Rozprawy doktorskie na temat „Mycobacterial Stress Response”

The success of M. tuberculosis lies in its ability to stay alive and persist in a potentially hostile environment represented by the macrophage phagosome. Hence there is a desperate need to identify the molecular mechanisms and associated proteins enabling mycobacterial survival and replication inside macrophages. Recent studies have shown that several mycobacterial proteins may play distinct roles during different stages of infection. This thesis was focused on investigation of the biological function of two mycobacterial proteins, Rv1219c and Rv3136 (PPE51 protein) during macrophage infection and stress response. A recent study has established that RaaS (Rv1219c in M. tuberculosis and BCG_1279c in M. bovis BCG) mediates mycobacterial survival in stationary phase and during mouse infection. RaaS (for regulator of antimicrobial-assisted survival) controls expression of ATP-dependent efflux pumps Bcg_1278c/1277c and DrrC and mediates survival of M. bovis BCG in growth non-permissive conditions. One of the aims of this project was investigating the role of RaaS in mycobacterial replication and persistence in macrophages. The result showed that ΔraaS mutant of M. bovis BCG was significantly impaired in initial survival in macrophages. Moreover, the mutant was extremely sensitive to H2O2 and low pH, the stress factors, which probably influence mycobacterial viability upon their uptake into macrophages. Treatment with reserpine, an inhibitor of ATP-dependent efflux pumps, prior to stress exposure had managed to improve the survival of the mutant suggesting that the impaired stress response of the raaS mutant was due to dysregulation of efflux pumps. While BCG_1279c and its orthologous in M. tuberculosis, Rv1219c, are important for survival inside macrophages and in stationary phase, published data has shown that Rv3136 is up-regulated during mycobacterial replication in macrophages. To establish the role of Rv3136 in macrophage infection and virulence, an rv3136 deletion mutant was generated in M. tuberculosis H37Rv. The mutant did not show any growth defect in laboratory medium. Although macrophage infection experiments did not link rv3136 to survival in macrophages, preliminary data from mouse infection indicated that Rv3136 could potentially play a role in survival inside the host.

Mycobacterium is one of the oldest known bacterial genera and its species are commonly associated with human and animal disease, although most are free-living saprophytes which form part of a balanced microbial community in natural habitats such as water and soil Mycobacteria are highly distributed in the environment and in man-made water infrastructures mainly due to its robustness and ability to adapt Until now there are only scarce reports of their presence in extreme environments but no methodic and large-scale survey of these environments has been done in order to truly understand their distribution in such environments. This thesis encompasses the isolation of strains from 263 different positive samples and their identification using different techniques. The most commonly isolated species were Mycobacterium gordonae in Glacier National Park and Mycobacterium parascrofulaceum and Mycobacterium avium in Yellowstone National Park. Isolates were tested for their resistance co temperature, pH and different stressors, in an attempt to explain the remarkable resistance for most of the tested conditions, several cell characteristics were tested, including fatty acids composition, polyphosphate accumulation and catalase activity. Scanning Electron Microscopy was performed to have an insight into the genera! structure of the cells. Results emphasize the highly resilience of mycobacteria to different types of environmental stress. From the tested conditions, the subtle differences In the fatty add composition, in the catalase activity and polyphosphate accumulation, demonstrates that the formation of biofilm-like structures helps to understand cite highly resistance of the members of this genus.

Mycobacterium tuberculosis is a remarkably successful human pathogen due to its ability to switch into dormancy or non-replicating persistence (NRP) phase driven by the host stress microenvironments. Identifying the panoply of genes or pathways involved in dormancy will progress our understanding on latent tuberculosis infection. Rv2660c and Rv2661c (conserved hypothetical proteins) and Rv1675c (transcriptional regulator) were implicated in mycobacterial stress response and transition to dormancy, hence their biological importance in M. tuberculosis biology was further explored. Rv2660c and rv2661c were highly upregulated in vitro starvation and in vivo infection model, however, recent high upregulation of a noncoding RNA, ncRv12659 in these models challenged the importance of these genes for NRP. A panel of single and double in-frame deletion mutants and over-expressing strains of rv2660c and rv2661c in M. tuberculosis were generated. A deletion of rv2660c and rv2661c also resulted in partial inactivation of ncRv12659 and rv2662 respectively. The deletion mutants exhibited normal growth in vitro and in mice. Furthermore, the strains showed unimpaired survival under nutrient starvation, hypoxia, oxidative and nitrosative stresses. Quantitative RT-PCR analysis revealed that neither target gene was highly expressed throughout starvation, oxidative and acidic pH stresses. Rv1675c (Cmr) is a redox sensor that regulates the DosR signalling pathway. Cmr binding to DNA was severely reduced by nitrosation of the two conserved cysteine residues. The cmr mutant displayed survival advantage during exposure to nitrosative stress. The over-expression of cmr or cmrC2A form (mutated cysteines) had a mild inhibitory effect on growth of M. tuberculosis. The over-expressing strain of cmrC2A was more resistant to hydrogen peroxide, suggesting that Cmr may also control the response to oxidative stress. Our study clarified the role of Rv2660c and Rv2661c in growth, NRP and infection, and further highlighted a novel Cmr-mediated regulatory network involved during nitrosative stress and transition to dormancy.

Attempts were made to activate human monocytes with immunomodulators and human macrophages with T-cell supernatants for in vitro antimycobacterial activity. Both these approaches failed. Alveolar macrophages from Mycobacterium bovis BCG-vaccinated guinea pigs have previously been shown to kill Mycobacterium tuberculosis in vitro. The guinea pig was therefore used to investigate macrophage antimycobacterial mechanisms. Tuberculocidal factors were found within lysosomes. Lysosomal fractions of macrophages from BCG-vaccinated guinea pigs significantly (P 0.001) killed M. tuberculosis. Tuberculocidal activity was not observed with macrophage lysosomal fractions from non-vaccinated guinea pigs. The specific activities of enzymes in macrophage homogenates were tested. None of the lysosomal enzymes had significantly different activities in macrophages from vaccinated and non-vaccinated guinea pigs. Strains of M. tuberculosis were sensitive to reactive nitrogen intermediates (RNI). However, guinea pig alveolar macrophages did not generate RNI on infection with mycobacteria. Macrophages from BCG-vaccinated guinea pigs killed M. tuberculosis in the presence of an inhibitor of RNI synthesis. Thus, no evidence was gained to suggest that RNI are responsible for the tuberculocidal activity of guinea pig macrophages. Aminoaldehydes have been shown to be toxic to M. tuberculosis. Ornithine decarboxylase (ODC) is the first enzyme in the pathway that synthesises aminoaldehydes. ODC activity was not elevated in macrophages twenty-four hours and six days after BCG vaccination. Increased ODC activity was not observed in macrophages infected with M. bovis BCG for twenty-four hours. ODC is therefore not responsible for any prolonged increase in aminoaldehyde production that may take place when macrophages are infected with mycobacteria. Mycobacterium tuberculosis was passaged once through the mouse. After passaging, the mycobacteria exhibited increased resistance to hydrogen peroxide but not RNI. This indicates that hydrogen peroxide is generated during the murine immune response to mycobacteria. Culturing Mycobacterium smegmatis at pH5.0 allowed the bacteria to survive subsequent incubation at pH3.5 better than bacteria grown solely at pH7.6. Mycobacterium smegmatis could therefore be adapted to lethal pH values by pre-exposing the bacteria to mildly acidic conditions.

Knowledge of the mechanisms by which Mycobacterium tuberculosis regulates its defence against oxidative stress will aid in the development of drugs and vaccines to combat the extensive morbidity and mortality caused by tuberculosis. In this thesis I have investigated the SenX3-RegX3 two component signal transduction system, one of 12 such systems in M. tuberculosis , which is essential for virulence in a mouse model and is similar to systems in other organisms that are involved in sensing oxygen. By complementing the attenuated growth of the senX3 and regX3 null mutants in mice I have shown that this phenotype is truly due to the targeted mutations in these strains. In vitro assays of oxygen stress have shown the senXS and regX3 null mutants to be less sensitive than the wild-type to superoxide and organic hydroperoxide stresses. Microarray analysis of mutants in the SenX3-RegX3 system after growth in microaerobic conditions suggested some functions for RegX3 but there was little overlap between strains. However, microarray comparison of a strain overproducing regX3 transcript demonstrated control of genes such as ahpC and cydB, indicating that this system may indeed play a role in the resistance of M. tuberculosis to oxidative stress. RegX3 was expressed and purified in E. coli and used to examine the interaction of this protein to different promoters, including that of senX3. Two further genes, which are probable one component regulators of oxidative stress in M. tuberculosis, Rv0465c and Rvl049, as judged by similarity to proteins in other organisms, were analysed through the isolation of null mutations. In vitro analysis suggests that Rvl049 is necessary for resistance to organic hydroperoxide stress, although neither the Rv0465c nor the Rvl049 null mutants were attenuated in macrophage or murine intravenous injection models of infection.

Mycobacterial populations are known for the heterogeneity in terms of cell size, morphology, and metabolic status, which are believed to help the population survive under stress conditions. Such population heterogeneity had been observed in TB patients, in animal models, and in in vitro cultures. Also, the physiological relevance of population heterogeneity under nutrient starvation has been studied. However, the physiological significance of population heterogeneity in oxidative and nitrite stress has not been addressed yet. Our laboratory had earlier shown that a subpopulation of mycobacterial mid-log phase cultures divide by highly deviated asymmetric division, generating short cells and normal-sized/long cells. This proportion has been found to be consistent and reproducible, and has been found in the freshly diagnosed pulmonary tuberculosis patients’ sputum, which is known to have high levels of oxidative stress. The highly deviated asymmetric cell division has been found to be one of the mechanisms that mycobacteria use to generate cell size heterogeneity in the population. However, the physiological significance of the population heterogeneity generated by the highly deviated asymmetric division remained to be addressed. Therefore, in the present study, we addressed the physiological significance of the generation of population heterogeneity in terms of cell size in Mycobacterium smegmatis and Mycobacterium tuberculosis. In this regard, we explored whether the minor subpopulation of short cells generated in the population has any relevance in the response of mycobacteria to oxidative and nitrite stress for survival. The Chapter 1, which forms the Introduction to the thesis, gives an extensive literature survey on the phenotypic heterogeneity in diverse bacterial systems and the physiological significance of such heterogeneity. Subsequently, an account of the phenotypic heterogeneity reported in mycobacteria is given, with examples of its significance implicated for survival under nutrient stress. Then an account of various studies on the oxidative and nitrite stress response of mycobacteria and on the genes involved in those processes are given. Further, the present study is justified by stating that so far there has not been any study to find out the physiological relevance of phenotypic heterogeneity on oxidative and nitrite stress response in mycobacteria. Finally, the Introduction is concluded by stating that the present study investigates and reports for the first time the physiological significance of the minor subpopulation of short cells for survival under oxidative and nitrite stress conditions. The Chapter 2 forms the Materials and Methods used in the present study. Here a detailed description of the methods used for the separation of the short cells, their characterisation, stress response, and so on are given in great detail. The Chapter 3 forms the first data chapter that presents results on the nature of response of Mycobacterium smegmatis and Mycobacterium tuberculosis against oxidative and nitrite stress. Here the cell size natural distribution, in terms of short cells and normal-sized/long cells in the mid-log phase population, the fractionation and enrichment of these subpopulations, differential susceptibility of the cells in the fractions to the stress conditions, the enhanced survival of the population upon mixing of these cell populations at the natural proportion, and the decreased survival upon mixing them at unnatural proportion are presented. The differential survival of the short cells and normal-sized/long cells was studied at a variety of stress concentrations for oxidative (H2O2) and nitrite (acidified sodium nitrite, pH 5), cell densities and exposure time to show the robustness of the phenomenon. Enhanced survival upon extended exposure to stress also has been documented. Essentially the data in this chapter shows that although the different sized populations show differential stress susceptibility to the stress conditions, their combined presence at the proportion that naturally exists in the mid-log phase population enhances the survival of the population, at the cost of the highly susceptible short cells for the enhanced survival of the less susceptible normal-sized/long cells, kin selection and altruism. The Chapter concludes with a discussion on the results. The Chapter 4 delineates the mechanism of the altruistic phenomenon that results in the enhanced survival of the population at the sacrifice of the minor subpopulation of short cells. Here we present evidence that hydroxyl radical generated through Fenton reaction is responsible for the enhanced survival through the induction of the synthesis of catalase-peroxidase (KatG) for the degradation of H2O2. The free iron deficient short cells acquire more iron, which in turn becomes stoichiometrically detrimental to them due to the high levels of hydroxyl generation in the presence of H2O2. On the contrary, the free iron containing normal-sized/long cells do not acquire iron and hence the hydroxyl radical produced in the population becomes stoichiometrically beneficial to them. Thus, the deficiency of free iron which consequentially necessitates the short cells to acquire more iron becomes a maladaptive trait in the presence of H2O2 but gets co-opted in kin selection, for the survival of the normal-sized/long cells that form major proportion of the population – a phenomenon reminiscent of altruism. The Chapter concludes with a model depicting the entire phenomenon and a discussion on the results and their implications.

Mycobacterial populations are known for the heterogeneity in terms of cell size, morphology, and metabolic status, which are believed to help the population survive under stress conditions. Such population heterogeneity had been observed in TB patients, in animal models, and in in vitro cultures. Also, the physiological relevance of population heterogeneity under nutrient starvation has been studied. However, the physiological significance of population heterogeneity in oxidative and nitrite stress has not been addressed yet. Our laboratory had earlier shown that a subpopulation of mycobacterial mid-log phase cultures divide by highly deviated asymmetric division, generating short cells and normal-sized/long cells. This proportion has been found to be consistent and reproducible, and has been found in the freshly diagnosed pulmonary tuberculosis patients’ sputum, which is known to have high levels of oxidative stress. The highly deviated asymmetric cell division has been found to be one of the mechanisms that mycobacteria use to generate cell size heterogeneity in the population. However, the physiological significance of the population heterogeneity generated by the highly deviated asymmetric division remained to be addressed. Therefore, in the present study, we addressed the physiological significance of the generation of population heterogeneity in terms of cell size in Mycobacterium smegmatis and Mycobacterium tuberculosis. In this regard, we explored whether the minor subpopulation of short cells generated in the population has any relevance in the response of mycobacteria to oxidative and nitrite stress for survival. The Chapter 1, which forms the Introduction to the thesis, gives an extensive literature survey on the phenotypic heterogeneity in diverse bacterial systems and the physiological significance of such heterogeneity. Subsequently, an account of the phenotypic heterogeneity reported in mycobacteria is given, with examples of its significance implicated for survival under nutrient stress. Then an account of various studies on the oxidative and nitrite stress response of mycobacteria and on the genes involved in those processes are given. Further, the present study is justified by stating that so far there has not been any study to find out the physiological relevance of phenotypic heterogeneity on oxidative and nitrite stress response in mycobacteria. Finally, the Introduction is concluded by stating that the present study investigates and reports for the first time the physiological significance of the minor subpopulation of short cells for survival under oxidative and nitrite stress conditions. The Chapter 2 forms the Materials and Methods used in the present study. Here a detailed description of the methods used for the separation of the short cells, their characterisation, stress response, and so on are given in great detail. The Chapter 3 forms the first data chapter that presents results on the nature of response of Mycobacterium smegmatis and Mycobacterium tuberculosis against oxidative and nitrite stress. Here the cell size natural distribution, in terms of short cells and normal-sized/long cells in the mid-log phase population, the fractionation and enrichment of these subpopulations, differential susceptibility of the cells in the fractions to the stress conditions, the enhanced survival of the population upon mixing of these cell populations at the natural proportion, and the decreased survival upon mixing them at unnatural proportion are presented. The differential survival of the short cells and normal-sized/long cells was studied at a variety of stress concentrations for oxidative (H2O2) and nitrite (acidified sodium nitrite, pH 5), cell densities and exposure time to show the robustness of the phenomenon. Enhanced survival upon extended exposure to stress also has been documented. Essentially the data in this chapter shows that although the different sized populations show differential stress susceptibility to the stress conditions, their combined presence at the proportion that naturally exists in the mid-log phase population enhances the survival of the population, at the cost of the highly susceptible short cells for the enhanced survival of the less susceptible normal-sized/long cells, kin selection and altruism. The Chapter concludes with a discussion on the results. The Chapter 4 delineates the mechanism of the altruistic phenomenon that results in the enhanced survival of the population at the sacrifice of the minor subpopulation of short cells. Here we present evidence that hydroxyl radical generated through Fenton reaction is responsible for the enhanced survival through the induction of the synthesis of catalase-peroxidase (KatG) for the degradation of H2O2. The free iron deficient short cells acquire more iron, which in turn becomes stoichiometrically detrimental to them due to the high levels of hydroxyl generation in the presence of H2O2. On the contrary, the free iron containing normal-sized/long cells do not acquire iron and hence the hydroxyl radical produced in the population becomes stoichiometrically beneficial to them. Thus, the deficiency of free iron which consequentially necessitates the short cells to acquire more iron becomes a maladaptive trait in the presence of H2O2 but gets co-opted in kin selection, for the survival of the normal-sized/long cells that form major proportion of the population – a phenomenon reminiscent of altruism. The Chapter concludes with a model depicting the entire phenomenon and a discussion on the results and their implications.

Mycobacterium tuberculosis (Mtb) has evolved as an important clinical pathogen due to its ability to gain multidrug resistance, to enter into latency to persist there and to get reactivated from the latent infection in aged, immunocompromised persons to cause the disease. Mtb, which can pesist within the granulomatous parenchymal lesions in a nonreplicating persistent (NRP) stage called dormancy (clinically called latency), experience hypoxic stress. In order to understand the response of the bacilli to the hypoxic environment, several in vivo and in vitro model systems have been designed and used. Amongst the in vitro model systems, the one which closely resembles the hypoxic stress induced within the host wherein the bacilli experience a gradual depletion of oxygen, was found to be the Wayne’s in vitro hypoxia model. Therefore, it is considered to be the best model system to study the response of M. tuberculosis to the hypoxic stress in vitro as here as well the bacilli experience a gradual depletion of oxygen to progress from the actively growing mid‐log phase to NRP‐I and NRP‐II stages. Even though, several works have been carried out at the gene expression and proteomic levels to know the response of the bacilli using Wayne’s in vitro hypoxia model, surprisingly there is hardly any information available on the morphological and cellular level changes, which occur in response to hypoxia, and their correlation to molecular changes. Therefore, in the present study, using Wayne’s in vitro hypoxia model, this lacuna of information was addressed by studying morphological and cellular changes occurring to NRPI and NRP‐II M. tuberculosis cells at the ultrastructural level, correlating the changes with respective gene expression level changes, and finding out their physiological significance. The Chapter 1, which forms the Introduction to the thesis, gives an extensive literature survey on all the different aspects of the research performed on mycobacterial cells under hypoxia, which are linked to the present study, and under other stress conditions such as nutrient depletion, pH and so on. The Chapter 2 presents in detail all the materials and methods used to perform the experiments. A large number of biochemical, biophysical, and molecular level methods, such as scanning and transmission electron microscopy, atomic force microscopy, Fourier transform infrared spectroscopy, dynamic light scattering, flow cytometry, fluorescence microscopy, real time RT‐PCR analysis, and others were used to perform the experiments. The data Chapter 3 presents ultrastructural analysis of the M. tuberculosis cells exposed to hypoxic stress condition using Wayne’s in vitro hypoxia model. Among the different ultra-structural changes observed, this study focused mainly on the unique thickening of the outer layer (OL), during the progression of the bacilli from mid‐log phase to NRP‐I and then to NRPII stages. On the contrary, the NRP‐I and NRP‐II cells of the saprophytic mycobacterial species, Mycobacterium smegmatis, did not show thickening of the OL. Dynamic light scattering experiments using Zetasizer showed the NRP‐I and NRP‐II M. tuberculosis cells to be longer in size. Experiments using Zeta potential analyzer revealed high level of negative charge on the surface of the NRP‐I and NRP‐II M. tuberculosis bacilli. Ultrastructural studies, using scanning and transmission electron microscopy and atomic force microscopy revealed that the surface of the NRP‐II M. tuberculosis bacilli was extensively rough and uneven, unlike the smooth and even surface of the mid‐log phase cells. Fourier transform infrared spectroscopy of the groups present in the polysaccharides extracted from thickened outer layer were found to be highly anionic in nature. Polysaccharide‐specific calcofluor white staining showed the thickened outer layer of M. tuberculosis to be rich in polysaccharides. Transcriptome analysis of the respective genes involved in the synthesis of capsular polysaccharides showed significant upregulation in comparison to the same genes in the MLP cells, thus supporting the observed hypoxic adaptation of the thickening of the OL in NRP‐II M. tuberculosis cells. The data Chapter 4 presents studies on the physiological significance of the thickening of the OL in terms of the permeability to the anti‐TB drug, rifampicin. Since it was necessary to choose a non‐bioactive variant of rifampicin in order to avoid the growth inhibitory antibiotic action on the cells, rifampicin conjugated to 5‐carboxyfluorescein (5‐FAMrifampicin), which showed only 2.5% bioactivity, was used for the permeability assay, instead of 14C‐rifampicin. The thickened OL of the NRP‐I and NRP‐II cells were found to significantly restrict the entry of 5‐FAM‐rifampicin into the cells. Mild bead beating of the NRP‐II cells, to remove the thickened OL without affecting the outer membrane integrity, as confirmed using transmission electron microscopy, restored the permeability of 5‐FAM‐rifampicin ascomparable to that into mid‐log phase cells. The entry of 5‐FAM‐rifampicin into the cells was monitored using flow cytometry analysis. M. tuberculosis cells, at 48 hrs. post‐release from= the NRP‐II stage, also showed restoration of the permeability of 5‐FAM‐rifampicin as comparable to that into mid‐log phase cells. These observations suggested that the recalcitrance of dormant bacilli to anti‐TB drugs might be due to the presence of thickened OL generated by the bacilli as a strategy to evade bactericidal effects. The data chapters in the thesis are concluded with discussion of the findings presented in the respective chapter. The thesis finally lists the highlights of the present study, followed up with an extensive bibliography.

The stringent response is a highly conserved physiological response mounted by bacteria under stress (Ojha and Chatterji, 2001; Magnusson et al., 2005; Srivatsan and Wang, 2007; Potrykus and Cashel, 2008). Until recently, the only known players in this pathway were the (p)ppGpp synthesizing and hydrolyzing long RSH enzymes (Mittenhuber, 2001; Atkinson et al., 2011) - RelA and SpoT in Gram negative bacteria and the bifunctional Rel in Gram positive bacteria including mycobacteria. The existence of Short Alarmone Synthetases (SAS) (Lemos et al., 2007, Nanamiya et al., 2008; Das et al., 2009; Atkinson et al., 2011) and Short Alarmone Hydrolases (SAH) (Sun et al., 2010, Atkinson et al., 2011), small proteins possessing a single functional (p)ppGpp synthetase or hydrolase domain respectively, is a recent discovery that has modified this paradigm. Around the same time that the presence of the SAS proteins was reported, we chanced upon such small (p)ppGpp synthetases in the genus Mycobacterium. The stringent response in the soil saprophyte Mycobacterium smegmatis was first reported by Ojha and co-workers (Ojha et al., 2000), and the bifunctional RSH, RelMsm, responsible for mounting the stringent response in this bacterium, has been characterized in detail (Jain et al., 2006 and 2007). RelMsm was the only known RSH enzyme present in M. smegmatis, and consequently, a strain of M. smegmatis deleted for the relMsm gene (ΔrelMsm) (Mathew et al., 2004), was expected to show a null phenotype for (p)ppGpp production. In this body of work, we report the surprising observation that the M. smegmatis ΔrelMsm strain is capable of synthesizing (p)ppGpp in vivo. This unexpected turn of events led us to the discovery of a second (p)ppGpp synthetase in this bacterium. The novel protein was found to possess two functional domains – an RNase HII domain at the amino-terminus, and a (p)ppGpp synthetase or RSD domain at the carboxy-terminus. We have therefore named this protein ‘MS_RHII-RSD’, indicating the two activities present and identifying the organism from which it is isolated. Orthologs of this novel SAS protein occur in other species of mycobacteria, both pathogenic and non-pathogenic. In this study, we report the cloning, purification and in-depth functional characterization of MS_RHII-RSD, and speculate on its in vivo role in M. smegmatis. Chapter 1 reviews the available literature in the field of stringent response research and lays the background to this study. A historical perspective is provided, starting with the discovery of the stringent response in bacteria in the early 1960s, highlighting the development in this area till date. The roles played by the long and short RSH enzymes, ‘Magic Spot’ (p)ppGpp, the RNA polymerase enzyme complex, and a few other RNA and proteins are described, briefly outlining the inferences drawn from recent global gene expression and proteomics studies. The chapter concludes with a description of the motivation behind, and the scope of the present study. Chapter 2 discusses the in vivo and in silico identification of MS_RHII-RSD in M. smegmatis. Experiments performed for the genotypic and phenotypic revalidation of M. smegmatis ΔrelMsm strain are described. Detailed bioinformatics analyses are provided for the in silico characterization of MS_RHII-RSD in terms of its domain architecture, in vivo localization, and protein structure prediction. A comprehensive list of the mycobacterial orthologs of MS_RHII-RSD from a few representative species of infectious and non-infectious mycobacteria is included. Chapter 3 summarizes the materials and methods used in the cloning, purification, and the biophysical and biochemical characterization of full length MS_RHII-RSD and its two domain variants – RHII and RSD, respectively. A detailed description of the purification protocols highlighting the specific modifications and changes made is given. Peptide mass fingerprinting to confirm protein identity, as well as preliminary mass spectrometric, chromatographic, and circular dichroism-based characterization of the proteins under study is also provided. Chapter 4 deals in detail with the in vivo and in vitro functional characterization of the RNase HII and (p)ppGpp synthesis activities of full length MS_RHII-RSD and its two domain variants - RHII and RSD, respectively. The RNase HII activity is characterized in vivo on the basis of a complementation assay in an E. coli strain deleted for the RNase H genes; while in vitro characterization is done by performing a FRET-based assay to monitor the degradation of a RNA•DNA hybrid substrate in vitro. The (p)ppGpp synthesis activity is characterized in terms of the substrate specificity, magnesium ion utilization, and a detailed analysis of the kinetic parameters involved. A comparison of the (p)ppGpp synthesis activity of MS_RHII-RSD vis-à-vis that of the classical RSH protein, RelMsm, is also provided. Inferences drawn from (p)ppGpp hydrolysis assays and the in vivo expression profile of MS_RHII-RSD in M. smegmatis wild type and ΔrelMsm strains are discussed. Based on the results of these functional assays, a model is proposed suggesting the probable in vivo role played by MS_RHII-RSD in M. smegmatis. Chapter 5 describes the attempts at generating MS_RHII-RSD overexpression and knockout strains in M. smegmatis, using pJAM2-based mycobacterial expression system, and mycobacteriophage-based specialized transduction strategy, respectively. The detailed methodology and the principle behind the techniques used are explained. The results obtained so far, and the future work and strain characterization to be carried out in this respect are discussed. Chapter 6 takes a slightly different route and summarizes the work carried out in characterizing the glycopeptidolipids (GPLs) from M. smegmatis biofilm cultures. A general introduction about the mycobacterial cell wall components, with special emphasis on GPLs, is provided. The detailed protocols for chemical composition and chromatographic analyses are mentioned, and the future scope of this work is discussed. Appendix-1 briefly revisits the preliminary studies performed to determine the pppGpp binding site on M. smegmatis RNA polymerase using a mass spectrometry-based approach. Appendices-2, 3, 4 and 5 give a comprehensive list of the bacterial strains; PCR primers; antibiotics, buffers and media used; and the plasmid and phasmid maps, respectively.

The stringent response is a highly conserved physiological response mounted by bacteria under stress (Ojha and Chatterji, 2001; Magnusson et al., 2005; Srivatsan and Wang, 2007; Potrykus and Cashel, 2008). Until recently, the only known players in this pathway were the (p)ppGpp synthesizing and hydrolyzing long RSH enzymes (Mittenhuber, 2001; Atkinson et al., 2011) - RelA and SpoT in Gram negative bacteria and the bifunctional Rel in Gram positive bacteria including mycobacteria. The existence of Short Alarmone Synthetases (SAS) (Lemos et al., 2007, Nanamiya et al., 2008; Das et al., 2009; Atkinson et al., 2011) and Short Alarmone Hydrolases (SAH) (Sun et al., 2010, Atkinson et al., 2011), small proteins possessing a single functional (p)ppGpp synthetase or hydrolase domain respectively, is a recent discovery that has modified this paradigm. Around the same time that the presence of the SAS proteins was reported, we chanced upon such small (p)ppGpp synthetases in the genus Mycobacterium. The stringent response in the soil saprophyte Mycobacterium smegmatis was first reported by Ojha and co-workers (Ojha et al., 2000), and the bifunctional RSH, RelMsm, responsible for mounting the stringent response in this bacterium, has been characterized in detail (Jain et al., 2006 and 2007). RelMsm was the only known RSH enzyme present in M. smegmatis, and consequently, a strain of M. smegmatis deleted for the relMsm gene (ΔrelMsm) (Mathew et al., 2004), was expected to show a null phenotype for (p)ppGpp production. In this body of work, we report the surprising observation that the M. smegmatis ΔrelMsm strain is capable of synthesizing (p)ppGpp in vivo. This unexpected turn of events led us to the discovery of a second (p)ppGpp synthetase in this bacterium. The novel protein was found to possess two functional domains – an RNase HII domain at the amino-terminus, and a (p)ppGpp synthetase or RSD domain at the carboxy-terminus. We have therefore named this protein ‘MS_RHII-RSD’, indicating the two activities present and identifying the organism from which it is isolated. Orthologs of this novel SAS protein occur in other species of mycobacteria, both pathogenic and non-pathogenic. In this study, we report the cloning, purification and in-depth functional characterization of MS_RHII-RSD, and speculate on its in vivo role in M. smegmatis. Chapter 1 reviews the available literature in the field of stringent response research and lays the background to this study. A historical perspective is provided, starting with the discovery of the stringent response in bacteria in the early 1960s, highlighting the development in this area till date. The roles played by the long and short RSH enzymes, ‘Magic Spot’ (p)ppGpp, the RNA polymerase enzyme complex, and a few other RNA and proteins are described, briefly outlining the inferences drawn from recent global gene expression and proteomics studies. The chapter concludes with a description of the motivation behind, and the scope of the present study. Chapter 2 discusses the in vivo and in silico identification of MS_RHII-RSD in M. smegmatis. Experiments performed for the genotypic and phenotypic revalidation of M. smegmatis ΔrelMsm strain are described. Detailed bioinformatics analyses are provided for the in silico characterization of MS_RHII-RSD in terms of its domain architecture, in vivo localization, and protein structure prediction. A comprehensive list of the mycobacterial orthologs of MS_RHII-RSD from a few representative species of infectious and non-infectious mycobacteria is included. Chapter 3 summarizes the materials and methods used in the cloning, purification, and the biophysical and biochemical characterization of full length MS_RHII-RSD and its two domain variants – RHII and RSD, respectively. A detailed description of the purification protocols highlighting the specific modifications and changes made is given. Peptide mass fingerprinting to confirm protein identity, as well as preliminary mass spectrometric, chromatographic, and circular dichroism-based characterization of the proteins under study is also provided. Chapter 4 deals in detail with the in vivo and in vitro functional characterization of the RNase HII and (p)ppGpp synthesis activities of full length MS_RHII-RSD and its two domain variants - RHII and RSD, respectively. The RNase HII activity is characterized in vivo on the basis of a complementation assay in an E. coli strain deleted for the RNase H genes; while in vitro characterization is done by performing a FRET-based assay to monitor the degradation of a RNA•DNA hybrid substrate in vitro. The (p)ppGpp synthesis activity is characterized in terms of the substrate specificity, magnesium ion utilization, and a detailed analysis of the kinetic parameters involved. A comparison of the (p)ppGpp synthesis activity of MS_RHII-RSD vis-à-vis that of the classical RSH protein, RelMsm, is also provided. Inferences drawn from (p)ppGpp hydrolysis assays and the in vivo expression profile of MS_RHII-RSD in M. smegmatis wild type and ΔrelMsm strains are discussed. Based on the results of these functional assays, a model is proposed suggesting the probable in vivo role played by MS_RHII-RSD in M. smegmatis. Chapter 5 describes the attempts at generating MS_RHII-RSD overexpression and knockout strains in M. smegmatis, using pJAM2-based mycobacterial expression system, and mycobacteriophage-based specialized transduction strategy, respectively. The detailed methodology and the principle behind the techniques used are explained. The results obtained so far, and the future work and strain characterization to be carried out in this respect are discussed. Chapter 6 takes a slightly different route and summarizes the work carried out in characterizing the glycopeptidolipids (GPLs) from M. smegmatis biofilm cultures. A general introduction about the mycobacterial cell wall components, with special emphasis on GPLs, is provided. The detailed protocols for chemical composition and chromatographic analyses are mentioned, and the future scope of this work is discussed. Appendix-1 briefly revisits the preliminary studies performed to determine the pppGpp binding site on M. smegmatis RNA polymerase using a mass spectrometry-based approach. Appendices-2, 3, 4 and 5 give a comprehensive list of the bacterial strains; PCR primers; antibiotics, buffers and media used; and the plasmid and phasmid maps, respectively.

Under hostile conditions, bacteria elicit stress response. Such stress response is regulated by a secondary messenger called (p)ppGpp. (p)ppGpp is involved in wide range of functions such as GTP homeostasis, biofilm formation and cell growth. Its regulation and mode of action is not well understood. This work has been initiated with an aim to gain insights into the molecular basis of stress response. (p)ppGpp was discovered on the chromatogram of cell extract from starved E. coli cells. (p)ppGpp is synthesized and hydrolyzed by Rel/SpoT in Gram negative bacteria (such as E. coli), and by bifunctional enzyme called Rel in Gram positive bacteria (such as Mycobacteria). The obvious question that comes in our mind is how bifunctional Rel enzyme decides on synthesis or hydrolysis in Gram positive bacteria such as Mycobacterium? In our laboratory, it has been shown that N-terminal domain of Rel shows unregulated (p)ppGpp synthesis implying regulatory role of C-terminal domain. Also, concurrent increase in anisotropy of Rel C-terminal domain with the increase in concentration of pppGpp has been observed indicating the binding of pppGpp to the C-terminal domain. We performed Isothermal Calorimetry experiment to confirm that pppGpp binds with C-terminal domain of Rel enzyme. For identification of the binding region, small molecule analogue 8-azido-pppGpp has been synthesized. This analogue is UV-crosslinked with C-terminal domain of Rel and specificity of the interaction has been determined by gel based crosslinking experiments. Crosslinked protein has been subjected to the ingel¬trypsin digestion and analyzed by mass spectrometry. We identified two crosslinked peptides in the mass spectra of trypsin digest in case of the crosslinked protein where identity of the parent peptide is confirmed by MS-MS analysis. Site directed mutagenesis has been carried out based on the conservation of residues in the crosslinked peptides. Isothermal Calorimetry analysis has been done where Rel C-terminal domain mutants are titrated with pppGpp in order to detect any defect in binding due to the mutations. Mutations leading to the reduced binding affinity of pppGpp to Rel C-terminal domain have been introduced in the full length Rel protein and activity assays are carried out so as to evaluate the effects of mutations on synthesis and hydrolysis activity. In mutants, synthesis activity is found to be increased with the concomitant reduction in hydrolysis activity. This indicates the feedback loop where pppGpp binds to Rel C-terminal domain to regulate it own synthesis and hydrolysis. In E. coli, pppGpp binds to RNA polymerase and modulates the transcription. The region where it binds is controversial. In addition, whether ppGpp and pppGpp have different binding site on RNA polymerase is not known. The latter question becomes important in the light of evidence where differential regulation of transcription by ppGpp and pppGpp have been indicated. We found that ppGpp and pppGpp have an overlapping binding site on RNA polymerase. The 8-azido-ppGpp has been mapped on β and β’ subunits whereas binding site of 8-azido-pppGpp has been located on the β’ subunit. We observed that the 8-azido¬pppGpp labels RNA polymerase more efficiently than ppGpp. pppGpp can compete out ppGpp as illustrated by DRaCALA assay and gel based crosslinking experiment. However, the RNAP from B. subtilis does not bind to (p)ppGpp. (p)ppGpp is ubiquitous in bacteria but absent in mammals. Thus, blocking (p)ppGpp synthesis would impede the survival of bacteria without having any effect on humans. Recently, Relacin compound has been synthesized by another group in order to inhibit (p)ppGpp synthesis. The limitations of this compound are the requirement of high concentration (5mM) for inhibition and low permeability across the membrane. Taking hints from the latter compound, we acetylated the nd 2’, 3’ and 5’ position of ribose ring and benzoylated the 2position of guanine moiety in guanosine molecule. We observed significant inhibition of in vitro pppGpp synthesis and biofilm formation. More studies will be conducted in near future to test these compounds for their plausible functions. In collaboration with Prof. Jayaraman (Organic Chemistry, IISc), many artificial glycolipids are synthesized and tested for biological function. We observed that synthetic glycolipids exhibit a profound effect as inhibitors of the key mycobacterial functions. These analogs impede biofilm formation and can plausibly affect long term survival. Glycolipid analogs can compete with natural glycolipids, thus may help in understanding their functions. Our past and recent studies have showed that the synthetic glycolipids act as inhibitors of mycobacterial growth, sliding motility and biofilm formation. The major lacuna of these glycolipid inhibitors is the requirement of high concentration. Their inhibitions at nanomolar concentrations remain to be achieved. Issues surrounding the thick, waxy mycobacterial cell wall structures will continue to be the focus in manifold approaches to mitigate detrimental effects of mycobacterial pathogens. In chapter 1, introduction to the research work has been written and role of (p)ppGpp and its functions have been discussed. In chapter 2, novel binding site of pppGpp on Rel C-terminal domain and its regulatory role have been discussed. In chapter 3, differential binding of ppGpp and pppGpp to RNA polymerase has been discussed. In chapter 4, studies on natural and synthetic analogues of pppGpp have been presented. In chapter 5, synthetic glycolipids studies have been described. Chapter 6 summarizes all the chapters.

Under hostile conditions, bacteria elicit stress response. Such stress response is regulated by a secondary messenger called (p)ppGpp. (p)ppGpp is involved in wide range of functions such as GTP homeostasis, biofilm formation and cell growth. Its regulation and mode of action is not well understood. This work has been initiated with an aim to gain insights into the molecular basis of stress response. (p)ppGpp was discovered on the chromatogram of cell extract from starved E. coli cells. (p)ppGpp is synthesized and hydrolyzed by Rel/SpoT in Gram negative bacteria (such as E. coli), and by bifunctional enzyme called Rel in Gram positive bacteria (such as Mycobacteria). The obvious question that comes in our mind is how bifunctional Rel enzyme decides on synthesis or hydrolysis in Gram positive bacteria such as Mycobacterium? In our laboratory, it has been shown that N-terminal domain of Rel shows unregulated (p)ppGpp synthesis implying regulatory role of C-terminal domain. Also, concurrent increase in anisotropy of Rel C-terminal domain with the increase in concentration of pppGpp has been observed indicating the binding of pppGpp to the C-terminal domain. We performed Isothermal Calorimetry experiment to confirm that pppGpp binds with C-terminal domain of Rel enzyme. For identification of the binding region, small molecule analogue 8-azido-pppGpp has been synthesized. This analogue is UV-crosslinked with C-terminal domain of Rel and specificity of the interaction has been determined by gel based crosslinking experiments. Crosslinked protein has been subjected to the ingel¬trypsin digestion and analyzed by mass spectrometry. We identified two crosslinked peptides in the mass spectra of trypsin digest in case of the crosslinked protein where identity of the parent peptide is confirmed by MS-MS analysis. Site directed mutagenesis has been carried out based on the conservation of residues in the crosslinked peptides. Isothermal Calorimetry analysis has been done where Rel C-terminal domain mutants are titrated with pppGpp in order to detect any defect in binding due to the mutations. Mutations leading to the reduced binding affinity of pppGpp to Rel C-terminal domain have been introduced in the full length Rel protein and activity assays are carried out so as to evaluate the effects of mutations on synthesis and hydrolysis activity. In mutants, synthesis activity is found to be increased with the concomitant reduction in hydrolysis activity. This indicates the feedback loop where pppGpp binds to Rel C-terminal domain to regulate it own synthesis and hydrolysis. In E. coli, pppGpp binds to RNA polymerase and modulates the transcription. The region where it binds is controversial. In addition, whether ppGpp and pppGpp have different binding site on RNA polymerase is not known. The latter question becomes important in the light of evidence where differential regulation of transcription by ppGpp and pppGpp have been indicated. We found that ppGpp and pppGpp have an overlapping binding site on RNA polymerase. The 8-azido-ppGpp has been mapped on β and β’ subunits whereas binding site of 8-azido-pppGpp has been located on the β’ subunit. We observed that the 8-azido¬pppGpp labels RNA polymerase more efficiently than ppGpp. pppGpp can compete out ppGpp as illustrated by DRaCALA assay and gel based crosslinking experiment. However, the RNAP from B. subtilis does not bind to (p)ppGpp. (p)ppGpp is ubiquitous in bacteria but absent in mammals. Thus, blocking (p)ppGpp synthesis would impede the survival of bacteria without having any effect on humans. Recently, Relacin compound has been synthesized by another group in order to inhibit (p)ppGpp synthesis. The limitations of this compound are the requirement of high concentration (5mM) for inhibition and low permeability across the membrane. Taking hints from the latter compound, we acetylated the nd 2’, 3’ and 5’ position of ribose ring and benzoylated the 2position of guanine moiety in guanosine molecule. We observed significant inhibition of in vitro pppGpp synthesis and biofilm formation. More studies will be conducted in near future to test these compounds for their plausible functions. In collaboration with Prof. Jayaraman (Organic Chemistry, IISc), many artificial glycolipids are synthesized and tested for biological function. We observed that synthetic glycolipids exhibit a profound effect as inhibitors of the key mycobacterial functions. These analogs impede biofilm formation and can plausibly affect long term survival. Glycolipid analogs can compete with natural glycolipids, thus may help in understanding their functions. Our past and recent studies have showed that the synthetic glycolipids act as inhibitors of mycobacterial growth, sliding motility and biofilm formation. The major lacuna of these glycolipid inhibitors is the requirement of high concentration. Their inhibitions at nanomolar concentrations remain to be achieved. Issues surrounding the thick, waxy mycobacterial cell wall structures will continue to be the focus in manifold approaches to mitigate detrimental effects of mycobacterial pathogens. In chapter 1, introduction to the research work has been written and role of (p)ppGpp and its functions have been discussed. In chapter 2, novel binding site of pppGpp on Rel C-terminal domain and its regulatory role have been discussed. In chapter 3, differential binding of ppGpp and pppGpp to RNA polymerase has been discussed. In chapter 4, studies on natural and synthetic analogues of pppGpp have been presented. In chapter 5, synthetic glycolipids studies have been described. Chapter 6 summarizes all the chapters.

Tuberculosis (TB) is an infectious disease caused by the micro-organism Mycobacterium tuberculosis (MTB) that currently represents a global health emergency with around 9 million people affected and nearly 2 million deaths each year (1). The present therapeutic treatment for TB consists in the use of multiple anti-mycobacterial drugs such as rifampicin and isoniazid. This treatment is carried out for a long time leading to the onset of drug- and multidrug-resistant strains of MTB. Therefore new therapeutic models are needed to overcome this problem (2). During MTB infection, the host antimicrobial response generates several metabolically activated DNA alkylating agents leading to severe DNA-damaging injuries on MTB cells (3, 4). Protection of the DNA molecule from chemical damages then strictly depends on the MTB repair mechanisms. Recent studies identified an adaptive response mechanism in MTB homologous to that already described in E.coli (5). Being the adaptive response a fundamental biological process for MTB viability whereas it is missing in human, this process may represent a putative therapeutic target to explore in search for new TB treatments. Previous studies in MTB identified four genes which encode the proteins constituent of the adaptive response mechanism homologous to the same process already defined in E. coli. Exposure of E. coli to sublethal concentrations of alkylating agents induces the expression of four genes (ada, alkA, alkB and aidB). The activation of these genes increases the resistance to the cytotoxic and mutagenic effects of alkylating molecules (6). Unlike E. coli, the DNA repair/protection systems in MBT have not been investigated and it is still largely unknown. Aims This thesis project focuses on the investigation of the adaptive response to DNA methylation stress in MTB, in order to identify possible innovative therapeutic targets against tubercular infection. A non-pathogenic, reference strain of Mycobacteria, M. smegmatis, was used to perform all the experiments. The effect of methylating molecules on M. smegmatis was evaluated using MMS, a common laboratory methylating molecule. Moreover, Busulfan was chosen as methylating reference drug. First the effect of alkylating agents was investigated on M. smegmatis cells by evaluating the effect of MMS on cell survival, DNA alkylation and on the global proteomic response. Second, structural and functional characterization of the recombinant Ada-Alka and Ogt proteins was carried out because of their involvement in the cellular response to alkylating agents. Finally, functional proteomic experiments on M.smegmatis Ogt protein was performed in order to shine a light on its biological function through the identification of its protein partners in vivo.

Mycothiol (MSH), produced in actinomycetes including mycobacteria, is functionally analogous to glutathione (GSH) in other organisms, replacing G S H as the main systemic protectant against oxidative stress. In this work, we investigated two possible control points in the regulation of MSH in response to oxidative stress: 1) transcriptional upregulation of MSH biosynthesis mediated by Rv0485 & Rv0818 (putative transcriptional regulators located directly upstream of some MSH biosynthesis genes); and 2) maintenance of the MSH:MS=SM redox balance upon oxidative stress. To monitor the changes in redox state and total MSH levels in Mycobacterium smegmatis mc²155 and M. bovis BCG cultures upon exposure to diamide (a thiol-specific oxidative agent), H₂O₂, or gaseous nitric oxide, we performed mycothiol assays and developed a novel, modified mycothiol assay to detect MSH oxidized as MS=SM. We found that diamide and H₂O₂-induced oxidative stress in M. bovis BCG induces partial depletion of MSH to the oxidized form MS=SM, while treatment with gNO does not. M. smegmatis, an environmental saprophyte, displays a greater tolerance to these oxidative stresses than M. bovis BCG, as reflected by the lesser magnitudes in changes in redox state and total MSH levels upon treatment. We also investigated gene expression of Rv0485 and Rv0818 in M. bovis BCG upon exposure to diamide and upon infection of J774A.1 murine macrophages, using quantitative real-time reverse-transcriptase PCR. We found that although expressions of Rv0485 and Rv0818 were unchanged in diamide-treated bacteria, they were increased about 8-fold in bacteria harvested 6 and 18 hours after macrophage infections, hi addition, we conducted protein-protein binding assays to investigate if Rv0818 protein binds to the SigH RNA polymerase subunit specifically under oxidizing conditions in vitro, as would be expected if Rv0818 is involved in the transcriptional regulation of msh biosynthesis genes upon oxidative stress. As an addendum to this thesis, we looked at two potential GSH-dependent genes in mycobacteria, ggtA & ggtB, which code for putative gammaglutamyltranspeptidases and might have a role in the recently described phenomenon of mycobacterial sensitivity to GSH and GSNO.
Medicine, Faculty of
Medicine, Department of
Experimental Medicine, Division of
Graduate

A distinctive feature of host-pathogen interactions in the case of Mycobacterium tuberculosis is the asymptomatic latent phase of infection. The ability of the bacillus to survive for extended periods of time in the host suggests an adaptive mechanism in M. tuberculosis that can cope with a variety of environmental stresses and other host stimuli. Extensive genomic studies and analysis of knock-out phenotypes revealed elaborate cellular machinery in M. tuberculosis that ensures a rapid cellular response to host stimuli. Prominent amongst these are two-component systems and σ factors that exclusively govern transcription re-engineering in response to environmental stimuli. M. tuberculosis σK is a σ factor that was demonstrated to control the expression of secreted antigenic proteins. The study reported in this thesis was geared to understand the molecular basis for σK activity as well as to explore conditions that would regulate σK activity. Transcription in bacteria is driven by the RNA polymerase enzyme that can associate with multiple σ factors. σ factors confer promoter specificity and thus directly control the expression of genes. The association of different σ factors with the RNA polymerase is essential for the temporal and conditional re-engineering of the expression profile. Environment induced changes in expression rely on a subset of σ factors. This class of σ factors (also referred to as Class IV or Extra-cytoplasmic function (ECF) σ factors) is regulated by a variety of mechanisms. The regulation of an ECF σ factor activity at the transcriptional, translational or posttranslational steps ensures fidelity in the cellular concentration of free, active ECF σ factors. In general, ECF σ factors associate with an inhibitory protein referred to as an anti-σ factor. The release of a free, active σ factor from a σ /anti-σ complex is thus a mechanism that can potentially control the cellular levels of an active σ factor in the cell. M. tuberculosis σK is associated with a membrane bound anti-σK (also referred to as RskA) (Said-Salim et al., Molecular Microbiology 62: 1251-1263: 2006). The extracellular stimulus that is recognized by RskA remains unclear. However, recent studies have suggested the possibility of a regulated proteolytic cascade that can selectively degrade RskA and other membrane associated anti-σ factors. The goal of the study was to understand this regulatory mechanism with a specific focus on the M. tuberculosis σK/RskA complex. The structure of the cytosolic σK/RskA complex and the associated biochemical and biophysical characteristics revealed several features of this /anti-σ complex that were hitherto unclear. In particular, these studies revealed a redox sensitive regulatory mechanism in addition to a regulated proteolytic cascade. These features and an analysis of the M. tuberculosis σK/RskA complex vis-à-vis the other characterized σ/anti σfactor complexes are presented in this thesis. This thesis is organized as follows- Chapter 1 provides an overview of prokaryotic transcription. A brief description of the physiology of M. tuberculosis is presented along with a summary of characterized factors that contribute to the pathogenecity and virulence of this bacillus. The pertinent mechanistic issues of σ/anti-σ factor interactions are placed in the context of environment mediated changes in M. tuberculosis transcription. A summary of studies in this area provides a background of the research leading to this thesis. Chapters 2 and 3 of this thesis describe the structural and mechanistic studies on the σK/RskA complex. The crystal structure of the σK/RskA complex revealed a disulfide bond in domain 4 (σK4). σK4 interacts with the -35 element of the promoter DNA. The disulfide forming cysteines were seen to be conserved in more than 70% of σK homologs, across both gram-positive and gram-negative bacteria. The conservation of the disulfide-forming cysteines led us to further characterize the role of this disulfide in σK/RskA interactions. These were examined by several biochemical and biophysical experiments. The redox potential of these disulfide bond forming cysteine residues were consistent with the proposed role of a sensor. The crystal structure and biochemical studies thus suggest that M. tuberculosis σK is activated under reducing conditions. Chapter 4 of this thesis describes the progress made thus far in the structural and biochemical characterization of an intra-membrane protease, M. tuberculosis Rip1 (Rv2869c). This protein is an essential component of the proteolytic cascade that selectively cleaves RskA. The proteolytic steps that govern the selective degradation of an anti-σ factor were first characterized in the case of E. coli σE (Li, X. et al. Proc. Natl. Acad. Sci. USA, 106:14837-14842, 2009). This cascade is triggered by the concerted action of a secreted protease (also referred to as a site-1 protease) and a trans-membrane protease (also referred to as a site-2 protease). M. tuberculosis Rip1 was demonstrated to be bona-fide site 2 protease that acts on three anti-σ factors viz., RskA, RslA and RsmA (Sklar et al., Molecular Microbiology 77:605-617; 2010). To further characterize the role of Rip1 in the proteolytic cascade, this intra-membrane protease was cloned, expressed and purified for structural, biochemical and biophysical analysis. The preliminary data on this membrane protein is described in this chapter. The conclusions from the studies reported in this thesis and the scope for future work in this area is described in Chapter 5. Put together, the σK/RskA complex revealed facets of σ/anti-σ factor interactions that were hitherto unrecognized. The most prominent amongst these is the finding that an ECF σfactor can respond to multiple environmental stimuli. Furthermore, as seen in the case of the σK/RskA complex, the σ factor can itself serve as a receptor for redox stimuli. Although speculative, a hypothesis that needs further study is whether these features of the σK/RskA complex contribute to the variable efficacy of the M. bovis BCG vaccine. In this context it is worth noting that σK governs the expression of the prominent secreted antigens- MPT70 and MPT83. The studies reported in this thesis thus suggest several avenues for future research to understand mycobacterial diversity, immunogenicity and features of host-pathogen interactions. The appendix section is divided into two subparts- Appendix 1 of the thesis is a review on peptidase V. This is a chapter in The Handbook of Proteolytic enzymes (Elsevier Press, ISBN:9780123822192). Appendix 2 of the thesis includes technical details and an extended materials and methods section.

A distinctive feature of host-pathogen interactions in the case of Mycobacterium tuberculosis is the asymptomatic latent phase of infection. The ability of the bacillus to survive for extended periods of time in the host suggests an adaptive mechanism in M. tuberculosis that can cope with a variety of environmental stresses and other host stimuli. Extensive genomic studies and analysis of knock-out phenotypes revealed elaborate cellular machinery in M. tuberculosis that ensures a rapid cellular response to host stimuli. Prominent amongst these are two-component systems and σ factors that exclusively govern transcription re-engineering in response to environmental stimuli. M. tuberculosis σK is a σ factor that was demonstrated to control the expression of secreted antigenic proteins. The study reported in this thesis was geared to understand the molecular basis for σK activity as well as to explore conditions that would regulate σK activity. Transcription in bacteria is driven by the RNA polymerase enzyme that can associate with multiple σ factors. σ factors confer promoter specificity and thus directly control the expression of genes. The association of different σ factors with the RNA polymerase is essential for the temporal and conditional re-engineering of the expression profile. Environment induced changes in expression rely on a subset of σ factors. This class of σ factors (also referred to as Class IV or Extra-cytoplasmic function (ECF) σ factors) is regulated by a variety of mechanisms. The regulation of an ECF σ factor activity at the transcriptional, translational or posttranslational steps ensures fidelity in the cellular concentration of free, active ECF σ factors. In general, ECF σ factors associate with an inhibitory protein referred to as an anti-σ factor. The release of a free, active σ factor from a σ /anti-σ complex is thus a mechanism that can potentially control the cellular levels of an active σ factor in the cell. M. tuberculosis σK is associated with a membrane bound anti-σK (also referred to as RskA) (Said-Salim et al., Molecular Microbiology 62: 1251-1263: 2006). The extracellular stimulus that is recognized by RskA remains unclear. However, recent studies have suggested the possibility of a regulated proteolytic cascade that can selectively degrade RskA and other membrane associated anti-σ factors. The goal of the study was to understand this regulatory mechanism with a specific focus on the M. tuberculosis σK/RskA complex. The structure of the cytosolic σK/RskA complex and the associated biochemical and biophysical characteristics revealed several features of this /anti-σ complex that were hitherto unclear. In particular, these studies revealed a redox sensitive regulatory mechanism in addition to a regulated proteolytic cascade. These features and an analysis of the M. tuberculosis σK/RskA complex vis-à-vis the other characterized σ/anti σfactor complexes are presented in this thesis. This thesis is organized as follows- Chapter 1 provides an overview of prokaryotic transcription. A brief description of the physiology of M. tuberculosis is presented along with a summary of characterized factors that contribute to the pathogenecity and virulence of this bacillus. The pertinent mechanistic issues of σ/anti-σ factor interactions are placed in the context of environment mediated changes in M. tuberculosis transcription. A summary of studies in this area provides a background of the research leading to this thesis. Chapters 2 and 3 of this thesis describe the structural and mechanistic studies on the σK/RskA complex. The crystal structure of the σK/RskA complex revealed a disulfide bond in domain 4 (σK4). σK4 interacts with the -35 element of the promoter DNA. The disulfide forming cysteines were seen to be conserved in more than 70% of σK homologs, across both gram-positive and gram-negative bacteria. The conservation of the disulfide-forming cysteines led us to further characterize the role of this disulfide in σK/RskA interactions. These were examined by several biochemical and biophysical experiments. The redox potential of these disulfide bond forming cysteine residues were consistent with the proposed role of a sensor. The crystal structure and biochemical studies thus suggest that M. tuberculosis σK is activated under reducing conditions. Chapter 4 of this thesis describes the progress made thus far in the structural and biochemical characterization of an intra-membrane protease, M. tuberculosis Rip1 (Rv2869c). This protein is an essential component of the proteolytic cascade that selectively cleaves RskA. The proteolytic steps that govern the selective degradation of an anti-σ factor were first characterized in the case of E. coli σE (Li, X. et al. Proc. Natl. Acad. Sci. USA, 106:14837-14842, 2009). This cascade is triggered by the concerted action of a secreted protease (also referred to as a site-1 protease) and a trans-membrane protease (also referred to as a site-2 protease). M. tuberculosis Rip1 was demonstrated to be bona-fide site 2 protease that acts on three anti-σ factors viz., RskA, RslA and RsmA (Sklar et al., Molecular Microbiology 77:605-617; 2010). To further characterize the role of Rip1 in the proteolytic cascade, this intra-membrane protease was cloned, expressed and purified for structural, biochemical and biophysical analysis. The preliminary data on this membrane protein is described in this chapter. The conclusions from the studies reported in this thesis and the scope for future work in this area is described in Chapter 5. Put together, the σK/RskA complex revealed facets of σ/anti-σ factor interactions that were hitherto unrecognized. The most prominent amongst these is the finding that an ECF σfactor can respond to multiple environmental stimuli. Furthermore, as seen in the case of the σK/RskA complex, the σ factor can itself serve as a receptor for redox stimuli. Although speculative, a hypothesis that needs further study is whether these features of the σK/RskA complex contribute to the variable efficacy of the M. bovis BCG vaccine. In this context it is worth noting that σK governs the expression of the prominent secreted antigens- MPT70 and MPT83. The studies reported in this thesis thus suggest several avenues for future research to understand mycobacterial diversity, immunogenicity and features of host-pathogen interactions. The appendix section is divided into two subparts- Appendix 1 of the thesis is a review on peptidase V. This is a chapter in The Handbook of Proteolytic enzymes (Elsevier Press, ISBN:9780123822192). Appendix 2 of the thesis includes technical details and an extended materials and methods section.