Studies reported in this thesis address the question of how pre-termination ribosomal complexes stalled during translation of mRNA are recycled. The process of recycling of the stalled ribosomes involves many translational factors. During the course of my studies, I have uncovered new roles of SsrA (tmRNA), IF3 and ribosome recycling factor (RRF) in recycling stalled ribosomes. These findings are summarized as follows: (i) A physiological connection between tmRNA and peptidyl-tRNA hydrolase functions in Escherichia coli The bacterial ssrA gene codes for a dual function RNA, tmRNA, which possesses tRNA-like and mRNA-like regions. The tmRNA appends an oligopeptide tag to the polypeptide on the P-site tRNA by a trans-translation process that rescues ribosomes stalled on mRNAs and targets the aberrant protein for degradation. In cells, processing of the stalled ribosomes is also pioneered by drop-off of peptidyl-tRNAs. The ester bond linking the peptide to tRNA is hydrolyzed by peptidyl-tRNA hydrolase (Pth), an essential enzyme, which releases the tRNA and the aberrant peptide. As the trans-translation mechanism utilizes the peptidyl-transferase activity of the stalled ribosomes to free the tRNA (as opposed to peptidyl-tRNA drop-off), the need for Pth to recycle such tRNAs is bypassed. Thus, we hypothesized that tmRNA may rescue a defect in Pth. The findings of the experiments detailed in this thesis show that SsrA rescues a defect in Pth by reducing the peptidyl-tRNA load on Pth. (ii) Evidence for a role of initiation factor 3 in recycling ribosomal complexes stalled on mRNAs in Escherichia coli. Specific interactions between ribosome recycling factor (RRF) and EF-G mediate disassembly of post-termination ribosomal complexes for new rounds of initiation. The interactions between RRF and EF-G are also important in peptidyl-tRNA release from pre-termination complexes. Unlike the post-termination complexes (harboring tRNA), the pre-termination complexes (harboring peptidyl-tRNA) are not recycled by RRF and EF-G in vitro, suggesting participation of additional factor(s) in the process. Using a combination of biochemical and genetic approaches, we show that, 1. Inclusion of IF3 with RRF and EF-G results in recycling of the pre-termination complexes; 2. IF3 overexpression in Escherichia coli LJ14 rescues its temperature sensitive phenotype for RRF; (3) Transduction of infC135 (encoding functionally compromised IF3) in E. coli LJ14 generates a ‘synthetic severe’ phenotype; (4) The infC135 and frr1 (a promoter down RRF gene) alleles synergistically rescue a temperature sensitive mutation in peptidyl-tRNA hydrolase in E. coli; and (5) IF3 facilitates ribosome recycling by Thermus thermophilus RRF and E. coli EFG in vivo and in vitro. These lines of evidence clearly demonstrate the physiological importance of IF3 in the overall mechanism of ribosome recycling in E. coli. (iii) The role of RRF in dissociating of pre-termination ribosomal complexes stalled during elongation Translating ribosomes often stall during the repetitive steps of elongation for various reasons. The stalled ribosomes are rescued by the process of trans-translation involving tmRNA (SsrA) or by a factor mediated dissociation of the stalled ribosome into its subunits leading to the drop-off of the peptidyl-tRNA. The mechanistic details of how the factor mediated dissociation is carried out, is not well studied. Studies described in the above section have highlighted the role of RRF in dissociating stalled pre-termination complexes. However, the in vivo studies in this area have been limited for lack of defined pre-termination complexes. Two in vivo systems based on translation of AGA minigene and the ung gene (EcoUngstopless) transcripts were designed. Evidence is presented to show that translation of both of these transcripts is toxic to E. coli because of the accumulation of the transcript specific stalled pre-termination complexes. Availability of these model systems has allowed us to address the role of RRF in dissociating stalled ribosomes. We show that RRF rescues stalled ribosomes on these constructs and its overexpression can rescue the toxicity. The physiological importance of this observation is highlighted by the rescue of AGA minigene inhibitory effect on λimmP22 hybrid phage growth upon RRF overexpression.

Thesis addresses the physiological role of formyl tetrahydrofolate synthetase (Fhs) and bifunctional folate dehydrogenase (FolD) in folate mediated one carbon metabolism in bacteria. Thesis consists of 5 chapters. First chapter provides the details of the literature on folate metabolism, enzymes involved the synthesis and physiological roles various folate co-factors. Second chapter discusses the study of Clostridium perfringens Fhs generation of folD deletion in the support of fhs. Third chapter explores the characterization of the folD deletion strain. Fourth chapter presents the characterization of monofunctional versions of FolD from Clostridium perfringens. Fifth chapters talks about anti-correlation existence of Fhs and PurT (phosphoribosyl glycinamide formyl transferase II) The detailed experimental study is discussed below i. Characterization of Clostridium perfringens Formyl Tetrahydrofolate Synthetase (Fhs) In this chapter we have characterized Fhs from pathogenic Clostridium perfringens. Fhs catalyzes the formation of N10-formyl THF from THF and formate. Previously Fhs has been characterized from various non-pathogenic species of Clostridium. In addition, the detailed kinetic parameters are not known. In this report we have characterized the Fhs Clostridium perfringens and detailed kinetic parameters were determined. We have also shown the biological function by rescue of UV photorepair sensitive strain. ii. One-carbon metabolic pathway rewiring in Escherichia coli reveals an evolutionary advantage of 10-formyltetrahydrofolate synthetase (Fhs) in survival under hypoxia In cells, N10-formyltetrahydrofolate (N10-formyl THF) required for formylation of eubacterial/organeller initiator tRNA and purine biosynthesis is produced by methylene- tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) and/or 10-formyltetrahydrofolate synthetase (Fhs). folD is present in all organisms, where as fhs shows mixed distribution. We show that in E. coli, which naturally lacks fhs, essential function of folD could be replaced with fhs of Clostridium perfringens when provided on a medium copy plasmid or integrated as single copy gene in the chromosome of the ∆folD strains, for their growth in a complex medium. However, these strains require purines and glycine as supplements for growth in M9 minimal medium. The in vivo levels of N10-formyl THF in the ∆folD strains (harboring fhs) were limiting despite their high enzymatic capacity to synthesize the same. Auxotrophy for purines could be alleviated by adding formate to the medium, and that for glycine by engineering THF import into the cells. The ∆folD strains showed high NADP+/NADPH ratio and were hypersensitive to trimethoprim (TMP). Further, the presence of fhs was disadvantageous to E. coli under aerobic growth. However, under hypoxia, E. coli strains harboring fhs outcompeted those lacking it. And, the computational analysis revealed a predominant natural occurrence of fhs in anaerobic and facultative anaerobic bacteria. We also propose that inhibitors aimed at folD could potentiate the effect TMP drugs. iii. 5, 10-methylene-THF dehydrogenase (DH) and 5, 10-methenyl-THF cyclohydrolase (CH) activities of FolD are essential to maintain folate homeostasis and anti-folate resistance While E. coli and many other organisms have folD alone or folD and fhs, Clostridium species possess an annotated bi-functional FolD and an annotated methenyl tetrahydrofolate cyclohydrolase (FchA). Simultaneous presence of 3 enzymes for the synthesis of N10-formyl THF was intriguing. To understand this unusual feature we have cloned Clostridium perfringens CpeFolD and CpeFchA, over expressed and purified to near homogeneity. Biochemical analyses revealed that CpeFolD possess only dehydrogenase activity as opposed to in silico prediction, while CpeFchA possess cyclohydrolase activity as expected. We also show that expression of both proteins together allowed folD deletion in E. coli. From this study we found that presence of dehydrogenase and cyclohydrolase functions are very important in the maintenance of folate homeostasis and anti-folate resistance. iv. Analysis of distribution of fhs and purT genes in the organisms While analysing distribution of fhs across genomes, serendipitously we also found that large number of organism which have fhs lack purT(phosphoribosyl glycinamide formyl transferase II), in short where ever purT was present fhs was absent. This kind of anti-correlation was strictly conserved in Bacillus genes as well. Growth competition experiments were done to address anti-correlation between fhs and purT. Growth competition experiments revealed that simultaneous presence of both purT and fhs is disadvantageous, when compared to presence of either one gene.

Cardiovascular diseases are one of the leading causes of mortality. A common denominator across most of the cardiovascular diseases like diabetic cardiomyopathy, hypertrophic cardiomyopathy, myocardial infarction and dilated cardiomyopathy is the pathological remodelling of heart leading to fibrosis. Cardiac fibrosis is characterized by the excessive production and deposition of extracellular matrix components due to unwarranted proliferation of fibroblasts. Under normal conditions, following cardiac remodelling, my fibroblasts undergo programmed cell death. However, this does not happen under pathological conditions ultimately leading to fibrosis. Although the molecular events and signalling pathways that contribute to the development of cardiac fibrosis is well established, there are limited studies which try to understand the mechanisms by which fibroblasts persist and resist programmed cell death. Here we demonstrate that SIRT6, one of the members of sirtuin family of histone deacetylases, plays an important role in regulating my fibroblast cell death. When we analysed the mice hearts and isolated fibroblasts deficient in SIRT6, we observed increased expression of my fibroblast markers, suggesting that SIRT6 deficient hearts might have a high proportion of resident my fibroblasts. Also, when SIRT6 deficient fibroblasts were subjected to genotoxic stress, they showed reduced cell death and impaired mitochondrial to nuclear AIF translocation as compared to WT controls. An important regulator of AIF mediated cell death is the protein PARP-1. When we checked the expression levels of this protein under SIRT6 deficient conditions, it was found to be low. PARP-1 was also found to degrade faster under SIRT6 deficient conditions. Further qPCR analysis revealed that the transcript levels of PARP-1 were unaffected by SIRT6 suggesting that the regulation might not be at the transcriptional level. When we studied the acetylation of PARP-1 under SIRT6 deficient conditions we found the protein to be hypo-acetylated indicating a more complex mechanism of regulation.

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.

Tee PhD thesis presents the study of the response of Mycobacterium tuberculosis, the causative agent of tuberculosis, upon prolonged exposure to lethal concentrations of the first line anti-tuberculosis drug, rifampicin. The study shows that prolonged exposure to lethal concentration of rifampicin causes cell death initially by several log orders of cells, followed by a persistence phase, from where rifampicin resisters emerge carrying mutation in the RRDR locus of the rpoB gene. This phenomenon was found to occur even when the drug concentration is well above MBC levels. Luria-Delbruck experiment, in a modified format, showed that the resisters emerged as fresh mutants, and not due to the growth of pre-existing natural resisters to rifampicin. The per sister cells, which showed high levels of hydroxyl radical generation, were found to have thickened outer layer, unlike the mid-log phase cells, which restricts the permeability of a fluorescent-conjugate of rifampicin, 5-FAM-rifampicin, 10-fold less in per sister cells, as compared to mid-log phase cells. The thickened outer layer has high negative surface charge and is hydrophilic in nature. It is proposed that the hydrophilic-natured thickened outer layer might have restricted the permeability of lethal concentrations of hydrophobic-natured rifampicin. This, in turn, might have ensured the presence of sub-lethal concentration rifampicin inside the per sisters, which in turn might have generated the hydroxyl radical that caused mutagenesis to generate rifampicin resisters. The Chapter 1 is the Introduction to the thesis presented in 4 parts – Part 1.1, 1.2, 1.3 and 1.4, introducing briefly the history of antibiotics, antibiotic resistance, antibiotic persistence and a brief history of tuberculosis respectively. It is concluded with a rationale behind the present study. The Chapter 2 presents the entire Materials and Methods used in the experiments described in the thesis. The Chapter 3 presents the data on the response of M. tuberculosis to rifampicin upon extended exposure. Rifampicin exposure of susceptible M. tuberculosis H37Ra cells showed a decrease in their CFU/ml followed by a persistence phase wherein the CFU/ml remained constant. However, prolonged exposure of rifampicin even at higher concentrations showed regrowth in the culture, which was found to be due to the emergence of rifampicin-resistant bacteria. Screening of rifampicin-resistant mutants showed point mutations in the rifampicin resistance determining region (RRDR) of the rpoB gene in all the mutants. In parallel, using trans formants of M. tuberculosis expressing unstable GFP under the respective native ribosomal RNA promoter, the metabolic status of the per sister cells was determined. When actively growing highly fluorescing cells were exposed to lethal concentration of rifampicin, their metabolism diminished, as illustrated by the decrease in their fluorescence during persistence phase, followed by the emergence of a sub-population of bacteria which were again metabolically active. In order to verify whether the rifampicin resisters are freshly formed mutants or have come from the naturally existing resisters, Luria-Delbruck fluctuation test was performed in a modified manner. The number of rifampicin resisters that emerged from the persistent phase was found to vary amongst different cultures from different days and different times of exposure, showing fluctuation. However, the addition of theorem before persistence phase almost abolished the generation of the rifampicin-resistant bacilli, indicating the role of hydroxyl radical in the emergence of rifampicin resisters. The generation of hydroxyl radical in mycobacteria exposed to rifampicin was confirmed using electron para-magnetic resonance spectrometry (EPR), with the spin trap agent, 5,5- Dimethyl-1-pyrroline N-oxide (DMPO) specific for hydroxyl radical. An increase in the formation DMPO-OH adduct in the persistence phase cells was observed, in comparison to mid-log phase cells. Exposure to theorem significantly diminished the adduct formation. The persistence phase cells also showed significantly high levels of signal specific to the hydroxyl-specific fluorescent dye, hydroxyphenyl fluorescein (HPF), as compared to the mid-log phase cells. In addition we have determined the oxidative stress in the bacilli upon rifampicin exposure using a redox biosensor (Mrx1-roGFP) which also showed high oxidative stress in the persistent phase. These observations confirmed the presence of high levels of oxidative stress and hydroxyl radical in the rifampicin persistent cells, in comparison with mid-log phase cells. Whole genome sequencing of the four independently isolated rifampicin resistant M. tuberculosis showed genome wide mutations having less common mutations with respect to the wild type genome, indicative of the occurrence of random mutagenesis. In addition mutation frequencies were comparable between the samples with respect to the wild type sample. About 69% of the mutations were A-C or T-G, followed by A-T or T-A, which is known to be due to oxidative stress in the cells. Variations in the colony morphology were also observed on the persistent phase cells, indicating the occurrence of mutagenesis in the bacterial genome during rifampicin treatment. The Chapter 4 is on the morphological and ultrastructural studies on rifampicin-exposed M. tuberculosis cells. Transmission electron micrographs of rifampicin per sisters showed significant thickening of the outermost capsular layer (OL), as compared to the mid-log phase cells. This observation was verified by staining the cells with ruthenium red, which specifically stains anionic polysaccharide of the OL. Zeta potential (ZP) measurement of the surface charge of the persistent cells showed high negative ZP, as compared to the mid-log phase cells. The negative ZP value was found to gradually increase during the course of rifampicin treatment and to decrease during the regrowth phase. Hexadecane assay showed larger proportion of per sister cells being retained in the aqueous phase, as compared to the mid-log phase cells. This indicated the higher hydrophilicity of the per sister cells, which was in agreement with the higher surface negative charge of the cells. The permeability of rifampicin per sister cells to 5-FAM-rifampicin (rifampicin conjugated to 5-carboxy fluorescein, which is as hydrophobic as rifampicin) was found to be 10-fold less than that of the mid-log phase cells. However, removal of the thick OL by bead beating (BB) with 4 mm glass beads significantly improved the permeability of per sister cells to 5-FAM-rifampicin. On the contrary, no difference in the 5-FAM-rifampicin uptake was observed in mid-log phase cells, with or without BB. These observations implied that the thick hydrophilic-natured OL with high negative surface charge may be playing a significant role in limiting the permeability of hydrophobic-natured rifampicin entry into the persisted cells. This in turn may ensure the presence of sub-lethal concentration of rifampicin inside the persisted cells that are exposed to lethal concentration of the antibiotic. Exposure of bacteria to sub-lethal concentration of antibiotics has been reported to generate oxidative stress in the bacteria, leading to mutagenesis. A model has been proposed based on these observations in which the persistent mycobacteria are protected from lethal concentrations of the rifampicin by the thick OL which in turn ensures sub-lethal intracellular antibiotic concentration, leading to the generation of hydroxyl radical mediated mutagenesis and thereby emergence of rifampicin resisters. This thesis is concluded with the list of salient findings, publications and references.

Hepatitis C virus (HCV), a blood-borne pathogen, is a small enveloped RNA virus belonging to the Hepacivirus genus of the Flaviviridae family. HCV infection represents one of the major health concerns affecting approximately 170 million people globally. Patients with chronic HCV infection are at risk of developing hepatic fibrosis, cirrhosis and hepatocellular carcinoma. No protective anti-HCV vaccine is available yet. Until recently, standard therapy based on pegylated interferon plus ribavirin, was inadequate in treating all the patients as it results in a sustained virological response in only 40 to 50 percent of patients infected with the most common genotype (gt 1). Advances in understanding host-HCV interactions have helped developing newer anti-HCV agents such as telaprevir and boceprevir. However, treatment success is still limited due to different factors including genotype specificity, high cost, potential drug-drug interactions, substantial side effects etc. The positive-sense single-stranded RNA genome of HCV is approximately 9.6kb long which is flanked by highly structured and conserved 5’ and 3’ untranslated regions (UTRs) at both ends. Unlike cap-dependent translation of host cell mRNAs, HCV translation is mediated by an internal ribosomal entry site (IRES) present majorly within the 5’UTR. Several reports have demonstrated the interaction of different cellular proteins with HCV-5’UTR and/or 3’UTR, which include human La protein, polypyrimidine tract binding protein (PTB), poly (rC)-binding protein 2 (PCBP2) etc. These interactions of trans-acting factors with the UTRs may be important for HCV translation and/or replication. Earlier study from our laboratory revealed the importance of interaction of human La protein, by its central RNA recognition motif (RRM), with the HCV IRES around a tetranucleotide sequence GCAC near initiator AUG in influencing HCV translation. However, the role of this interaction, if any, in HCV RNA replication was not known. In the first part of the thesis, we characterized the interaction between human La protein and the GCAC to understand its role in HCV replication. We incorporated mutation, which altered the binding of La, in the GCAC motif in HCV monocistronic replicon and checked HCV RNA replication by reverse transcriptase polymerase chain reaction (RT-PCR). The mutation drastically inhibited HCV replication. Interestingly, overexpression of La could reverse the effect of this mutation and significantly enhanced HCV RNA levels. Using a bicistronic replicon, we observed that decrease in replication was independent of translation inhibition. Furthermore, mutation at the GCAC motif reduced the association between La and viral polymerase, NS5B as seen in co-immunoprecipitation assays. Moreover, this mutation affected translation to replication switch regulated by the interplay between HCV-NS3 protease and human La protein. Our analyses of point mutations, based on RT-PCR and luciferase assays, revealed distinct roles of each nucleotide of the GCAC motif in HCV replication and translation. Finally, 5’-3’ crosslink assays revealed that specific interaction of the GCAC motif with human La protein is important for linking 5’ and 3’ends of HCV genome. Results clearly demonstrate the mechanism of regulation of HCV replication by interaction of cis-acting element GCAC within the HCV IRES with human La protein. HCV is highly species-specific. Under natural conditions, HCV infects only humans and chimpanzees. This restricted host-tropism has prevented the development of a small animal model to study HCV infection in vivo. Although several human-specific entry factors have been identified to be responsible for this species selectivity, full multiplication of the HCV in animals (other than humans and chimpanzees) is still not possible. In the second part of the thesis, we showed that a post-entry host factor –‘La protein’ may also contribute in determining HCV host tropism. We aligned La protein sequences from different species and interestingly we found that HCV RNA interacting beta-turn sequence (KYKETDL) in central RRM (residues 112-184) is conserved only in human and chimpanzee. Earlier, it was shown from our laboratory that a heptameric peptide comprising of this sequence (derived from human La) could inhibit HCV translation by competing with La interaction with the IRES element. However, in the current study, another peptide corresponding to the mouse La sequence (KYKDTNL) was unable to inhibit HCV RNA translation. Similarly, wild-type mouse La (mLa) failed to stimulate HCV IRES function, but addition of chimeric mouse La protein bearing human beta-turn sequence (mLahN7) significantly increased HCV IRES mediated translation in vitro. Also, exogenous supplementation of mLahN7 enhanced HCV translation in cell culture system. Moreover, quantitative as well as tagged RT-PCR analyses showed an enhanced HCV replication upon overexpression of mLahN7. The findings obtained in this part raise a possibility of creating HCV mouse model using human specific cellular entry factors and a humanized form of La protein. Hepatitis C has emerged as a major challenge to the medical community. Developing more potent and safe anti-HCV regimens is need of the hour. As described above, a linear hepatapeptide (KYKETDL) was synthesized and shown to reduce HCV translation. However, this linear peptide was stable only for a shorter time scale. Therefore, in the third part of the thesis, effect of a more stable cyclic form of this peptide has been described. NMR spectroscopy suggested that the beta turn conformation is preserved in cyclic peptide as well. Also, using in vitro bicistronic reporter assay, we demonstrated that cyclic peptide inhibits HCV translation in a dose dependent manner. In fact, due to its higher stability, cyclic peptide reduced HCV translation and replication more efficiently than the corresponding linear peptide at longer post-treatment time point. Additionally, we observed that cyclic peptide is non-toxic in cell culture system. Our results suggest that cyclic peptide might emerge as a promising lead compound against hepatitis C. Due to availability of only partially effective liver protective drugs in modem medicine, complementary and alternative medicine approach, based on plant derived compounds, is also being utilised against HCV. Plant derived compounds have advantages of having high chemical diversity, drug-likeliness properties and ability of being metabolized by the body with little or no toxicity than synthetic ones. Different studies have shown that phytochemicals may exert anti-HCV activities by acting as direct-acting antivirals and play a potential therapeutic role in treating HCV infection. Also, from our laboratory, it was shown that methanolic extract of Phyllanthus amarus (P. amarus) plant inhibited HCV replication. The fourth part of the thesis describes the study on the anti-HCV properties of several bioactive components from P. amarus extract. Using a fluorimetric assay, we demonstrated that two principal components of this extract, phyllanthin and corilagin reduced the HCV NS3 protease activity significantly in vitro. We also observed a sharp reduction in HCV negative sense RNA levels in cell culture system. Structural knowledge-based molecular docking studies showed interactions of phyllanthin and corilagin with the amino acid residues of the catalytic triad of NS3 protease. Further, these compounds were found to be non-toxic in cell culture. Also, phyllanthin and corilagin displayed antioxidant properties by blocking HCV induced oxidative stress generated by reactive oxygen species suggesting their hepatoprotective nature. More importantly, our in vivo toxicity analyses and pharmacokinetics studies proved their safety, tolerability, metabolic stability, and systemic oral bioavailability and support their potential as novel anti-HCV therapeutic candidates. Altogether, the study deciphers mechanistic details of translation and replication of HCV RNA and demonstrates novel antiviral agents targeting these important viral processes.

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.

Gene deletions are a powerful method to uncover the cellular functions of a given gene in living systems. A limitation to this methodology is that it is not applicable to essential genes. Even for non-essential genes, gene knockouts cause complete absence of gene product thereby limiting genetic analysis of the biological pathway. Alternatives to gene deletions are mutants that are conditional, for e.g, temperature sensitive (ts) mutants are robust tools to understand temporal and spatial functions of genes. By definition, products of such mutants have near normal activity at a lower temperature or near-optimal growth temperature which is called as the permissive temperature and reduced activity at a higher, non-optimal temperature called as the non-permissive temperature. Generation of ts alleles in genes of interest is often time consuming as it requires screening a large population of mutants to identify those that are conditional. Often many essential proteins do not yield ts such alleles even after saturation mutagenesis and extensive screening (Harris et al., 1992; Varadarajan et al., 1996). The limited availability of such mutants in many essential genes prompted us to adopt a biophysical approach to design temperature-sensitive missense mutants in an essential gene of fission yeast. Several studies report that mutations in buried or solvent-inaccessible amino acids cause extensive changes in the thermal stability of proteins and specific substitutions create temperature-sensitive mutants (Rennell et al., 1991; Sandberg et al., 1995). We used the above approach to generate conditional mutants in the fission yeast gene spprp18+encoding an essential predicted second splicing factor based on its homology with human and S. cerevisiae proteins. We have used a missense mutant coupled with a conditional expression system to elucidate the cellular functions of spprp18+. Further, we have employed the same biophysical principle to generate a missense mutant in spago1+ RNA silencing factor that is non-essential for viability but has critical functions in the RNAi pathway of fission yeast. Fission yeast pre-mRNA splicing: cellular functions for the protein factor SpPrp18 Pre-mRNA splicing is an evolutionarily conserved process that excises introns from nascent transcripts. Splicing reactions are catalyzed by the large ribonuclear protein machinery called the spliceosome and occur by two invariant trans-esterification reactions (reviewed in Ruby and Abelson, 1991; Moore et al., 1993). The RNA-RNA, RNA–protein and protein-protein interactions in an assembly of such a large protein complex are numerous and highly dynamic in nature. These interactions in in vitro splicing reactions show ordered recruitment of essential small nuclear ribonucleic particles snRNPs and non–snRNP components on pre-mRNA cis-elements. Further these trans acting factors recognize and poise the catalytic sites in proximity to identify and excise introns. The precision of the process is remarkable given the diversity in architecture for exons and introns in eukaryotic genes (reviewed in Burge et al., 1999; Will and Luhrmann, 2006). Many spliceosomal protein components are conserved across various organisms, yet introns have diverse features with large variations in primary sequence. We hypothesize that co-evolution of splicing factor functions occurs with changes in gene and intron architectures and argue for alternative spliceosomal interactions for spliceosomal proteins that thus enabling splicing of the divergent introns. In vitro biochemical and genetic studies in S. cerevisiae and biochemical studies with human cell lines have indicated that ScPRP18 and its human homolog hPRP18 function during the second catalytic reaction. In S. cerevisiae, ScPrp18 is non-essential for viability at growth temperatures <30°C (Vijayraghavan et al., 1989; Vijayraghavan and Abelson, 1990; Horowitz and Abelson, 1993b). The concerted action of ScSlu7 - ScPrp18 heteromeric complex is essential for proper 3’ss definition during the second catalytic reaction (Zhang and Schwer, 1997; James et al., 2002). These in vitro studies also hinted at a possible intron -specific requirement for ScPrp18 and ScSlu7 factors as they were dispensable for splicing of intron variants made in modified ACT1 intron containing transcripts (Brys and Schwer, 1996; Zhang and Schwer, 1997). A short spacing distance between branch point adenosine to 3’splice site rendered the substrate independent of Prp18 and Slu7 for the second step (Brys and Schwer, 1996; Zhang and Schwer, 1997). Extensive mutational analyses of budding yeast ScPrp18 identified two functional domains and suggested separate roles during splicing (Bacikova and Horowitz, 2002; James et al., 2002). Fission yeast with its genome harboring multiple introns and degenerate splice signals has recently emerged as a unique model to study relationships between splicing factors and their role in genomes with short introns. Previously, studies in our lab had initiated genetic and mutational analysis of S. pombe Prp18, the predicted homolog of budding yeast Prp18. Genetic analysis showed its essentiality, but a set of missense mutants based on studies of budding yeast ScPrp18 (Bacikova and Horowitz, 2002) gave either inactive null or entirely wild type phenotype for the fission yeast protein. In this study, we have extended our previous mutational analysis of fission yeast Prp18 by adopting biophysical and computational approaches to generate temperature-sensitive mutants. A missense mutant was used to understand the splicing functions and interactions of SpPrp18 and the findings are summarized below. Fission yeast SpPrp18 is an essential splicing factor with transcript-specific functions and links efficient splicing with cell cycle progression We initiated our analysis of SpPrp18 by adopting a biophysical approach to generate ts mutants. We used the PREDBUR algorithm to predict a set of buried residues, which when mutated could result in a temperature-sensitive phenotype that complements the null allele at permissive temperature. These predictions are based upon two biophysical properties of amino acids: 1) Hydrophobicity, which is calculated in a window of seven amino acids 2) Hydrophobic moment, which is calculated in a sliding window of nine amino acids in a given protein sequence. Several studies correlate these properties to protein stability and function (Varadarajan et al., 1996). One of the buried residue mutants V194R, in helix 1 of SpPrp18 conferred weak temperature- sensitivity and strong cold-sensitivity even when the protein was over expressed from a plasmid. Through semi-quantitative RT-PCR we showed splicing-defects for tfIId+ intron1 in these cells even when grown at permissive temperature. The primary phenotype was the accumulation of pre-mRNA. Further, we showed this splicing arrest is co-related with reduced levels of SpPrp18 protein, linking protein stability and splicing function. Next we examined the effects of this mutation on function by further reduction of protein levels. This was done by integrating the expression cassette nmt81:spprp18+/spprp18V194R at the leu1 chromosomal locus and by metabolic depletion of the integrated allele. Through RT-PCRs we demonstrated that depletion of wild type or missense protein has intron specific splicing defects. These findings showed its non-global and possibly substrate-specific splicing function. In the affected introns, precursor accumulation is the major phenotype, confirming prior data from our lab that hinted at its likely early splicing role. This contrasts with the second step splicing role of the human or budding yeast Prp18 proteins. Previous data from our lab showed loss of physical interaction between SpPrp18 and SpSlu7 by co-immunoprecipitation studies. This again differs from the strong and functionally important ScPrp18 and ScSlu7 interaction seen in budding yeast. We show the absence of charged residues in SpSlu7 interaction region formed by SpPrp18 helix1 and helix2 which can explain the altered associations for SpPrp18 in fission yeast. Importantly, as the V194R mutation in helix 1 shows splicing defects even at permissive temperature, the data indicate a critical role for helix 1 for splicing interactions, possibly one that bridges or stabilizes the proposed weak association of SpPrp18-SpSlu7 with a yet unknown splicing factor. We also investigated the effects of mutations in other helices; surprisingly we recovered only mutations with very subtle growth phenotypes and very mild splicing defects. Not surprisingly, stop codon at L239 residue predicted to form a truncated protein lacking helices 3, 4 and 5 conferred recessive but null phenotype implicating essential functions for other helices. Other amino acid substitutions at L239 position had near wild type phenotype at 30°C and 37°C. Helix 3 buried residue mutant I259A conferred strong cold-sensitivity when over expressed from plasmid, but semi quantitative analysis indicated no splicing defects for intron1 in the constitutively expressed transcript tfIId+. These findings indicate cold sensitivity either arises due to compromised splicing of yet unknown transcripts or that over-expressed protein has near wild type activity. We find mutations in the helix 5 buried residues L324 also conferred near WT phenotype. Earlier studies in the lab found that substitution of surface residues KR that are in helix 5 with alanine lead to null phenotypes (Piyush Khandelia and Usha Vijayraghavan unpublished data). We report stable expression of all of these mutant proteins; L239A, L239P, L239G, I259A, I259V, L324F, L324A as determined by our immunoblot analysis at 30°C and 37°C. The mild phenotypes of many buried residues can be attributed to orientation of their functional groups into a protein cavity between the helices. Lastly, our microscopic cellular and biochemical analysis of cellular phenotypes of spprp18 mutant provided a novel and direct role of this factor in G1-S transition of cell cycle. Our RT-PCR data suggest spprp18+ is required for efficient splicing of several intron containing transcripts involved in G1-S transition and subsequent activation of MBF complex (MluI cell cycle box-binding factor complex) during S-phase and shows a mechanistic link between cell cycle progression and splicing. A tool to study links between RNA interference, centromeric non-coding RNA transcription and heterochromatin formation S.pombe possesses fully functional RNA interference machinery with a single copy for essential RNAi genes ago1+, dcr1+ and rdp1+. Deletion of any of these genes causes loss of heterochromatinzation with abnormal cytokinesis, cell-cycle deregulation and mating defects (Volpe et al., 2002). In S.pombe, exogenous or endogenously generated dsRNA’s from transcription of centromeric repeats are processed by the RNaseIII enzyme dicer to form siRNA. These siRNA’s are loaded in Ago1 to form minimal RNA induced silencing complex (RISC) complex or specialized transcription machinery complex RNA induced transcriptional silencing (RITS) complex and target chromatin or complementary mRNAs for silencing. Thus as in other eukaryotes, fission yeast cells deploy RNAi mediated silencing machinery to regulate gene-expression and influence chromatin status. Several recent studies point to emerging new roles of RNAi and its association with other RNA processes (Woolcock et al., 2011; Bayane et al., 2008; Kallgren et al., 2014). Many recent reports suggest physical interactions of RISC or RITS and RNA dependent RNA polymerase complex (RDRC) with either some factors of the spliceosomal machinery, heterochromatin machinery (CLRC complex) and the exosome mediated RNA degradation machinery (Bayne et al., 2008 and Chinen et al., 2010 ; Hiriart et al., 2012; Buhler et al., 2008; Bayne et al., 2010 ). Thus we presume conditional alleles in spago1+ will facilitate future studies to probe the genetic network between these complexes as most analyses thus far rely on ago1∆ allele or have been based on proteomic pull down analyses of RISC or RITS complexes. In this study, we employed biophysical and modeling approaches described earlier to generate temperature sensitive mutants in spago1+ and spdcr1+. We tested several mutants for their ability to repress two reporter genes in a conditional manner. Our modeling studies on SpAgo1 PAZ domain indicated structural similarities with human Ago1 PAZ domain. We created site-directed missense mutants at predicted buried residues or in catalytic residues. We also analyzed the effects of random amino acid replacements in specific predicted buried or catalytic residues of SpAgoI. These ago1 mutants were screened as pools for their effects on silencing of GFP or of ura4+ reporter genes. These assays assessed post transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) activity of these mutants. We obtained three temperature sensitive SpAgo1 mutants V324G, V324S and L215V while the V324E replacement was a null allele. Based upon our modeling, a likely explanation for the phenotype of these mutants is structural distortion or mis-orientation of the functional groups caused due to these mutations, which affect activity in a temperature dependent manner. This distortion in the PAZ domain may affect binding of siRNA and thereby lead to heterochromatin formation defects that we observed. Our data on the SpAgo1 V324 mutant shows conditional centromeric heterochromatin formation confirmed by semi quantitative RT-PCR for dh transcripts levels that shows temperature dependent increase in these transcripts. We find reduced H3K9Me2 levels at dh locus by chromatin immunoprecipitation (ChIP) assay, linking the association of siRNAs for establishment of heterochromatin at this loci. The data on PTGS of GFP transcripts show SpAgo1 V324G mutation has decreased slicing activity as semi-quantitative RT-PCR for GFP transcripts show increased levels at non permissive temperature. These studies point out the importance of siRNA binding to the PAZ domain and its effect on slicing activity of SpAgo1. The mutations in Y292 showed residue loss of centromeric heterochromatin formation phenotype. Thus, we ascribe critical siRNA binding and 3’ end recognition functions to this residue of SpAgo1. These studies point out functional and structural conservation across hAgo1 and SpAgo1. Adopting the aforementioned biophysical mutational approach, we generated mutants in spdcr1+ and screened for those with conditional activity. Our modeling studies on SpDcr1 helicase domain shows it adopts the conserved helicase domain structure seen for other DEAD Box helicases. Our data on mutational analysis of a conserved buried residue I143 in the walker motif B created inactive protein. The data confirm critical functions for dicer in generation of siRNAs and also in recognition of dsRNA ends. Mutants in buried residues L1130 and I1228 of RNase IIIb domain were inactive and the proximity of these residues to the catalytic core suggest that the critical structural alignment of catalytic residues is indispensable for carrying out dsRNA cleavage to generate siRNAs. We also attribute critical catalytic functions to SpDcr1 D1185 residue for generation of siRNA and heterochromatin formation as measured by our transcriptional gene silencing assay. Our studies employing biophysical and computational approaches to design temperature-sensitive mutants have been successfully applied to an essential splicing factor SpPrp18, which was refractory for ts mutants by other methods. Using a missense mutant, we showed its intron-specific splicing function for subsets of transcripts and deduced that its ubiquitous splicing role is arguable. We have uncovered a link between the splicing substrates of SpPrp18 and direct evidence of splicing based cell cycle regulation, thus providing a mechanistic link to the cell cycle arrest seen in some splicing factor mutants. The same methodology was applied to another important biological pathway, the RNAi machinery, where central factors SpAgoI and SpDcrI were examined We report the first instance of conditional gene silencing tool by designing Ago1 ts mutants which will be useful for future studies of the global interaction network between RNAi and other RNA processing events.

Phenotypic heterogeneity in terms of cell size, morphology, and metabolic status, which are believed to help the population survive under stress conditions, is known in mycobacterial populations. Such population heterogeneity had been observed in in vitro cultures, TB patients, and in animal models. Our laboratory had earlier shown that about 20-30% of the 15% septating cells of Mycobacterium smegmatis, Mycobacterium tuberculosis and Mycobacterium xenopi mid-log phase cultures divide by highly deviated asymmetric cell division (ACD), generating subpopulations of short cells and normal-sized/long cells. The remaining 70-80% of the septating cells divide by symmetric cell division (SCD) with 5-10% deviation of the division site constriction from the median. The proportion of short cells amounted to about 3-5% of the total population, while the remaining 97-98% of the population was constituted by normal-sized/long cells. This proportion of short cells has been found to be consistent and reproducible irrespective of culture media. Comparable proportion of short cells of tubercle bacilli has been found in the freshly diagnosed pulmonary tuberculosis patients’ sputum also. It indicated that such processes must be occurring in the tubercle bacillary population in the TB patients too and that the presence of short cells has some physiological relevance. Thus, ACD has been found to be one of the mechanisms that mycobacteria use to generate cell size heterogeneity in the population. However, there has not been any study on the mechanism that generate such subpopulation of short cells through ACD. Therefore, in the present study, we investigated the mechanism behind the ACD that generates short cells in the Mycobacterium smegmatis and Mycobacterium tuberculosis populations. The Chapter 1, which forms the Introduction to the thesis, gives an extensive literature survey on all the different areas of research in bacterial physiology that are linked by the present study. These areas of research include bacterial cell division process per se, the proteins involved in bacterial cell division, cell division in general in mycobacteria, different modes of cell division in mycobacteria, generation of cell size heterogeneity through symmetric and asymmetric cell division in mycobacteria, cell-cell communication in bacterial systems, and the characteristics and physiological significance of diadenosine polyphosphates from bacteria to humans. The complete account of the research in these areas that are linked in the present study thus justifies the introduction to the results that are given in the ensuing chapters. The Chapter 2 forms the Materials and Methods used in the present study. Here a detailed description of the methods used for the cell division bioassay used to score for the proportion of cells undergoing symmetric and asymmetric divisions, the biochemical assays performed to find out the biochemical identity of the molecule, the isolation and fractionation methods for the ACD-IM from the concentrated culture supernatants (CS), and finally the mass spectrometric methods used for the elucidation of the structure of the molecule are given The Chapter 3 forms the first data chapter that presents results on the detection of the presence of the asymmetric cell division inducing molecule (ACD-IM) from the concentrated culture supernatant (CS) of Mycobacterium smegmatis and Mycobacterium tuberculosis, as inducing ACD in higher proportions of cells. Further, it shows that the levels of ACD-IM increase at late growth phases of 0.8 and 1.0 OD600 nm. The chapter is concluded with a discussion of the results. The Chapter 4 describes the biochemical analyses of the CS to find out the chemical nature of the ACD-IM. DNase I, snake venom phosphodiesterase (SVP), RNase A, lipase, and proteinase K were used to find whether exposure of CS of M. smegmatis and M. tuberculosis cells to these enzymes could abolish the ACD inducing activity of the molecule in the CS on the respective cells. These experiments showed that the ACD-IM was susceptible to DNase I and SVP, but not to RNase A, lipase, or proteinase K. However, proteinase K showed direct effect on the cells by decreasing the proportion of cells dividing by ACD, indicating the probable presence of the receptor to the molecule on the external cell surface. The data also shows the conservation of the molecule in M. smegmatis and M. tuberculosis by demonstrating that the CS of M. smegmatis could induce ACD in higher proportions of M. tuberculosis cells and vice versa. The structural identification of ACD-IM present in the concentrated CS of M. smegmatis and M. tuberculosis using LC-ESI-MS and MS-MS analyses as diadenosine hexaphosphate (Ap6A). The structure of the natural Ap6A molecule was confirmed using synthetic Ap6A molecule subjected to LC-ESI-MS and MS-MS analyses. Further, the ACD inducing activity of the synthetic Ap6A molecule, its susceptibility to DNase I and SVP, but not to RNase A, lipase and proteinase K were verified to establish that the structural, biochemical, and functional properties of the naturally occurring Ap6A in the CS of M. smegmatis and M. tuberculosis were identical to those of synthetic Ap6A. The chapter is concluded with a discussion of the results. The Chapter 5 presents the data on the genes involved in the degradation and synthesis of Ap6A in M. smegmatis and M. tuberculosis. The knockout of MSMEG_2936 gene of M. smegmatis resulted with significant increase in the ACD proportion. The quantitation of Ap6A in the CS obtained from this strain found significantly higher concentration than wild type. These results showed that MSMEG_2936 protein might catalyse the degradation of Ap6A in mycobacteria. The knockout of MSMEG_2932 gene of M. smegmatis resulted with significant reduction in the ACD proportion. The quantitation of Ap6A in the CS obtained from this strain found comparable to wild type. These results showed that MSMEG_2932 protein might not involve in the synthesis of Ap6A in mycobacteria. The chapter is concluded with a discussion of the results. Thus, the present study, for the first time, establishes the identity, structure, and function of Ap6A as the molecule that induces asymmetric cell division in mycobacteria.

The process of translation initiation is the most highly regulated step of protein synthesis. In bacteria, three initiation factors (IF1, IF2 and IF3) play crucial roles during initiation. IF3 acts as an anti-association factor for the two ribosomal subunits. Eubacterial IF3 also permits initiator tRNA (i-tRNA) selection at the P site of the ribosome. Two features of i-tRNA, i. e. the characteristic 3GC base pairs in the anticodon stem and the cognate interaction of the anticodon sequence with the initiation codon of the mRNA contribute to IF3 based selection and/or proofreading. However, the exact mechanism of this discrimination and the contribution of the individual domains towards this process of selection/ proofreading are unclear. Further, there are exceptional instances in the natural world where either the codon-anticodon interaction or the anticodon stem composition deviates from the norm. For instance, in mammalian mitochondria, non-AUG codons such as AUU and AUA are present in the genome although they are notoriously poor initiation codons. In addition, some species of Mycoplasma have i-tRNAs with variations in the typically conserved 3GC base pairs of the anticodon stem. In this study, we have investigated the mechanism of proofreading activity of IF3 of E. coli, mitochondrial and mycoplasmal origins. Part I: Proofreading function of IF3 in E. coli IF3 is composed of N and C terminal domains joined by a flexible linker region. By means of complete and partial IF3 knockouts, we show that the C-terminal domain (CTD) is essential for the survival of E. coli while the N-terminal (NTD) is required for cellular fitness. Using reporter assays, we have established the role of the NTD in proofreading, while polysome profile analyses reaffirm that the CTD alone can bind to the 30S and carry out ribosome anti-association. Therefore, we show that the CTD is the ribosome binding and anti-association domain, while the NTD is the major proofreading domain. Unpublished cryoEM structures from Prof. Ramakrishnan’s lab indicate that the NTD of IF3 pushes the i-tRNA at its elbow and helps in P site accommodation of the i-tRNA. We propose that when the codon-anticodon interaction is non-cognate or if the 3GC base pairs of the anticodon stem are not intact, then the dynamic action of the NTD destabilises the tRNA at the P site and leads to its rejection. Part II: Proofreading function of mitochondrial IF3 (IF3mt) Of the 13 protein-coding genes in mammalian mitochondria, 3 utilise the non-canonical AUA codon and one utilises the non-canonical start codon AUU. Since IF3mt does not possess many of the generally conserved residues implicated in proofreading, we decided to characterise the proofreading function of IF3mt and its role in initiation with non-canonical start codons. Structurally, IF3mt is similar to EcoIF3 with its N and C terminal domains joined by a linker region. However, IF3mt additionally possesses N- and C-terminal extensions which are generally disordered in structure. In vivo studies of mitochondrial translation factors have been mired by the lack of methodologies to manipulate mitochondria. We have developed an E. coli strain to study the proofreading functions of mitochondrial IF3 (IF3mt) with the help of reporter genes. Consistent with its function in mitochondria, IF3mt allowed promiscuous initiation from non-AUG codons. However, IF3mt avoided initiation with i-tRNAs lacking evolutionarily conserved 3GC pairs in anticodon stems. Interestingly, expression of IF3mt N-terminal domain or IF3mt devoid of its typical N-, and C-terminal extensions significantly improved its proofreading activity. Our immunoblot assays from polysome profile fractions indicate that the IF3mt derivative lacking extensions is capable of superior 30S ribosome binding. The two derivatives of IF3mt missing the Next (IF3mtΔNext) or both the Next and Cext (IF3mtΔNextCext) display an affinity for the 50S ribosome. We propose that the extensions of IF3mt may have evolved to reduce the affinity of IF3mt to the ribosome and thereby permit initiation with non-canonical start codons like AUU and AUA. Our studies suggest that E. coli provides an excellent heterologous model to study distinctive features of mitochondrial factors. Part III: Fidelity of translation initiation in mycoplasma One of the many singular features of mycoplasma is the presence of many anticodon stem variants of the i-tRNA across different species. In general, i-tRNAs are characterized by the presence of the typical feature of the conserved 3 consecutive GC base pairs (GC/GC/GC) in the anticodon stem. However, many mycoplasmal species have i-tRNAs with AU/GC/GC, GC/GC/GU or AU/GC/GU sequences. Interestingly, the mycoplasmal species which harbour the AU/GC/GU i-tRNA are also human pathogens. Therefore, we decided to investigate whether these organisms possess any unique features to accommodate the i-tRNA variants, by investigating the usage of Shine Dalgarno sequences and by carrying out multiple sequence alignments of genes encoding initiation factors, ribosomal proteins S9 and S13 and 16S rRNA. Since IF3 plays a crucial role in i-tRNA selection, we carried out computational analysis of mycoplasmal IF3 sequences, which revealed many interesting features. Most striking amongst them was the variation of the highly conserved R at position 131 in some species. Interestingly, these were the very mycoplasmal species which possessed the anticodon stem variant AU/GC/GU, suggesting a strong correlation between these two features. It is known that the R131P mutation of EcoIF3 is characterised by an enormous loss of proofreading activity. It seemed unusual that such compromised proofreading would be tolerated in the cell, so we decided to investigate other components of the translational machinery as well. The C-terminal SKR tail of the ribosomal protein S9, which contacts the P-site tRNA, is highly conserved across bacteria. Analysis of the C-terminal sequences of S9 proteins in various mycoplasmal species revealed a surprising variation- the presence of a TKR tail in strains with the AU/GC/GU tRNA. In this study we have investigated the co-occurrence of S9 and IF3 variations in i-tRNA selection in E. coli. We see that the R131P polymorphism of IF3 leads to a tremendous loss of proofreading, but this loss is significantly tempered by the presence of the S9 TKR variation. Our bioinformatics studies revealed that the mycoplasmal species which are sustained on AU/GC/GU i-tRNAs also tend to use a higher percentage of non-AUG codons. By means of our reporter assays in E. coli, we have shown once again that the R131P polymorphism of IF3 leads to a tremendous increase in initiation with the non-canonical start codon AUA, but this increase is significantly tempered by the presence of the S9 TKR variation.