Chapter: 1 Introduction Genus Salmonella is a Gram-negative rod shaped facultative anaerobic bacteria that can survive inside the host macrophages and cause persistent infection. Salmonella Typhimurium, Salmonella Typhi and Salmonella Enteritidis are the serovars belonging to Salmonella enterica. S. Typhi causes typhoid fever in humans. S. Typhimurium is one of the important causes for food poisoning in humans. It causes typhoid like fever in mice and serves as a good model system to study Salmonella pathogenesis. Upon entry Salmonella resides in an intracellular phagosomal compartment called Salmonella containing vacuole (SCV). It eventually uncouples from the endocytic pathway to avoid lysosomal fusion and ultimately reaches the golgi apparatus achieving a perinuclear position. Professional phagocytes like macrophages generate nitric oxide (NO) that acts as a potent agent to limit the growth of many intracellular pathogens including Salmonella. Upon activation of the inducible nitric oxide synthase (iNOS), NO is produced continuously at a high rate in the presence of adequate Larginine supply. Nitric oxide synthases catalyze the oxidation of one of the guanidino nitrogens of larginine to nitric oxide (NO). Of the multiple NOS isoforms that can catalyze NO synthesis, iNOS is mostly associated with antimicrobial activity. Host expression of iNOS is primarily regulated at the transcriptional level and can be stimulated following interaction with microbial products or in response to cytokines such as interleukin 1 (IL1), tumor necrosis factor α (TNFα) and interferon γ (IFNγ). To date, mutations that inactivate iNOS in humans have not been described. The importance of iNOS in human infection can therefore only be understood from indirect evidence and experimental models. Despite initial difficulty in demonstrating iNOS expression and NO production by human mononuclear phagocytes, an increasing body of evidence has identified a number of chronic inflammatory conditions, infectious diseases and in vitro treatments that stimulate iNOS mRNA expression and protein synthesis associated with NO bioactivity in human macrophages. Numerous studies have documented the production of RNIs in rodent models of Salmonella infection. Plasma nitrite and nitrate levels, a measure of RNI generation, have been shown to rise significantly after systemic infection of mice with S. Typhimurium. Chapter:2 Role of nirC in Salmonella infection-Nitrosative stress response. Activation of macrophages by interferon gamma (IFNγ) and the subsequent production of nitric oxide (NO) are critical for the host defense against Salmonella enterica serovar Typhimurium infection. We report here the inhibition of IFNγ induced nitric oxide production in RAW264.7 macrophages infected with the wild type Salmonella. This phenomenon was shown to be dependent on the nirC gene, which encodes a potential nitrite transporter. We observed a higher NO output from the IFNγ treated macrophages infected with the nirC mutant Salmonella. The nirC mutant also showed significantly decreased intracellular proliferation in a NO dependent manner in the activated RAW264.7 macrophages and in liver, spleen and secondary lymph nodes of mice, which was restored by complementing the gene in trans. Under acidified nitrite stress, a 2fold more pronounced NO mediated repression of SPI2 was observed in the nirC knockout strain when compared to the wild type. This enhanced SPI2 repression in the nirC knockout led to a higher level of STAT1 phosphorylation and iNOS expression than the wild type strain. In the iNOS knockout mice, the organ load of the nirC knockout strain was similar to the wild type strain indicating the fact that the mutant is exclusively sensitive towards the host nitrosative stress. Taken together, these results reveal that intracellular Salmonella evade their killing in the activated macrophages by down regulating IFNγ induced NO production and highlights the critical role of nirC as a virulence gene. Chapter:3 Salmonella mediated utilization of the host Arginine pool for intracellular growth -a novel strategy to survive. Cationic amino acid transporters (CAT) are crucial regulators of both the nitric oxide synthase and arginase activity in the host cells as they regulate the Larginine availability. In this study, we show that Salmonella induces arginase activity in both the bone marrow derived macrophages and in dendritic cells in a LPS dependent manner. Further evidence is provided suggesting that the Salmonella mediated arginase induction is accompanied by an enhanced arginine uptake in the infected cells by up regulation of the expression of both mouse cationic amino acid transporters mCAT1 and mCAT2B. The bacterial growth was reduced in the presence of inhibitors of both arginase and arginine transport. We also observed that the argT knockout strain in Salmonella coding for an arginine permease was defective in the Larginine uptake and was also attenuated for growth in the mice model of infection. By utilizing both host and bacterial arginine transporters, Salmonella can access the host Larginine pool in the cytosol. The host CAT transporters co localize with the Salmonella containing vacuole in both the bone marrow derived macrophages and in dendritic cells. Thus the host arginine is channelized to the intracellular Salmonella for its growth and this novel strategy plays a pivotal role to counteract the stringent nutrient condition for the intracellular bacteria. On the other hand this channelization should ultimately decrease the substrate for NO production and serve as a survival strategy of the pathogenic Salmonella under host nitrosative stress.

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.