O Mycobacterium tuberculosis é o agente causador da tuberculose, doença responsável por 26% das mortes passíveis de prevenção no mundo todo. No Brasil, são notificados anualmente 85 mil casos novos, e estima-se que 50 milhões de pessoas estejam infectadas pelo M. tuberculosis. A tuberculose é considerada prioritária para o controle de doenças e agravos pelo Ministério da Saúde. Para esse controle, é fundamental disponibilizar métodos e recursos para o pronto diagnóstico laboratorial. Os métodos utilizados para o diagnóstico da doença são bacterioscopia, análise histológica ou cultivo do micro-oganismo a partir de amostras de escarro. A bacterioscopia apresenta baixa sensibilidade, e o resultado da cultura demanda um período de tempo de até oito semanas. A PCR é uma técnica de amplificação de ácidos nucléicos que tem se mostrado promissor instrumento para o diagnóstico da tuberculose. O M. tuberculosis é um micro-organismo que tem tropismo pelas células (micro-organismo intracelular), e pode estar presente nas células do trato respiratório e da mucosa bucal. O esfregaço bucal, ao contrário do escarro, é obtido de forma fácil, sem constrangimentos, por procedimento não invasivo, e oferece menores riscos de contaminação por outros micro-organismos. Foram analisadas 80 amostras de esfregaço bucal de pacientes com diagnóstico confirmado de tuberculose, das quais 78 (97,4%) tiveram resultado positivo na PCR. Esse resultado permite concluir que a aplicação da PCR em amostras de esfregaço bucal é um método efetivo e confiável para detecção do M. tuberculosis. / Mycobacterium tuberculosis is the causing agent of the tuberculosis, responsible illness for 26% of the prevention deaths in the entire world. In Brazil 85000new cases are notified annually, being esteem 50 million people contaminated by the M. tuberculosis. It is considered priority disease for the control of illnesses for the Health department. For this control, it has to be reliable methods and resources for the ready laboratorial diagnosis. Bacterioscopiv, histological analysis or culture of the microrganism from samples of sputum are techniques normally used. The limitation of these methods is low sensitivity and long-winded 8 weeks. The PCR is one technique of amplification of DNA, that if has shown promising instrument for the diagnosis of the tuberculosis. The M. tuberculosis is a microorganism that has affinity for the cells (intracellular microorganism) and can be present in the cells of the respiratory treat and the oral mucosa. Oral swab, in contrast of sputum, is easily taken, not invasive and offering lesser risks of contamination for other microorganisms. We analyze 80 samples of oral swab of patients with confirmed diagnosis of tuberculosis, of these, 78 (97,4%) had resulted positive in the PCR. We conclude that the oral swab use and the application of the PCR are an effective and trustworthy method for tuberculosis detention of the M. tuberculosis.

O Mycobacterium tuberculosis é o agente causador da tuberculose, doença responsável por 26% das mortes passíveis de prevenção no mundo todo. No Brasil, são notificados anualmente 85 mil casos novos, e estima-se que 50 milhões de pessoas estejam infectadas pelo M. tuberculosis. A tuberculose é considerada prioritária para o controle de doenças e agravos pelo Ministério da Saúde. Para esse controle, é fundamental disponibilizar métodos e recursos para o pronto diagnóstico laboratorial. Os métodos utilizados para o diagnóstico da doença são bacterioscopia, análise histológica ou cultivo do micro-oganismo a partir de amostras de escarro. A bacterioscopia apresenta baixa sensibilidade, e o resultado da cultura demanda um período de tempo de até oito semanas. A PCR é uma técnica de amplificação de ácidos nucléicos que tem se mostrado promissor instrumento para o diagnóstico da tuberculose. O M. tuberculosis é um micro-organismo que tem tropismo pelas células (micro-organismo intracelular), e pode estar presente nas células do trato respiratório e da mucosa bucal. O esfregaço bucal, ao contrário do escarro, é obtido de forma fácil, sem constrangimentos, por procedimento não invasivo, e oferece menores riscos de contaminação por outros micro-organismos. Foram analisadas 80 amostras de esfregaço bucal de pacientes com diagnóstico confirmado de tuberculose, das quais 78 (97,4%) tiveram resultado positivo na PCR. Esse resultado permite concluir que a aplicação da PCR em amostras de esfregaço bucal é um método efetivo e confiável para detecção do M. tuberculosis. / Mycobacterium tuberculosis is the causing agent of the tuberculosis, responsible illness for 26% of the prevention deaths in the entire world. In Brazil 85000new cases are notified annually, being esteem 50 million people contaminated by the M. tuberculosis. It is considered priority disease for the control of illnesses for the Health department. For this control, it has to be reliable methods and resources for the ready laboratorial diagnosis. Bacterioscopiv, histological analysis or culture of the microrganism from samples of sputum are techniques normally used. The limitation of these methods is low sensitivity and long-winded 8 weeks. The PCR is one technique of amplification of DNA, that if has shown promising instrument for the diagnosis of the tuberculosis. The M. tuberculosis is a microorganism that has affinity for the cells (intracellular microorganism) and can be present in the cells of the respiratory treat and the oral mucosa. Oral swab, in contrast of sputum, is easily taken, not invasive and offering lesser risks of contamination for other microorganisms. We analyze 80 samples of oral swab of patients with confirmed diagnosis of tuberculosis, of these, 78 (97,4%) had resulted positive in the PCR. We conclude that the oral swab use and the application of the PCR are an effective and trustworthy method for tuberculosis detention of the M. tuberculosis.

L'accentuation des phénomènes de résistance aux antibiotiques augmente la difficulté d'enrayer les infections bactériennes. Afin de palier ce problème, la mise au point de nouvelles stratégies, telle la stratégie antivirulence, est essentielle. Ainsi, ce manuscrit propose une nouvelle approche thérapeutique contre les bactéries à développement intracellulaire. Les analyses génomiques ont permis de mettre en évidence l'histidinol déshydrogénase (HDH, EC 1.1.1.23), enzyme impliquée dans la biosynthèse de l'histidine, comme cible biologique pour la conception de nouveaux agents antibactériens. L'étude de l'inhibition de cette dernière permet une validation de l'approche sur Brucella suis, agent responsable de la brucellose, et un élargissement du spectre d'action des composés mis au point est envisagé par l'inhibition de HDH de Mycobacterium tuberculosis. Les travaux préliminaires nécessaires a cet élargissement sont présentés dans ce manuscrit. / The raise of antibiotic resistances increases the difficulty to eradicate bacterial infections. The development of new therapeutic approaches, such as the antivirulence strategy, is essential to limitate the impact of this phenomenon. This manuscript details a new therapeutic approach against intracellular pathogens. Genomic analyses allowed to discover new targets. The histidinol dehydrogenase (HDH, EC 1.1.1.23), which is an enzyme involved in histidine biosynthesis, has therefore be chosen for the conception of new antibacterial compounds. Inhibition studies of HDH of Brucella suis allows a validation of the strategy. In order to confirm the width of the therapeutic spectrum of synthesised compounds, the inhibition of HDH from Mycobacterium tuberculosis is envisaged and preliminary experiments are presented.

Mycobacterium tuberculosis, the principal causative agent of tuberculosis (TB) in humans, is considered to be a successful pathogen owing to the elicitation of multidrug resistance, ability to survive inside macrophage phagosomes by taking nutrients from host cell cytoplasm, and the capacity to alternate between proliferating and dormant (nonproliferating) conditions of growth. Thus, whether one looks at tubercle bacillus from the standpoint of regulation of cell division in the host system, or uptake of nutrients from the host cell cytoplasm or elicitation of drug resistance, the requirement for ATP Binding Cassette (ABC) transporter type protein complexes, which might be involved in the transport of drugs, nutrients or proteins, could be of critical importance to the pathogen. Therefore the present study was initiated to characterize ABC transporter type proteins, FtsE and FtsX of M. tuberculosis (MtFtsE and MtFtsX), and their interaction with FtsZ and FtsQ, which are the septation proteins that are recruited respectively before and after the localization of FtsE and FtsX proteins. The study was carried out in 3 parts. 1. Cloning, overexpression and purification of MtFtsE and MtFtsX proteins and elucidation of ATP binding activity of MtFtsE There exists considerable extent of homology between the FtsE and FtsX proteins of M. tuberculosis and E. coli. Therefore, in order to verify whether the structural homology is reflected in functional homology, complementation of growth defect of E. coli ftsE (Ts) by MtFtsE and MtFtsX was carried out. The MtFtsE protein could partially complement growth defect of E. coli ftsE temperature sensitive strain MFT1181, whereas co-expression of MtFtsE and MtFtsX efficiently complemented growth defect, indicating that the MtFtsE and MtFtsX proteins functionally complement E. coli FtsE and FtsX and that the two proteins together might be performing an associated function. Subsequently, in order to biochemically characterize MtFtsE and MtFtsX proteins of M. tuberculosis, MtftsE gene was cloned in pQE30, overexpressed, purified by Ni2+-NTA agarose affinity chromatography under denaturing conditions and refolded. MtFtsX protein, being toxic to E. coli cells, could not be expressed to sufficient amounts. Western blotting with anti-MtFtsE antibody showed that the recombinant 6xHis-MtFtsE protein and the native MtFtsE protein were localized to the membrane of E. coli and M. tuberculosis cells respectively. 6xHis-MtFtsE protein showed ATP binding in vitro, whereas K42R mutation abolished ATP binding. Thus, like in the case of E. coli FtsE, the K42 residue, which is positionally equivalent to K41 in EcFtsE in Walker A motif, was found to be essential for ATP binding. At 1.3 nM concentration of [α32P] ATP,70 molar excess of ATP, ADP, AMP, and GTP competed out respectively 97%, 87%, 73% and 57% of the [α32P] ATP bound to 6xHis-MtFtsE. 2. Biochemical characterization of MtFtsE protein The functional architecture of an ABC transporter consists of two each of nucleotide binding domain (NBD) and transmembrane domain (TMD), which are either part of a single polypeptide chain or individual subunits. The functional NBD is a ‘nucleotide-sandwich dimer’ with ATP flanked by the Walker A and B motifs of one NBD and the signature motif and D-loop of the other. NBD, through ATPase activity, is involved in energizing the transport of substrates namely drugs, proteins, ions, and solutes across the membrane. Since MtFtsE possesses Walker A and Walker B motifs that constitute NBD, and MtFtsX possesses TMD (four transmembrane segments), the two proteins together might constitute an ABC transporter type complex. Therefore, we wanted to know whether MtFtsE could hydrolyze ATP. MtFtsE not only could bind ATP with high affinity but could hydrolyse it also (Km, 1.5 µM; Vmax, 0.87 nmole/mg/min). It could bind and hydrolyse GTP as well, but not CTP, albeit with lower affinity and rate (Km, 25 µM; Vmax, 0.54 nmole/mg/min). The ATPase activity is strongly dependent on Mg2+ or Mn2+, with a pH optimum of 6.5 – 8.0 and temperature range of 27oC - 40oC. Kinetic analysis of ATPase and GTPase activities indicated nucleotide- dependent cooperativity (Hill coefficient for ATP is 1.7 and for GTP, 2.1). Inhibition of ATPase activity, to almost similar extent, in the presence of 10-fold excess of ATPγS, ADP, AMP, GTP, and CTP, but not TTP, indicated that nucleotide binding is through nitrogenous base of the nucleotide. Inhibition of MtFtsE by orthovanadate classified the enzyme as a P-type ATPase. Partially purified MtFtsE in soluble fraction also showed ATPase activity. The ATPase-active form of MtFtsE is a dimer with the sole cysteine (C84) at the dimer interface. Homology modeling of MtFtsE, using MalK (the NBD component of an ABC transporter for maltose) as the template, supported this observation. Stabilization of the dimer through cys-cys disulphide bond increased ATPase activity by 3.7-fold, although C84 does not have any role in ATPase activity. 3. Identification and elucidation of interaction among cell division proteins FtsE, FtsX, FtsQ and FtsZ of Mycobacterium tuberculosis Septum synthesis in E. coli is mediated by a dozen of proteins, among which the bacterial cytoskeletal protein FtsZ is the first molecule to localise to the mid-cell site, where it forms a scaffold for the localization of downstream cell division proteins namely, FtsA /ZipA < FtsE / FtsX < FtsK < FtsQ < FtsL < FtsB < FtsW < FtsI < FtsN and AmiC. If the above order of recruitment of proteins holds true for M. tuberculosis as well, the immediate proteins recruited to the mid-cell site after MtFtsZ in M. tuberculosis would be MtFtsE and MtFtsX, followed with MtFtsK and MtFtsQ. Thus it is possible that MtFtsE and MtFtsX could be interacting with MtFtsZ and MtFtsQ. Therefore attempts were made to delineate the interaction network among MtFtsE, MtFtsX, MtFtsQ and MtFtsZ of M. tuberculosis. Ni2+-NTA agarose pulldown, co-immunoprecipitation and bacterial two-hybrid assays using wild type and deletion mutants of the proteins showed that MtFtsE interacts with MtFtsQ and MtFtsX through its C-terminus. In addition, MtFtsX could interact with MtFtsZ and MtFtsQ. MtFtsX was found to homodimerise and interact with MtFtsQ in vivo. The ATPase-active of MtFtsE in vivo being a dimer, a hypothetical model for the translocation of MtFtsQ into the membrane at mid-cell site was proposed. According to this model, MtFtsQ might be inserted into the membrane at the mid-cell site by (MtFtsX)2 functioning as the membrane channel for the transport, which could be energized by the ATPase subunit (MtFtsE)2 of the (MtFtsE)2(MtFtsX)2 complex. MtFtsX might have a role in tethering the FtsZ-ring with the membrane at the mid-cell site. An altogether different possibility could be that the (FtsE)2(FtsX)2 complex might have a role in the stabilization or constriction of FtsZ-ring during the inward growth of septum.

Living organisms employ different kinds of mechanisms, to regulate the functions of genes or their products, which may help in maintaining homeostasis inside the cell or may help in fighting hostile environment in the case of pathogenic organisms. These mechanisms act at the transcriptional, post-transcriptional, translational, and post-translational levels. In order to understand the physiology of an organism, it is essential to obtain an in-depth knowledge of such mechanisms, in which several proteins participate in interlinked pathways. In this regard, the present study focuses on two such proteins: (i). the newly identified Fic (Filamentation induced by cAMP) protein; and (ii). NDK (Nucleoside Diphosphate Kinase), which had been studied for decades. Fic protein and NDK share several common features: (i). both use nucleoside triphosphate (NTPs) or nucleoside diphosphate (NDPs) or their derivatives as one of their substrates; (ii). they have been found to be involved in diverse cellular pathways, involving different types of substrates that form the second substrate of these proteins; (iii). both are ubiquitously present in all the living organisms - from bacteria to humans to plants. However, there is very little information on these proteins from mycobacterial systems, which include some major human pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, which are the causative agents of Tuberculosis and Leprosy, respectively. In view of these reasons, in the present study, the structural and/or functional features of the Fic and NDK proteins from Mycobacterium tuberculosis, were analysed, as it might be of medical significance for effectively combating the pathogen. The Chapter 1 of the thesis contains the Introduction to the research work and Chapter 2 is on the overall Materials and Methods. The remaining chapters pertain to the data obtained on the structural and/or functional features of the Fic and NDK proteins from Mycobacterium tuberculosis. Chapter 3. Cloning, Expression and Purification of Mycobacterium tuberculosis Fic The role of FIC (Filamentation induced by cAMP) domain containing proteins in the regulation of many vital pathways, mostly through the transfer of NMPs from NTPs to specific target proteins (NMPylylation), in microorganisms, higher eukaryotes, and plants is emerging. In order to understand the biological role of FIC domain containing proteins in mycobacteria, the gene for the FIC domain containing protein of the human pathogen, Mycobacterium tuberculosis, MtuFic, was cloned, overexpressed, purified to homogeneity, and biochemically characterised. Neither the His-tagged nor the GST-tagged MtuFic protein, overexpressed in Escherichia coli, nor expression of Mtufic in Mycobacterium smegmatis, yielded the protein in the soluble fraction. However, the maltose binding protein (MBP) tagged MtuFic (MBP-MtuFic) could be obtained partly in the soluble fraction. Denatured-refolded protein was used for the antibody generation in mice and rabbit. The cellular localisation and secretion of MtuFic were characterised using the antibody. Chapter 4. Biochemical Characterisation of Mycobacterium tuberculosis Fic Sequence alignment with several FIC motif containing proteins, complemented with homology modeling on the FIC motif containing protein, VbhT of Bartonella schoenbuchensis as the template, showed conservation and interaction of residues constituting the FIC domain. MtuFic, possesses the critical His144 residue, in the characteristic FIC Motif, HPFREGNGRSTR (HPFxxGNGRxxR), spanning 144th to 155th residue. Site-specific mutagenesis of the His144, or Glu148, or Asn150 of the FIC motif, or of Arg87 residue that constitutes the FIC domain, or complete deletion of the FIC motif, abolished the NTP to NMP conversion activity. The activity of MtuFic was consistent with the biochemical activities hitherto reported for a variety of bacterial FIC domain containing proteins. Studies were also carried out on NMPylylation in the presence of eukaryotic proteins and eukaryotic and mycobacterial cell lysates. Although formation of NMPs from NTPs mediated by MBP-MtuFic could be detected, we could not identify any protein as the target substrate either in the human macrophage (THP1) cells or in the M. tuberculosis cells. VopSΔ30 (kind gift from Dr. Kim Orth), along with human G proteins as targets, were used as the positive controls. Various possibilities for the inability to detect a protein target substrate are discussed. Chapter 5. Transcriptional Analysis of Mycobacterium tuberculosis fic Gene (Mtufic) In parallel, in order to understand the transcriptional regulation of Mtufic, primer extension analysis was carried out. The Transcription Start Site (TSS; +1 site) of Mtufic were mapped under different growth/stress conditions, which tubercle bacilli encounter in human host. Mtufic got expressed mainly through two transcripts, T1 and T2, arising from two different transcription start sites (TSS). Putative promoter regions were cloned in a promoter probe vector, which expresses a GFP protein of very high intensity, in order to qualitatively detect the activity of the promoters. The half-life of the gfp mRNA was determined to be 4 min and therefore justifiably quantitated the Mtufic promoter activity by determining the gfp mRNA levels. The levels of Mtufic mRNA were two-fold higher under nutrient-depleted stationary phase of growth, as compared to the levels at mid-log phase. The activity of P1 and P2, as quantitated real-time using the short half-life gfpm2+ mRNA levels in Mycobacterium smegmatis transformants, showed that the activity of P2 was upregulated two-fold under nutrient-depleted stationary phase of growth, while that of P1 remained unaltered while of P1 and P2 were low under hypoxia. Co-transcription of Mtufic, with the immediate upstream gene, Rv3642c, of unknown function, was observed. Taken together, the data strongly indicated that the expression of Mtufic gets altered under nutrient-depleted and hypoxic conditions, which are the stress conditions experienced by tubercle bacilli in granuloma in tuberculosis patients. Chapter 6. Functional Characterisation of Mycobacterial FtsZ-NDK Interaction During the past few decades, our laboratory has been carrying out extensive molecular and functional studies on the cytokinetic protein, FtsZ, of different mycobacterial species, and of a variety of other mycobacterial proteins that are believed to be interacting with the cell division machinery. In this regard, in parallel to the work on MtuFic, we carried out work on the identification and characterisation of the proteins that interact with mycobacterial FtsZ. In this context, we found for the first time that the nucleoside diphosphate kinase (NDK), which can generate NTPs from ATP/GTP and NDPs, interacts with FtsZ and that the interaction was conserved across several mycobacterial species. Therefore, the FtsZ-NDK interaction was extensively characterised in vitro, using the recombinant, purified FtsZ and NDK proteins from different mycobacterial species. This novel finding on the interaction of NDK with FtsZ adds another role to NDK, namely in bacterial cell division.