Orientador: Fernando Rogério Pavan / Resumo: A Tuberculose é uma doença infecto contagiosa causada pelo agente etiológico Mycobacterium tuberculosis, sendo este a causa de 1,3 milhões de mortes ao redor do mundo no ano de 2018. O tratamento preconizado pela Organização Mundial da Saúde é eficaz para cepas sensíveis, porém os efeitos adversos dos fármacos levam muitos pacientes ao abandono da terapia e consequente surgimento de resistência micobacteriana. Dentro desse contexto e mediante o reduzido número de fármacos disponíveis para o tratamento se faz necessário o desenvolvimento de novas alternativas terapêuticas. Bacteriófagos, vírus que infectam bactérias, têm sido sugeridos como importantes agentes terapêuticos no combate a bactérias multirresistentes, os quais podem ser encapsulados em lipossoma objetivando a proteção contra a degradação pelo sistema imune. O objetivo deste trabalho foi avaliar o perfil do micobacteriófago D29 livre e encapsulado em lipossoma frente a cepa padrão de M. tuberculosis (H37Rv). Os lipossomas apresentaram tamanho, PDI e valores de potencial zeta pelo DLS (Dynamic Light Scaterring). De acordo com imagens obtidas por microscopia de transmissão, foram classificados com vesículas unilamelares gigantes com uma eficiência de encapsulação fágica de 9,4 % ± 0,023. Para garantir mais de 50 % em viabilidade celular, com a linhagem MRC-5 e J774A.1, respectivamente o volume máximo de 80% e 60% das amostras (tampão de fago, micobacteriófago D29, micobacteriófago D29 encapsulado em lipossoma e lipos... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

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.

The ability to fine-tune gene-expression in the adverse conditions during pre and post infectious stages has contributed in no small measure to the success of Mycobacterium tuberculosis as the deadly pathogen. Multiple sigma factors, transcription regulators, and diverse two component systemshave facilitated tailoring the metabolic pathways to meet the challenges faced by the pathogen. Over the last decade, studies have been initiated to understand the various facets of transcription in mycobacteria. Although not as extensive as the work in other model systems, such as Escherichia coli and eukaryotes, it is evident from these initial studies that the machinery is conserved,yetmany aspects of transcription and its regulation seem to be different in mycobacteria.The work presented in the thesis deals with some of the steps in the process, primarily initiation in the context of the distinct physiology of M. tuberculosis. The detailed kinetic and equilibrium study of a few selected promoters of M. tuberculosis viz.PgyrB1, PgyrR, PrrnPCL1 and PmetU is described in Chapter 2.Different stages of transcription initiation that have been analyzed include promoter specific binding of RNAP, isomerization, abortive initiation and promoter clearance.The equilibrium binding and kinetic studies of various steps reveal distinct rate limiting events for each of the promoter, which also differed markedly in their characteristics from the respective promoters of Mycobacterium smegmatis. In addition, a novel aspect of the transcription initiation at the gyr promoter was unraveled. The marked differences in the transcription initiation pathway seen with rrn and gyr promoters of M. smegmatis and M. tuberculosis suggest that such species specific differences in the regulation of expression of the crucial housekeeping genes could be one of the key determinants contributing to the differences in growth rate and lifestyle of the two organisms. In Chapter 3, the mechanism of growth phase dependent control (GPDC) at a few of the M. tuberculosis promoters has been investigated. The experiments described in the chapter are carried out to demonstrate a different pattern of interaction between the promoters and sigma A (SigA) of M. tuberculosis to facilitate the iNTPs and pppGpp mediated regulation. Instead of cytosine and methionine, thymine at three nucleotides downstream to -10 element and leucine232 in SigA are found to be essential for iNTPs and pppGpp mediated response at the rrn and gyr promoters of the organism. The specificity of the interaction is substantiated by mutational replacements, either in the discriminator or in SigA, which abolish the nucleotide mediated regulation in vitro or in vivo. In chapter 4, the long standing hypothesis that deals with interdependence of the transcription elongation kinetics and the growth rates has been addressed. Previous studies suggest that the rate of synthesis of the key molecules in cells affects the growth kinetics. In order to validate, the kinetics of elongation of RNAPs from M. tuberculosis, M. smegmatis and E. coli whose growth rates vary from very slow to fast is measured. Surface Plasmon Resonance (SPR) is used to monitor the transcription in real time and kinetic equations are applied to calculate the elongation rates. Further, the effects of the composition of the template DNA on the elongation rates of RNAP from E. coli and M. smegmatis, whose genomes show difference in the GC content are explored. The results obtained from the analysis support the hypothesis and also reveal the effect of template composition on elongation rates of RNAP.

The eubacterial genome is maintained in a negatively supercoiled state which facilitates its compaction and storage in a small cellular space. Genome supercoiling can potentially influence various DNA transaction processes such as DNA replication, transcription, recombination, chromosome segregation and gene expression. Alterations in the genome supercoiling have global impact on the gene expression and cell growth. Inside the cell, the genome supercoiling is maintained judiciously by DNA topoisomerases to optimize DNA transaction processes. These enzymes solve the problems associated with the DNA topology by cutting and rejoining the DNA. Due to their essential cellular functions and global regulatory roles, DNA topoisomerases are fascinating candidates for the study of the effect of topology perturbation on a global scale. Genus Mycobacterium includes a large number of species including the well-studied Mycobacterium smegmatis (Msm) as well as various pathogens–Mycobacterium leprae, Mycobacterium abscessus and Mycobacterium tuberculosis (Mtb), the last one being the causative agent of the deadly disease Tuberculosis (TB), which claims millions of lives worldwide annually. The organism combats various stresses and alterations in its environment during the pathogenesis and virulence. During such adaptation, various metabolic pathways and transcriptional networks are reconfigured. Considering their global regulatory role, DNA topoisomerases and genome supercoiling may have an influence on the mycobacterial survival and adaptation. Biochemical studies from our laboratory have revealed several distinctive characteristics of mycobacterial DNA gyrase and topoisomerase I. DNA gyrase has been shown to be a strong decatenase apart from its characteristic supercoiling activity. Similarly, the mycobacterial topoisomerase I exhibits several distinct features such as the ability to bind both single- as well as double-stranded DNA, site specific DNA binding and absence of Zn2+ fingers required for DNA relaxation activity in other Type I enzymes. Although, efforts have been made to understand the biochemistry and mechanism of mycobacterial topoisomerases, in vivo significance and regulatory roles remain to be explored. The present study is aimed at understanding the mechanism, in vivo functions, regulation and genome wide distribution of mycobacterial topoisomerases. Chapter 1 of the thesis provides introduction on DNA topology, genome supercoiling and DNA topoisomerases. The importance of genome supercoiling and its regulatory roles has been discussed. Further, the regulation of topoisomerase activity and the role in the virulence gene regulation is described. Finally, a brief overview of Mtb genome, disease epidemiology, and pathogenesis is presented along with the description of the work on mycobacterial topoisomerases. In Chapter 2, the studies are directed to understand the DNA relaxation mechanism of mycobacterial Type IA topoisomerase which lack Zn2+ fingers. The N-terminal domain (NTD) of the Type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn2+ finger motifs in the CTD. The Zn2+ finger motifs were found to be essential in Escherichia coli TopoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial TopoI lacks Zn2+ fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. It is elucidated that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the strand passage step of the catalysis. It is hypothesized that the loss of Zn2+ fingers from the mycobacterial TopoI could be associated with Zn2+ export and homeostasis. In Chapter 3, the studies have been carried out to understand the regulation of mycobacterial TopoI. Identification of Transcription Start Site (TSS) suggested the presence of multiple promoters which were found to be sensitive to genome supercoiling. The promoter activity was found to be specific to mycobacteria as the promoter(s) did not show activity in E. coli. Analysis of the putative promoter elements suggested the non-optimal spacing of the putative -35 and -10 promoter elements indicating the involvement of supercoiling for the optimal alignment during the transcription. Moreover, upon genome relaxation, the occupancy of RNA polymerase was decreased on the promoter region of topoI gene implicating the role of DNA topology in the Supercoiling Sensitive Transcription (SST) of TopoI gene from mycobacteria. The involvement of intrinsic promoter elements in such regulation has been proposed. In Chapter 4, the importance of TopoI for the Mtb growth and survival has been validated. Mtb contains only one Type IA topoisomerase (Rv3646c), a sole DNA relaxase in the cell, and hence a candidate drug target. To validate the essentiality of Mtb topoisomerase I for bacterial growth and survival, conditionally regulated strain of topoI in Mtb was generated. The conditional knockdown mutant exhibited delayed growth on agar plate and in liquid culture the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the Mtb growth and open up new avenues for targeting the enzyme. In Chapter 5, the influence of perturbation of TopoI activity on the Msm growth and physiology has been studied. Notably, Msm contains an additional DNA relaxation enzyme– an atypical Type II topoisomerase TopoNM. The TopoI depleted strain exhibited slow growth and drastic change in phenotypic characters. Moreover, the genome architecture was disturbed upon depletion of TopoI. Further, the proteomic and transcript analysis indicated the altered expression of the genes involved in central metabolic pathways and core DNA transaction processes in the mutant. The study suggests the importance of TopoI in the maintenance of cellular phenotype and growth characteristics of fast growing mycobacteria having additional topoisomerases. In Chapter 6, the ChIP-Seq method is used to decipher the genome wide distribution of the DNA gyrase, topoisomerase I (TopoI) and RNA polymerase (RNAP). Analysis of the ChIP-Seq data revealed the genome wide distribution of topoisomerases along with RNAP. Importantly, the signals of topoisomerases and RNAP was found to be co-localized on the genome suggesting their functional association in the twin supercoiled domain model, originally proposed by J. C. Wang. Closer inspection of the occupancy profile of topoisomerases and RNAP on transcription units (TUs) revealed their co-existence validating the topoisomerases occupancy within the twin supercoiled domains. On the genomic scale, the distribution of topoisomerases was found to be more at the ori domains compared to the ter domain which appeared to be an attribute of higher torsional stress at ori. The reappearance of gyrase binding at the ter domain (and the lack of it in the ter domain of E. coli) suggests a role for Mtb gyrase in the decatenation of the daughter chromosomes at the end of replication. The eubacterial genome is maintained in a negatively supercoiled state which facilitates its compaction and storage in a small cellular space. Genome supercoiling can potentially influence various DNA transaction processes such as DNA replication, transcription, recombination, chromosome segregation and gene expression. Alterations in the genome supercoiling have global impact on the gene expression and cell growth. Inside the cell, the genome supercoiling is maintained judiciously by DNA topoisomerases to optimize DNA transaction processes. These enzymes solve the problems associated with the DNA topology by cutting and rejoining the DNA. Due to their essential cellular functions and global regulatory roles, DNA topoisomerases are fascinating candidates for the study of the effect of topology perturbation on a global scale. Genus Mycobacterium includes a large number of species including the well-studied Mycobacterium smegmatis (Msm) as well as various pathogens–Mycobacterium leprae, Mycobacterium abscessus and Mycobacterium tuberculosis (Mtb), the last one being the causative agent of the deadly disease Tuberculosis (TB), which claims millions of lives worldwide annually. The organism combats various stresses and alterations in its environment during the pathogenesis and virulence. During such adaptation, various metabolic pathways and transcriptional networks are reconfigured. Considering their global regulatory role, DNA topoisomerases and genome supercoiling may have an influence on the mycobacterial survival and adaptation. Biochemical studies from our laboratory have revealed several distinctive characteristics of mycobacterial DNA gyrase and topoisomerase I. DNA gyrase has been shown to be a strong decatenase apart from its characteristic supercoiling activity. Similarly, the mycobacterial topoisomerase I exhibits several distinct features such as the ability to bind both single- as well as double-stranded DNA, site specific DNA binding and absence of Zn2+ fingers required for DNA relaxation activity in other Type I enzymes. Although, efforts have been made to understand the biochemistry and mechanism of mycobacterial topoisomerases, in vivo significance and regulatory roles remain to be explored. The present study is aimed at understanding the mechanism, in vivo functions, regulation and genome wide distribution of mycobacterial topoisomerases. Chapter 1 of the thesis provides introduction on DNA topology, genome supercoiling and DNA topoisomerases. The importance of genome supercoiling and its regulatory roles has been discussed. Further, the regulation of topoisomerase activity and the role in the virulence gene regulation is described. Finally, a brief overview of Mtb genome, disease epidemiology, and pathogenesis is presented along with the description of the work on mycobacterial topoisomerases. In Chapter 2, the studies are directed to understand the DNA relaxation mechanism of mycobacterial Type IA topoisomerase which lack Zn2+ fingers. The N-terminal domain (NTD) of the Type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn2+ finger motifs in the CTD. The Zn2+ finger motifs were found to be essential in Escherichia coli TopoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial TopoI lacks Zn2+ fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. It is elucidated that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the strand passage step of the catalysis. It is hypothesized that the loss of Zn2+ fingers from the mycobacterial TopoI could be associated with Zn2+ export and homeostasis. In Chapter 3, the studies have been carried out to understand the regulation of mycobacterial TopoI. Identification of Transcription Start Site (TSS) suggested the presence of multiple promoters which were found to be sensitive to genome supercoiling. The promoter activity was found to be specific to mycobacteria as the promoter(s) did not show activity in E. coli. Analysis of the putative promoter elements suggested the non-optimal spacing of the putative -35 and -10 promoter elements indicating the involvement of supercoiling for the optimal alignment during the transcription. Moreover, upon genome relaxation, the occupancy of RNA polymerase was decreased on the promoter region of topoI gene implicating the role of DNA topology in the Supercoiling Sensitive Transcription (SST) of TopoI gene from mycobacteria. The involvement of intrinsic promoter elements in such regulation has been proposed. In Chapter 4, the importance of TopoI for the Mtb growth and survival has been validated. Mtb contains only one Type IA topoisomerase (Rv3646c), a sole DNA relaxase in the cell, and hence a candidate drug target. To validate the essentiality of Mtb topoisomerase I for bacterial growth and survival, conditionally regulated strain of topoI in Mtb was generated. The conditional knockdown mutant exhibited delayed growth on agar plate and in liquid culture the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the Mtb growth and open up new avenues for targeting the enzyme. In Chapter 5, the influence of perturbation of TopoI activity on the Msm growth and physiology has been studied. Notably, Msm contains an additional DNA relaxation enzyme– an atypical Type II topoisomerase TopoNM. The TopoI depleted strain exhibited slow growth and drastic change in phenotypic characters. Moreover, the genome architecture was disturbed upon depletion of TopoI. Further, the proteomic and transcript analysis indicated the altered expression of the genes involved in central metabolic pathways and core DNA transaction processes in the mutant. The study suggests the importance of TopoI in the maintenance of cellular phenotype and growth characteristics of fast growing mycobacteria having additional topoisomerases. In Chapter 6, the ChIP-Seq method is used to decipher the genome wide distribution of the DNA gyrase, topoisomerase I (TopoI) and RNA polymerase (RNAP). Analysis of the ChIP-Seq data revealed the genome wide distribution of topoisomerases along with RNAP. Importantly, the signals of topoisomerases and RNAP was found to be co-localized on the genome suggesting their functional association in the twin supercoiled domain model, originally proposed by J. C. Wang. Closer inspection of the occupancy profile of topoisomerases and RNAP on transcription units (TUs) revealed their co-existence validating the topoisomerases occupancy within the twin supercoiled domains. On the genomic scale, the distribution of topoisomerases was found to be more at the ori domains compared to the ter domain which appeared to be an attribute of higher torsional stress at ori. The reappearance of gyrase binding at the ter domain (and the lack of it in the ter domain of E. coli) suggests a role for Mtb gyrase in the decatenation of the daughter chromosomes at the end of replication.

Structural investigations on two mycobacterial enzymes were carried out. Tuberculosis still remains a major threat to mankind even though drugs against it have been in use for many decades. The emergence of drug resistant strains of the bacteria calls for the identification of new targets based on which new drugs can be developed to combat the disease. A thorough understanding of the functioning of the target molecules is essential for this approach. We have taken up the structural studies on two such molecules, aspartic proteinases and adenylyl cyclases, of Mycobacterium tuberculosis with a view to obtain insights into their mechanisms of action at the atomic level. The work presented in the thesis includes (i) the identification, cloning, expression, purification and structure determination of a putative aspartic proteinase domain of M. tuberculosis and (ii) the crystal structure of an adenylyl cyclase of M. tuberculosis and its mutant; and also of an adenylyl cyclase from M. avium. Chapter 1 presents an overview of aspartic proteinases and nucleotide cyclases with an emphasis on their structural features. The methods employed during the course of the present work are described in Chapter 2. Work on the putative aspartic proteinase domain identified in M. tuberculosis is presented in Chapter 3. The structure of the aspartic proteinase domain is the first structural report of such domain from any bacteria. A search in the genome of M. tuberculosis showed a weak similarity to the HIV aspartic proteinase sequence. This region corresponds to the C-terminal domain of a PE family protein in M. tuberculosis. The presence of two signature motifs, DTG and DSG, of aspartic proteinases in the full sequence of this domain encouraged us to take up further studies on this domain. Previous reports identifying the protein as a surface antigen and our findings on the occurrence of similar domains in two other PE proteins of M. tuberculosis and also in other pathological strains of Mycobacteria indicated that these domains probably play an important role in infecting the host. The crystal structure of one of the domains showed that it has a pepsin-like fold and the catalytic site architecture of known aspartic proteinases. However, no proteolytic activity was detected. The size of the molecule is intermediate to eukaryotic pepsins and the homodimeric retroviral pepsins. A close examination of the binding site revealed subtle differences when compared to the active enzyme structures. Appropriate mutations of some of the residues in this region to convert it to an active enzyme did not make it active. Once the in vivo function of these putative aspartic proteinase domains is established, their potential to act as drug targets can be probed as the PE proteins are present exclusively in pathogenic Mycobacteria. As part of an ongoing project on adenylyl cyclases of Mycobacteria, we have taken up the structure analysis of the catalytic domains of two adenylyl cyclases; Rv1625c from M. tuberculosis and Ma1120 from M. avium. This work is described in Chapter 4. The wild-type of Rv1625c crystallized as a domain swapped head to head inactive dimer even though it is an active dimer in solution and expected to have a head to tail arrangement as in the previously reported structures of the active forms of the enzyme. Mutation of a phenylalanine residue presumed to occur at the subunit interface of the active dimeric structure of the enzyme to an arginine residue, a conserved residue of guanylyl cyclases, resulted in reduced adenylyl cyclase activity. This mutant crystallized as a monomer though it was expected to be an active dimer. Similarly, Ma1120 also has a monomeric structure in the crystal in spite of showing activity in solution. Though our aim was to capture the active dimers in the crystalline state we did not succeed in this effort in any of the three cases. The catalytic reaction probably takes place very rapidly with the formation of a transient active form of the dimer which cannot be easily crystallized. However, the analysis revealed new structures which are likely to represent the stable states of the enzyme when it is required to stay inactive in certain conditions. We have also established that the N-terminal segments of the enzyme, a loop at the dimeric interface and external factors like pH are involved in determining the oligomeric status of the enzyme thereby regulating its function. Publications 1 Crystal structure of a putative aspartic proteinase domain of the Mycobacterium tuberculosis cell surface antigen PE_PGRS16; Deivanayaga V. Barathy and K. Suguna; FEBS Open Bio (In Press) 2 New structural forms of mycobacterial adenylyl cyclases (in preparation)

The genus Mycobacterium represents more than 120 species of bacteria including the pathogenic M. tuberculosis, the etiological agent of tuberculosis. The host mounts a robust inflammatory and cell-mediated response to contain the spread of pathogenic mycobacteria. While macrophages, dendritic cells (DCs) and neutrophils are known to facilitate early responses, the effector functions of CD4+ and CD8+ T cells are critical for containment of the mycobacteria. The type I T helper (Th1) subset of CD4+ T cell population orchestrates the protective immunity through cytokines like interferon (IFN)-γ, interleukin (IL)-12, IL-23 and tumor necrosis factor (TNF)-α However, it is known that despite such responses, host can only contain but not eradicate the infection. Additionally, infection of over one-third of the world’s population with pathogenic mycobacteria is a testimony of its success as a pathogen. Much of its success is attributed to the multiple evasion strategies employed such as inhibition of phagosome-lysosome fusion, secretion of reactive oxygen intermediates antagonistic proteins like superoxide dismutase and catalase, downregulation of antigen presentation to T cells, downregulation of the pro-inflammatory cytokines, skewing the immune balance toward the less effective Th2 responses, inhibition of autophagy, induction of regulatory T cells (Tregs) and immunosuppressive cytokines etc. Thus, an effective check on the infection would be possible if we understand the mechanisms underlying such evasion and survival strategies. In this perspective, evaluation of the host-pathogen interactions in terms of integration of key signaling centers, particularly that during mycobacteria-macrophage or mycobacteria-DC interactions, would underscore as a critical requisite to detail the immune responses and its regulation. This study addresses three such immune evasion and survival strategies employed by the mycobacteria; downregulation of IFN-γ-induced autophagy in macrophages, expansion of Tregs by modulating DC phenotype and finally epigenetic regulation of genes involved in foamy macrophage generation. Autophagy is one of the major immune mechanisms engaged to clear intracellular infectious agents. It contributes to both innate and adaptive immune responses to infections and plays an essential role in restricting intracellular pathogens and delivering pathogen-derived antigens for major histocompatibility complex class II presentation. Nonetheless, several pathogens, especially viruses such as herpes simplex virus, human immunodeficiency virus, influenza; and bacteria like Mycobacteria, Shigella and Listeria exhibit multiple mechanisms to evade autophagy. However, the identities and contributions of host signaling molecules and mechanisms by which pathogens modulate autophagy have not been explored in depth. Here, we demonstrate that M. bovis BCG, Shigella flexneri and Listeria monocytogenes but not Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli inhibit IFN-γ-induced autophagy in macrophages by evoking selective and robust activation of WNT and sonic hedgehog (SHH) pathways via mechanistic target of rapamycin (mTOR). Utilization of macrophages derived from mir155-null mice or by conventional siRNA or miRNA mimics emphasized the role for mTOR-responsive epigenetic modifications in the induction of microRNAs, miR-155 and miR-31 to fine-tune autophagy. Importantly, cellular levels of PP2A, a phosphatase, were regulated by miR-155 and miR-31. Diminished expression of PP2A led to inhibition of glycogen synthase kinase (GSK)-3β, a negative regulator and a nodal link that regulate WNT and SHH pathways. This facilitated the prolonged activation of WNT and SHH signaling pathways. Further, sustained WNT and SHH signaling effectuated the expression of anti-inflammatory lipoxygenases (ALOX5 and ALOX15), which in tandem inhibited IFN-γ-induced janus kinase (JAK)- signal transducer of activated (STAT) signaling and contributed to evasion of autophagy. Together, we have identified novel molecular mechanisms and host factors that are crucial to control autophagy and help the bacterial pathogens like mycobacteria to evade the host immune responses. Much of the protective immunity against mycobacterial infection is mediated by Th1 CD4+ T cells. However, suppressive T cell populations such as CD4+CD25+FoxP3+ Tregs or a less effective Th2 cells are exploited by mycobacteria to subvert the protective host immune response. In this perspective, the molecular mechanisms underlying mycobacteria-induced Treg expansion are unclear. Utilizing cues from the previous reports from others’ and our laboratory, we explored the role for host signaling pathways such as SHH, WNT and NOTCH1 signaling during mycobacteria-mediated DC maturation and Treg generation/expansion. We demonstrate that while inhibition of SHH signaling markedly reduced the ability of the infected DCs to expand Tregs, NOTCH1 signaling functioned to suppress M. bovis BCG-induced Treg expansion. Though SHH and NOTCH1 signaling did not regulate the DC maturation during infection in terms of the maturation markers CD1a, HLA-DR, CD40, CD83, CD80 and CD86, pro-inflammatory cytokines such as TNF-α, IL-2, IL-1β and IL-6 were moderately NOTCH1-responsive and suppressed by SHH signaling. Further, M. bovis BCG-induced SHH signaling and Treg expansion was mediated by the classical phosphoinositide 3-kinase (PI3K)-mTOR-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) cascade. Recent studies have attributed the role for programmed death ligand (PD-L)1 and cyclooxygenase (COX)-2-catalyzed prostaglandin (PG)E2 during expansion of Tregs. Experiments utilizing pharmacological inhibitors and conventional siRNAs indicated that both PD-L1 and COX-2/PGE2 were induced upon M. bovis BCG and M. tuberculosis infection in DCs and were regulated by SHH signaling. While SHH-responsive transcription factor, GLI1 arbitrated COX-2 expression, mycobacteria-stimulated SHH signaling was found to suppress miR-324 and miR-338, bonafide miRNAs that target PD-L1, to aid increased expression of PD-L1 and Treg expansion. This highlights the bi-functional role of SHH signaling during mycobacterial infection of DCs. Further, we found interesting cross-regulation of NOTCH and SHH pathway functions during M. bovis BCG infection of DCs. Inhibition of NOTCH1 signaling resulted in elevated expression of infection-induced PD-L1 whereas inhibition of SHH signaling showed increased transcripts of JAGGED2 (JAG2), a NOTCH1 ligand, and NOTCH intracellular domain (NICD), a marker for NOTCH activation. Thus, our results demonstrate that Mycobacterium directs a fine-balance of host signaling pathways and molecular regulators in DCs to determine the functional outcome of the immune responses including Tregs expansion that favours its survival. Foamy macrophages (FMs) are integral components of granulomas during mycobacterial pathogenesis. FMs are one of the morphotypes differentiated from macrophages characterized by the presence of lipid bodies (LBs)/droplets. The lipids provide nutrients to mycobacteria, leading to an enhanced ability to survive and replicate in host FMs. LBs are also known to regulate lipid metabolism, membrane trafficking, intracellular signaling and inflammatory mediator production. Interestingly, LBs are stores for various immune mediators including arachidonic acid, COX-2, ALOX5, ALOX15 and leukotrienes, underscoring the significance of FMs in the current study. However, molecular mechanisms that regulate intracellular lipid accumulation in FMs in the course of mycobacterial infection are not clear. Here, we analyzed the role for one of the histone modifications widely implicated in shaping the immune responses, Histone H3 lysine 27 trimethylation (H3K27me3), a known marker for gene silencing. While the trimethylation of H3K27 is catalyzed by EZH2, a component of Polycomb-repressive complex (PRC)2, Jumonji C (JmjC) domain protein (JMJD3) is a well-established H3K27me3 demethylase. Unlike M. smegmatis, infection of macrophages with M. tuberculosis or M. bovis BCG displayed JMJD3-dependent LB formation. Supporting this observation, the genes involved in lipid biosynthesis (Ascl1, Adrp, Psap) and uptake (Fat (CD36) and Msr1) were significantly upregulated with M. tuberculosis or M. bovis BCG infection of macrophages in a JMJD3- and TLR2-dependent manner. Abca1 and Abcg1, genes assisting in lipid export were downregulated or remained unchanged with M. tuberculosis or M. bovis BCG infection. Chromatin immunoprecipitation analysis revealed a reduced H3K27me3 mark on the promoters of the selected genes that were upregulated on mycobacterial infections. Corresponding, elevated recruitment of JMJD3 to these promoters was observed. Interestingly, NOTCH1 signaling-responsive MUSASHI (MSI), an evolutionarily conserved RNA-binding protein that inhibits translation of the mRNA, was found to positively regulate infection-induced JMJD3 expression. MSI targeted a transcriptional repressor of JMJD3, Msx2-interacting nuclear target protein (MINT/ SPEN), in the infected macrophages to aid in FM formation. Immunohistochemistry and immunofluorescence experiments utilizing in vivo murine granuloma model using M. bovis BCG substantiated these observations. Thus, our study has unveiled novel roles for JMJD3 and its regulators in epigenetic regulation of LB generation in FMs. Altogether, we have established significant roles for several new host factors and inhibitory, survival mechanisms employed by pathogenic mycobacteria. Emphasis on functions of miRNAs and epigenetic regulation in the study has underscored the importance of fine-tuning immune responses during mycobacterial pathogenesis to determine the cell-fate and shape the course of infection. Further understanding and evaluation of these molecular regulators bears potential importance in disease control by aiding the search for effective drugs and therapeutics.

Philosophiae Doctor - PhD (Biochemistry) / 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.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 –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. / South Africa

Homologous recombination is a fundamental cellular process evolved to maintain genomic integrity and to generate genetic diversity. It plays a crucial role in DNA repair, correct segregation of meiotic chromosomes and resumption of the stalled replication forks. In vitro, the homologous recombination pathway is kinetically separable into a four step process involving initiation, homologous pairing, branch migration and junction resolution. The process of pairing and strand exchange between two homologous double-stranded DNA molecules leads to the formation of an intermediate structure called the Holliday junction (HJ). The crucial enzyme involved in this step in bacteria is RecA. In eubacteria, the junction is processed by three proteins, collectively referred to as the RuvABC protein complex. RuvA binds to the HJ, while RuvB, a helicase, binds to the RuvA-HJ complex and pumps the duplex DNA thus facilitating branch migration. The work reported here is concerned with structural studies on mycobacterial RecA and RuvA. X-ray crystallography was used to solve the protein crystal structures. The hanging drop vapour diffusion method was used for crystallization in all cases. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator except for two data sets collected using synchrotron radiation. The data were processed mostly using Mosflm and Scala and few data sets were processed using the HKL program suite. The molecular replacement method using programs Phaser and AMoRe was used for structure solution. Structure refinements were carried out using programs CNS and PHENIX. Model building was performed using COOT and O. PROCHECK, MOLPROBITY, ALIGN and NACCESS were used for structure validation and analysis of the refined structures. Mycobacterium smegmatis RecA (MsRecA) and its nucleotide complexes crystallize in three different, but closely related, forms characterized by specific ranges of unit cell dimensions. The six crystals discussed in the earlier part of the thesis and the five reported earlier, all grown under the same or very similar conditions, belong to these three forms, all in space group P61. They include one obtained by reducing the relative humidity around the crystal. In all crystals, RecA monomers form filaments around a 61 screw axis. Thus, the c-dimension of the crystal corresponds to the pitch of the RecA filament. As reported in the case of E.coli RecA, the variation in the pitch among the three forms correlate well with the motion of the C-terminal domain of the RecA monomers with respect to the main domain. The domain motion is compatible with formation of inactive as well as active RecA filaments involving monomers with a fully ordered C-domain. It does not appear to influence the movement upon nucleotide-binding of the switch residue Gln 196, which is believed to provide the trigger for transmitting the effect of nucleotide-binding to the DNA-binding region. Interestingly, partial dehydration of the crystal results in the movement of the residue, in a way similar to that caused by nucleotide-binding. The ordering of the DNA-binding loops L1 and L2, which present an ensemble of conformations, is also unaffected by domain motion. The conformation of loop L2 appears to depend upon nucleotide-binding presumably on account of the movement of the switch residue which forms part of the loop. The conformations of loops L1 and L2 are correlated and have implications to intermolecular communications within the RecA filament. The structures resulting from different orientations of the C-domain and different conformations of the DNA-binding loops appear to represent snapshots of the RecA molecule at different phases of activity and provide insights into the mechanism of action of RecA. Crystal structures of mutants of MsRecA involving changes of Gln 196 from glutamine to alanine, asparagine and glutamic acid, wild type MsRecA and several of their nucleotide complexes were subsequently determined using mostly low temperature and partly room temperature X-ray data. At both the temperatures, nucleotide binding results in a movement of Gln 196 towards the bound nucleotide in the wild type protein. This movement is abolished in the mutants, thus establishing the structural basis for the triggering action of the residue in terms of the size, shape and the chemical nature of the side chain. The 25 crystal structures reported in this thesis, along with the 5 MsRecA structures reported earlier, provide further elaboration of the relation among the pitch of the `inactive´ RecA filament, the orientation of the C-terminal domain with respect to the main domain and the location of the switch residue. The low temperature structures define one extreme of the range of positions the C-domain can occupy. The movement of the C-domain is correlated to those of the LexA binding loop and the loop that connects the main and the N-terminal domains. These elements of molecular plasticity are made use of in the transition to the `active´ filament, as evidenced by the recently reported structures of RecA-DNA complexes. The available structures of RecA resulting from X-ray and electron microscopic studies appear to represent different stages in the trajectory of the allosteric transformations of the RecA filament. This work contributes to the description of the early stages of this trajectory and provides insights into structures relevant to the later stages. The interesting results observed in the case of MsRecA prompted similar studies on the RecA from Mycobacterium tuberculosis (MtRecA). In this study, the crystals were grown at slightly different conditions and examined at different relative humidities and temperatures. Surprisingly, in spite of the 92% sequence identity between the two proteins, the structures indicated MtRecA to be substantially less plastic than MsRecA. The crystal structures do not provide an obvious explanation for this difference. Further studies are warranted to explain the molecular basis of the difference. RuvA, along with RuvB, is involved in branch migration of heteroduplex DNA in homologous recombination. The structures of four crystal forms of RuvA from Mycobacterium tuberculosis (MtRuvA) have been determined. The RuvB-binding domain is cleaved off in one of them. Detailed models of the complexes of octameric RuvA from different species with the Holliday junction have also been constructed. A thorough examination of the structures determined as part of the doctoral programme and those reported earlier bring to light the hitherto unappreciated role of the RuvB-binding domain in determining inter-domain orientation and oligomerization. These structures also permit an exploration of the interspecies variability of structural features such as oligomerization and the conformation of the loop that carries the acidic pin, in terms of amino acid substitutions. These models emphasize the additional role of the RuvB-binding domain in HJ binding. This role along with its role in oligomerization could have important biological implications. In addition to the work on RecA and RuvA, which forms the body of the thesis, the author was also involved in a structural bioinformatics study in which several carbohydrate binding proteins were probed to identify common minimum principles required for binding mannose, glucose and galactose. The study, presented in an Appendix, identified interactions that were specific to particular sugars, leading to individual fingerprints. These fingerprints were then used for exploring lead compounds, using a fragment based approach. This investigation helped the author to familiarize himself with the analysis of protein structures and ligand design based on them.

Genetic material of bacteria is subject to damage due to multitudinous factors, both extrinsic and intrinsic in origin. Mechanisms for the maintenance of genomic integrity are thus essential for a bacterium to survive. Bacterium also requires appropriate minor changes in the genetic material so as to adapt to the changing environments. Structural and related studies of two proteins from mycobacteria, one involved in recombinational DNA repair (RecA) and the other involved in SOS response which helps in adaptation to stress (LexA) form the subject matter of the thesis. The available literature on structural and related studies on RecA and LexA are reviewed in the introductory chapter. The action of RecA involves transition to an active filament formed in association with DNA and ATP, from an inactive filament in the absence of DNA. The structure of the inactive filament was first established in E. coli RecA (EcRecA). The interaction of RecA with non-hydrolysable ATP analogues and ADP were thoroughly characterised and the DNA binding loops were visualised in this laboratory using the crystal structures involving the proteins from Mycobacterium tuberculosis (MtRecA) and Mycobacterium smegmatis (MsRecA). A switch residue, which triggers the transformation of the information on ATP binding to the DNA binding regions, was identified. The 20 residue C-terminal stretch of RecA, which is disordered in all other relevant crystal structures, was defined in an MsRecA-dATP complex. The ordering of the stretch is accompanied by the generation of a new nucleotide binding site which can communicate with the original nucleotide binding site of an adjacent molecule in the filament. The plasticity of MsRecA and its mutants involving the switch residue was explored by studying crystals grown under different conditions at two different temperatures and, in one instance, at low humidity. The structures of these crystals and those of EcRecA and Deinococcus radiodurans RecA (DrRecA) provide information on correlated movements involving different regions of the molecule. MtRecA has an additional importance as an adjuvant drug target in Mycobacterium tuberculosis. Apart from recombination, another important property of RecA is its coprotease activity whereby it stimulates the inherent cleavage of a certain class of proteins. One of the substrates for the coprotease activity of RecA is LexA. LexA is a transcriptional repressor involved in SOS response in bacteria. LexA performs its function through an autoproteolysis stimulated by RecA, resulting in the derepression of the genes under its control. Structural studies on LexA from E. coli have shown that it has an N-terminal domain involved in binding to DNA and a C-terminal domain involved in catalysis and dimerisation. LexA mediated SOS response in bacteria has been shown in many cases to be responsible for the resistance gained by bacteria on treatment with antibiotics. In that respect, LexA is considered to be a potential drug target in Mycobacterium tuberculosis. Structures of crystals of Mycobacterium tuberculosis RecA, grown and analysed under different conditions and reported in the thesis, provide insights into hitherto underappreciated details of molecular structure and plasticity (Chapter 2). In particular, they yield information on the invariant and variable features of the geometry of the P-loop, whose binding to ATP is central for all the biochemical activities of RecA. The strengths of interaction of the ligands with the P-loop reveal significant differences. This in turn affects the magnitude of the motion of the ‘switch’ residue, Gln195 in M. tuberculosis RecA, which triggers the transmission of ATP-mediated allosteric information to the DNA binding region. M. tuberculosis RecA is substantially rigid compared with its counterparts from M. smegmatis and E. coli, which exhibit concerted internal molecular mobility. Details of the interactions of ligands with the protein, characterised in the structures, could be useful for design of inhibitors against M. tuberculosis RecA. Eleven independent simulations, each involving three consecutive molecules in the RecA filament, carried out on the protein from M. tuberculosis, M. smegmatis and E. coli and their ATP complexes, provide valuable information which is complementary to that obtained from crystal structures, in addition to confirming the robust common structural frame work within which RecA molecules from different eubacteria function (Chapter 3). Functionally important loops, which are largely disordered in crystal structures, appear to adopt in each simulation subsets of conformations from larger ensembles. The simulations indicate the possibility of additional interactions involving the P-loop which remains largely invariant. The phosphate tail of the ATP is firmly anchored on the loop while the nucleoside moiety exhibits substantial structural variability. The most important consequence of ATP binding is the movement of the ‘switch’ residue. The relevant simulations indicate the feasibility of a second nucleotide binding site, but the pathway between adjacent molecules in the filament involving the two nucleotide binding sites appears to be possible only in the mycobacterial proteins. As described in Chapter 4, full length LexA, the N-terminal and C-terminal segments defined by the cleavage site, two point mutants involving changes in active site residues (S160A and K197A) and another involving change at the cleavage site (G126D) were cloned, expressed and purified. The wild type protein cleaves at basic pH. The mutants do not autocleave at basic pH even after incubation for 12 hours. The wild type and the mutant protein dimerise and bind DNA with equal facility. The C-terminal segment also dimerises, but has a tendency to form tetramer as well. The full length proteins including the mutants and the C-terminal segment crystallised. The structure of the crystals obtained for mutant G126D could not be solved. Each of the other crystals, four in number, contained only the catalytic core and a few residues preceding it, indicating that the full length proteins underwent cleavage, at the canonical cleavage site or elsewhere, during the long period involved in the formation of the crystals. Crystals obtained from the solutions of the wild type protein and the C-terminal segment contains dimers of the catalytic core. Crystals obtained using the active site mutants appear to contain different type of tetramers. One of them involves the swapping of the peptide segment preceding the catalytic core. Models of tetramerisation of the full length protein similar to those observed for the catalytic core are feasible. A model of a complex of MtLexA with M. tuberculosis SOS box could be readily built. In this complex, the mutual orientation of the two N-domains of the dimer is different from that in the EcLexA-DNA complex.

RNA polymerase binding protein A (RbpA) was first discovered as a RNA polymerase binding protein from Streptomyces. coelicolor. It was shown to cause rifampicin tolerance to RNA polymerase in vitro and leads to basal level of rifampicin resistance in vivo. This protein is exclusively present in the actinobacteria family with the nearest neighbour in mycobacteria. When null mutant of RbpA in S. coelicolor were transformed with the rbpA gene from Mycobacterium tuberculosis the resistance level of rifampicin increased from 0.75 µgml-1 to 2 µg ml-1 suggesting analogous role of MtbRbpA (RbpA from M. tuberculosis). MsRbpA, RbpA from Mycobacterium smegmatis was found to interact with the β-subunit of RNAP and its binding location on M. smegmatis RNAP was shown to be 18 Å from the (i+1) site. MsRbpA was also shown to rescue the inhibitory effect of rifampicin in vitro. Furthermore, overexpression of MsRbpA in wild type M. smegmatis resulted in the increase in the MIC of rifampicin to 85 µg ml-1 from 20 µg ml-1, which is the MIC of rifampicin for the wild type M. smegmatis. On the other hand, MsRbpA was unable to augment transcription in the presence of rifampicin when the reaction was catalysed by rifampicin resistant RNAP. Recent reports have shown that MtbRbpA enhances the affinity σA to core RNAP thereby activates transcription. The N and C-termini of MtbRbpA interact with σA while the C-terminal region of MtbRbpA is required for the oligomerisation of MtbRbpA. However M. tuberculosis and S. coleicolor are part of same family actinobacteria, RbpA is essential for the former while it is dispensable in the later case.This work focuses on characterisation of rifampicin resistant RNAP from M. smegmatis and elaborates on the roles played by MsRbpA. These include its effect on transcription activation, transcription rescue, its role in RNAP promoter closed and open complex formation, characterisation of its site of interaction with RNAP and σA, finding critical functional residues and establishing the essentiality of MsRbpA in M. smegmatis. Chapter 1 deals with the literature survey on structure of bacterial RNAP, promoters, sigma factors, RNAP inhibitors, transcriptional activators with the emphasis on the Mycobacteria. Chapter 2 summarises the identification of the mutations in rpoB gene from the rifampicin resistant (RifR) mutant strains of M. smegmatis, purification of RNAP from these strain, determining IC50 values of these RifR RNAP for rifampicin, finding kinetic parameters for the interaction of RifR RNAP with 3-formyl rifampicin and evaluating their interaction with MsRbpA. Chapter 3 describes the function of MsRbpA in transcription initiation, particularly its role in RNAP-promoter closed and open complex formation. Furthermore, this chapter throws light on the role of MsRbpA in transcription activation vis a vis its effects on transcription rescue from the inhibitory effect of rifampicin. Chapter 4 elucidates the function of a segment of MsRbpA from Arg58 to Lys 73 in activation of transcription activity, transcription rescue from the inhibitory effect of rifampicin and its interaction with σA and core RNAP. Furthermore, the alanine scanning of the region and subsequent in vitro transcription studies revealed four important residues required for MsRbpA functions. Chapter 5 describes the generation of conditional knock down strain of MsRbpA in M. smegmatis and establishing its essentiality. Chapter 6 summarizes the work documented in the thesis.