The genus Salmonella includes facultative intracellular pathogens. Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever in humans killing about 2,00,000 people globally every year. Salmonella enterica serovars Typhimurium (S. Typhimurium) and Enteritidis (S. Enteritidis) cause food poisoning in humans. Salmonellae also cause disease in animals of economic importance like poultry and cattle. Treatment of diseases caused by these notorious pathogens is becoming more and more difficult because of the emergence of drug resistant strains. Thus, it is vital to understand the virulence mechanisms of Salmonella which can lead us to potential drug targets and also help us design effective vaccines. Salmonella has evolved many strategies to enter the host, to evade intracellular and extracellular antimicrobial activities of the host and to extract nutrition in the stringent and hostile environment of the host. These strategies have enabled Salmonella to survive and multiply in the host making it a successful pathogen. Present study deals with four such survival strategies of Salmonella. S. Typhimurium causes a systemic disease in mice that is similar to typhoid fever caused by serovar Typhi in humans. This serves as a good model system to study and understand the pathogenesis of Salmonellae. This model system has been used throughout this study. In the present thesis attempts have been made to identify some novel survival strategies of Salmonella. The thesis is divided into five chapters. Chapter 1 gives an introduction into the basic biology of these notorious pathogens. The diseases caused by Salmonellae are introduced in this chapter. Typhoid fever is discussed in detail covering its epidemiology, clinical features, diagnosis, treatment and prevention. Next section covers the virulence determinants of Salmonella. In this section, Salmonella pathogenicity islands are discussed in detail. This chapter concludes with an overview of molecular pathogenesis of Salmonella covering its invasion strategy and its dangerous life inside the host cell. Salmonella stays and multiplies inside a specialized endosomal compartment of the host cell known as Salmonella-containing vacuole (SCV). It is believed that Salmonella multiplies inside SCV resulting in single big vacuole containing multiple bacteria. The results of Chapter 2 challenge this notion. Using transmission electron microscopy and confocal laser scanning microscopy we show that SCV also divides along with the division of Salmonella resulting in multiple SCVs containing single bacterium per vacuole. We also show that this division is mediated by the molecular motor dynein. This chapter concludes with a discussion on the advantages of SCV division with respect to Salmonella. Successful intracellular pathogens must have some strategy either to avoid lysosomal fusion or to endure the toxic molecules of lysosomes. In case of Salmonella, it is well accepted that SCV-lysosome fusion is blocked. However, the exact mechanism of this process is still unclear. The results of Chapter 3 enhance our understanding of this issue. This chapter explores an interesting possibility of Salmonella reducing the lysosomal number and thereby reducing the chances of SCV-lysosome fusion. Using flowcytometry and confocal laser scanning microscopy, we show that Salmonella decreases the number of acidic lysosomes in murine macrophages. Thus, our results suggest that there is an imbalance in the ratio of vacuoles to acidic lysosomes which decreases the probability of SCV-lysosome fusion thereby helping Salmonella avoid lysosomes. Multicellular organisms use various defense strategies to protect themselves from microbial infections; production of antimicrobial peptides (AMPs) is one of them. Being cationic in nature, AMPs interact and cause pores in the bacterial membrane eventually killing the bacteria. Pathogenic micro-organisms like Salmonella have evolved many strategies to counteract the AMPs they encounter upon their entry into the host systems. S Typhimurium genome has a gene cluster consisting of yejA, yejB, yejE and yejF genes which encode a putative ABC transporter. Chapter 4 deals with the detailed characterization of these genes. Our study shows that these genes constitute an operon. We have deleted the yejF gene which encodes the ATPase component of this putative ABC transporter. The ΔyejF strain showed increased sensitivity to AMPs like protamine, melittin, polymyxin B and human defensins and was compromised to proliferate inside activated macrophages and epithelial cells. In murine typhoid model, the ΔyejF strain displayed decreased virulence when infected intragastrically. These findings suggest that the putative transporter encoded by the yejABEF operon is involved in counteracting AMPs and contributes to the virulence of Salmonella. An important biochemical property of Salmonella that distinguishes it from the closely related E. coli is its inability to ferment lactose. In E. coli, lactose fermentation is carried out by the products of lac operon which is regulated by a repressor encoded by lacI. Salmonella does not have the lac operon and lacI. It has been proposed that S.enterica has lost lac region (lacI and lacZYA) during its evolution. Chapter 5 deals with the evolutionary and physiological significance behind the loss of lac region by S.enterica. We show that expression of LacI in S. enterica suppresses its virulence by interfering with the expression of SPI-2 virulence genes. We also observed that the genome of S. bongori which does not have the virulence genes of SPI-2 has a homologue of LacI. Our results suggest that presence of lacI has probably hindered the acquisition of virulence genes of SPI-2 in S. bongori, whereas absence of lacI has facilitated the same in S. enterica making it a successful systemic pathogen. Thus, lacI has played a remarkable role in the evolution of Salmonella virulence. Brief summary of four studies that are not directly related to survival strategies of Salmonella are included in Appendix. First two studies analyze molecular evolution of SPIs to understand the mechanism of host specificity in Salmonella and the last two studies explore the signaling of lipopolysaccharide (LPS) derived from Salmonella.

Das Schicksal aktivierter T-Zellen wird durch eine Vielzahl NF-kappaB regulierter Ziel-Gene bestimmt, wobei aktivierende und deaktivierende Signale für die Ausbalancierung einer adäquaten T-Zell Antwort benötigt werden. Im Rahmen dieser Arbeit konnte gezeigt werden, dass die negativ-regulatorische Modulierung des Carma1-Bcl10-Malt1 (CBM)-Proteinkomplexes für die Steuerung der NF-kappaB Aktivität in T-Zellen von großer Bedeutung ist. Überraschenderweise ist die Bildung des CBM-Komplexes abhängig von IKKbeta, einer Kinase, die zuvor ausschließlich mit CBM-nachgelagerten Effektorfunktionen in Verbindung gebracht wurde. IKKbeta übernimmt eine duale Funktion bei der Regulation des CBM-Komplexes: Obwohl IKKbeta zunächst für die Bildung des CBM-Komplexes benötigt wird, führt die Phosphorylierung der CBM-Komplexkomponente Bcl10 durch IKKbeta bereits kurze Zeit nach Beginn der T-Zell Aktivierung zu einer Dämpfung der Signalübertragung. Biochemische Analysen zeigen, dass die Phosphorylierung von Bcl10 die Proteinaffinitäten innerhalb des CBM-Komplexes beeinflusst, wodurch es zu einer Umlagerung des Komplexes mit negativ-regulatorischem Effekt kommt. Weiterführende Experimente haben aufgedeckt, dass Bcl10 im Zuge anhaltender T-Zell Stimulation lysosomal degradiert wird. Die Degradation von Bcl10 führt zum Zerfall des CBM-Komplexes und unterbindet die weitere Signalübertragung trotz persistenter Stimulation. Die Tatsache, dass beide in dieser Arbeit identifizierten negativ-regulatorischen Mechanismen am CBM-Komplex angreifen, unterstreicht die Bedeutung dieses Komplexes für die Signalübertragung in T-Zellen. Weiterhin besteht aufgrund der präsentierten Daten Anlass zur Annahme, dass in aktivierten T-Zellen ein vielfältig positiv und negativ regulierter Multikomponentenkomplex gebildet wird, der eine nicht-hierarchische Signalübertragung unterstützt. / A multitude of NF-kappaB regulated target genes determines the fate of activated T cells, whereas activating and de-activating signals are crucial for balancing adequate T cell responses. The presented data illustrate that negative-regulatory modulation of the Carma1-Bcl10-Malt1 (CBM)-complex is of great importance for the control of NF-kappaB activity in T cells. Surprisingly IKKbeta, a kinase that so far was thought to be involved in CBM-downstream effector functions, is needed for CBM-complex formation. IKKbeta exhibits a dual function regulating the CBM-complex: while initially being essential for the formation of the CBM-complex, phosphorylation of the CBM-complex component Bcl10 by IKKbeta shortly after the onset of T cell activation leads to a damping of signal transduction. Biochemical analysis reveal that Bcl10 phosphorylation influences the intermolecular protein affinities of the CBM-complex components causing a remodeling of the complex with a negative-regulatory effect. Further experiments uncover that upon persistent T cell activation Bcl10 is degraded by the lysosome. Bcl10 degradation promotes the collapse of the CBM-complex and thereby interferes with ongoing signal transduction despite persistent stimulation. Considering the fact that both negative-regulatory processes affect CBM-complex activity underscores the important role of this complex in T cell signal transduction. Moreover, the presented data demonstrate that formation of a multi-component signaling complex in activated T cells facilitates versatile positive, negative and non-hierarchical regulation.

Metazoans have evolved conserved mechanisms to promote cell survival under low oxygen tensions by initiating a transcriptional cascade centered on the action of Hypoxia Inducible transcription Factors (HIFs). In aerobic conditions, HIFs are inactivated by ubiquitin-proteasome-mediated degradation of their a subunit, which is dependent on prolyl hydroxylation by 2-oxoglutarate (2-OG) and Fe(II)-dependent prolyl hydroxylases (PHDs). In hypoxia, HIF-$\alpha$ is no longer hydroxylated and is therefore stabilised, activating a global transcriptional response to ensure cell survival. Interestingly, HIFs can also be activated in aerobic conditions, however the mechanisms of this oxygen-independent regulation are poorly understood. Here, I have explored the role of the vacuolar H+-ATPase (V-ATPase), the major proton pump for acidifying intracellular vesicles and facilitating lysosomal degradation, in regulating HIF-$\alpha$ turnover. Unbiased forward genetic screens in near-haploid human cells identified that disruption of the V-ATPase leads to activation of HIFs in aerobic conditions. Rather than preventing the lysosomal degradation of HIF-$\alpha$, I found that V-ATPase inhibition indirectly affects the canonical proteasome-mediated degradation of HIF-$\alpha$ isoforms by altering the intracellular iron pool and preventing HIF-$\alpha$ prolyl hydroxylation. In parallel, I characterised two putative mammalian V-ATPase assembly proteins, TMEM199 and CCDC115, identified by the forward genetic screen and subsequent mass spectrometry analysis. I confirmed that both TMEM199 and CCDC115 are required for V-ATPase function, and established assays to determine how TMEM199 and CCDC115 associate with components of the core V-ATPase complex. Lastly, to measure how V-ATPase activity leads to changes in the labile iron pool, I developed an endogenous iron reporter using CRISPR-Cas9 knock-in technology. This approach confirmed that iron homeostasis is impaired during V-ATPase inhibition, and demonstrated that exogenous ferric iron can restore the labile iron pool in a transferrin-independent manner. Together my studies highlight a crucial link between V-ATPase activity, iron homeostasis, and the hypoxic response pathway.