1 (P6), TLR2 (P7), IL-12Rb2 (P8), IL-12Rb1 (P9), IRAK2 (P10), SOCS-1 (P11) e TLR10 (P12). MDMs dos pacientes fagocitaram normalmente M. tuberculosis, porém reduzida capacidade em inibir o crescimento da M. tuberculosis foi observada. Concluímos que os pacientes estudados possuem defeitos moleculares heterogêneos e que os MDMs desses indivíduos apresentam falhas no controle do crescimento da M. tuberculosis. Nossos dados sugerem que esses são fatores subjacentes à susceptibilidade a infecções por micobactérias nesses indivíduos.

La biosynthèse de phénol-glycolipides (PGL) par Mycobacterium tuberculosis et M. leprae favorise l’invasion des macrophages via l'interaction de la partie saccharidique des PGL avec le domaine lectine du récepteur cellulaire au complément CR3. Les PGL inhibent également la production de cytokines inflammatoires par la cellule hôte, par un mécanisme inconnu. J’ai observé que des bactéries BCG transgéniques exprimant les PGL de M. tuberculosis ou M. leprae avaient une capacité de survie accrue dans les macrophages. Cette persistance intracellulaire était dépendante de CR3 et associée à une diminution de la production d'oxyde nitrique dans les cellules infectées. L’addition de PGL purifié suffisait à inhiber la production d’oxyde nitrique par des macrophages stimulés avec LPS/IFN-γ. J’ai montré que la liaison de PGL-1 à CR3 provoquait la dégradation post-transcriptionnelle de TIR-domain-containing adapter-inducing interféron-β (TRIF) dans les macrophages, ce qui entraînait une réduction de la signalisation TRIF-dépendante. Dans les macrophages stimulés avec LPS/IFN-γ, la dégradation de TRIF réduisait la production d’oxyde nitrique synthase, et la production TRIF dépendante de cytokines inflammatoires et des chimiokines. Mes résultats ont donc permis d’identifier un nouveau mécanisme de virulence développé par les mycobactéries pathogènes pour réprimer conjointement les réponses inflammatoires et antimicrobiennes de l’hôte / Biosynthesis of phenolic glycolipids (PGL) by Mycobacterium tuberculosis and M. leprae promotes macrophage invasion, which proceeds through the interaction of the PGL sugar moieties with the lectin domain of cell-displayed complement receptor (CR3). PGL also limit host cell production of inflammatory cytokines by an unknown mechanism. I observed that transgenic BCG that express PGL specific to M. tuberculosis or M. leprae displayed enhanced survival within macrophages. Increased intracellular persistence of PGL-expressing BCG was CR3-dependent and correlated with the decreased production of nitric oxide in infected cells. Notably, the addition of soluble PGL to macrophages was sufficient to induce a reduction in nitric oxide production upon stimulation with LPS/IFN-γ. I showed that PGL-1 binding to CR3 causes the post-transcriptional degradation of TIR-domain-containing adapter-inducing interferon-β (TRIF) in macrophages, resulting in impaired TRIF-dependent signaling. Functionally, PGL-1-mediated degradation of TRIF resulted in the decreased induction of nitric oxide synthase, and TRIF-dependent inflammatory cytokines and chemokines in LPS/IFN-γ-stimulated macrophages. My results thus identified a virulence mechanism evolved by pathogenic mycobacteria to suppress both the inflammatory and antimicrobial responses of infected host cells

This thesis aimed to the identification of immune biomarkers of mycobacterial infection for better diagnosis of tuberculosis (TB) and also focused on new vaccination strategies with a particular emphasis on the immune responses in the respiratory tract using murine models. Since the lung is the natural habitat for the M. tuberculosis, we reasoned that immune responses detected locally in the lungs would be good correlates of infection (Paper I). Likewise, immune responses induced in the respiratory tract following immunization would be more effective against mycobacterial infection. We showed that cytokines (IL-12, TNF, and IFN-γ) and cytokine receptors (sTNFR1 and sTNFR2) together with specific antibodies in the respiratory tract correlated better with the bacterial burden in the organs. In Paper II, we investigated the role of the BCG vaccination as a priming vaccine in a heterologous prime-boost immunization protocol. The results showed that the neonatal BCG vaccination primed the immune system for a relevant antigen and showed a generalized adjuvant effect. Using this immunization protocol, protective immune responses in the lungs were generated independently of the route used for the booster immunization. In Paper III, We showed that exposure to mycobacterial antigens during the gestational period led to antigen transportation from the mother to the fetus and this resulted in an early priming of the fetal immune system. Immunization with the same antigen during the postnatal life increased antigen-specific recall IFN-γ responses and protection against infection. We examined the role of innate immunity for the induction of acquired immune responses upon immunization with mycobacterial antigens using TLR2 deficient mice (Paper IV). Our data indicated that suboptimal innate immune responses in the TLR2-/- mice might compromise the induction of acquired immune responses. Overall, the current findings suggested that a better understanding of the mucosal immunity would be useful for the improvement of diagnostic procedures and the development of efficient vaccines against TB. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript

<p>While the many completely sequenced genomes of bacterial pathogens contain all the determinants of the host-pathogen interaction, and also every possible drug target and recombinant vaccine candidate, computational tools for selecting suitable candidates for further experimental analyses are limited to date. The overall objective of my PhD project was to attempt to design reusable systems that employ the two most important features of bacterial evolution, horizontal gene transfer and adaptive mutation, for the identification of potentially novel virulence-associated factors and possible drug targets. In this dissertation, I report the development of two novel technologies that uncover novel virulence-associated factors and mechanisms employed by bacterial pathogens to effectively inhabit the host niche. More importantly, I illustrate that these technologies may present a reliable starting point for the development of screens for novel drug targets and vaccine candidates, significantly reducing the time for the development of novel therapeutic strategies. Our initial analyses of proteins predicted from the preliminary genomic sequences released by the Sanger Center indicated that a significant number appeared to be more similar to eukaryotic proteins than to their bacterial orthologs. In order determine whether acquisition of genetic material from eukaryotes has played a role in the evolution of pathogenic bacteria, we developed a system that detects genes in a bacterial genome that have been acquired by interkingdom horizontal gene transfer.. Initially, 19 eukaryotic genes were identified in the genome of Mycobacterium tuberculosis of which 2 were later found in the genome of Pseudomonas aeruginosa, along with two novel eukaryotic genes.</p> <p>Surprisingly, six of the M. tuberculosis genes and all four eukaryotic genes in P. aeruginosa may be involved in modulating the host immune response through altering the steroid balance and the production of pro-inflammatory lipids. We also compared the genome of the H37Rv M. tuberculosis strain to that of the CDC- 1551 strain that was sequenced by TIGR and found that the organisms were virtually identical with respect to their gene content, and hypothesized that the differences in virulence may be due to evolved differences in shared genes, rather than the absence/presence of unique genes. Using this observation as rationale, we developed a system that compares the orthologous gene complements of two strains of a bacterial species and mines for genes that have undergone adaptive evolution as a means to identify possibly novel virulence &ndash / associated genes. By applying this system to the genome sequences of two strains of Helicobacter pylori and Neisseria meningitidis, we identified 41 and 44 genes that are under positive selection in these organisms, respectively. As approximately 50% of the genes encode known or potential virulence factors, the remaining genes may also be implicated in virulence or pathoadaptation. Furthermore, 21 H. pylori genes, none of which are classic virulence factors or associated with a pathogenicity island, were tested for a role in colonization by gene knockout experiments. Of these, 61% were found to be either essential, or involved in effective stomach colonization in a mouse infection model. A significant amount of strong circumstantial and empirical evidence is thus presented that finding genes under positive selection is a reliable method of identifying novel virulence-associated genes and promising leads for drug targets.</p>

Thesis (M.S.)--University of Texas at El Paso, 2008. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

Study of cell signalling pathways affected by pathogen entry comprises a fundamental aspect of understanding host-pathogen interactions. In this respect, the current study attempted to ascribe novel roles to Bone Morphogenesis Protein (BMP) signaling during infection. BMP pathway has been majorly studied in context of development where it plays an imperative role and its contribution to immunity has been poorly documented. Subsequent narrative talks about the perturbation of BMP signaling in context of specific signaling networks and its collaboration with other molecular players of host innate armamentarium. There is a pressing need to develop effective chemotherapy against Mycobacterium tuberculosis, the causative agent of tuberculosis, which has garnered the world’s attention as a leading cause of public health emergency. The tyrosine kinase, c-Abl was previously reported to be activated in murine bone marrow derived macrophages infected with mycobacteria. Yet, the identities of host signaling players and mechanisms exploited by mycobacteria in association with c-Abl lacked identification. Here, we deciphered an intricate signaling mechanism linking tyrosine kinase c-Abl, chromatin modifier, lysine acetyl transferase KAT5 and transcription factor, TWIST1 acting at Bmp2 and Bmp4 promoters. This molecular circuitry was observed to affect mycobacterial survival. Emerging studies suggest repurposing of c-Abl inhibitor, Imatinib, as an adjunct to existing anti-tuberculosis therapy. Through the use of Imatinib in an established model of tuberculosis, we demonstrated the ability of c-Abl inhibitors in potentiating innate immune responses. Distinctive instances report the cross regulation among Pattern Recognition Receptors (PRRs). Interestingly, TLR3 signaling cascade induced in response to its cognate ligand was dampened through c-Abl-BMP induced miR27a. TLR3 is known to activate immune surveillance upon viral infections; however, recent studies also suggest its role in tumour regression and induction of apoptosis. Our observation of mycobacteria elicited down regulation of TLR3 pathway corroborated with increased incidences of lung cancer among TB patients and mycobacterial evasion of a well characterized form of cell-death i.e. apoptosis. Further, we utilized a panel of such Mtb mutants associated with virulence and questioned their relevance in the activation of c-Abl-dependent BMP signaling. We found that nitric oxide, hypoxia and carbon monoxide-responsive mycobacterial WhiB3 and DosR, but not the sec-dependent protein secretion pathway, orchestrate mycobacteria driven c-Abl-BMP signaling. Continuing with the theme of exploring roles for BMP signaling during infection, we identified an important role for the C-type Lectin Receptor (CLR), Dectin-2, in activating Candida albicans-driven BMP signaling. Mounting evidences suggest BMP antagonists promote repair and regeneration in cells of varied lineages. We observed a role for BMP signaling in aggravating MMP2 and MMP9, factors that result in chronic non-healing wounds. Wounds are now increasingly recognized as being colonized with fungi along with bacteria. We propose a role for C. albicans orchestrated BMP signaling in contributing to enriched repressive methylation at Egf, Pdgf and Tissue Inhibitors of Matrix Metalloproteases (Timp2/3/4) promoters. Repressive H3K27me3 at these loci impedes the reparative tissue homeostasis, resulting in C. albicans endorsed impaired wound healing. Altogether, we uncovered hitherto unknown roles of BMP signaling during mycobacterial and fungal infections, enabling a better understanding of lesser studied pathways in mediating pathogenesis.