Salmonellen spp. sind invasive, intrazelluläre Pathogene, die in einem membranumhüllten Kompartiment innerhalb der infizierten Wirtszelle überleben. Wie auch andere intrazelluläre Pathogene repliziert Salmonella in dieser intrazellulären Nische, obwohl es anscheinend von sowohl extra- als auch intrazellulären Nährstoffquellen isoliert ist. Wir zeigen hier, dass intrazelluläre Salmonella den Proteinabbau des Wirts ausnutzen, um Aminosäuren in Form von Peptiden zu erhalten. Dieser spezielle, auch als Chaperon-vermittelte Autophagie bekannte, Abbauweg spielt eine Rolle im Transport zytosolischer Proteine zum Abbau im Lysosom. Ein Salmonellenmutant, der nur in Anwesenheit von Peptiden im Medium als Aminosäurenquelle wächst, wies intrazellulär eine Wachstumsrate auf, die der des Wildtyps ähnlich war. Dies deutet darauf hin, dass Peptide intrazellulär für Salmonella zugänglich sind. Wir fanden heraus, dass die Salmonella-enthaltende Vakuole (SCV, Salmonella containing vacuole) die Wirtproteine LAMP-2A und Hsc73, Kernkomponenten von CMA, anzieht, jedoch nicht lysosomale Proteine wie LAMP-2B und LIMP-2. Im Gegensatz zum Salmonellawildtyp zeigte der peptidabhängige Mutantentstamm stark verringertes Wachstum, wenn die Wirtszellen mit CMA-Inhibitoren behandelt wurde. Diese Ergebnisse zeigen einen neuen Mechanismus auf, durch den ein intrazelluläres Pathogen vom membranumhüllten Kompartiment aus Zugriff auf Cytosol der Wirtzelle zur Beschaffung von Nährstoffen hat. Wir schlagen vor, dass diese Ergebnisse eine Erklärung für die Rückfälle von persistenten Salmonellainfektionen liefern können. Des Weitern schlagen wir diesen Mechanismus als moegliches Ziel antibakterieller Therapeutika zur Bekämpfung intrazellulärer Pathogene vor. / Salmonella spp. are invasive, intracellular pathogens which survive and proliferate within a membrane-bound compartment inside infected host cells. Like other intracellular pathogens, Salmonella replicates within this intracellular niche, despite its apparent isolation from both extra- and intracellular sources of nutrients. Here, we show that intracellular Salmonella acquire amino acids in the form of peptides by co-opting the host protein degradation pathway known as chaperone-mediated autophagy (CMA) involved in the transport of cytosolic proteins to the lysosome for degradation. A mutant of Salmonella strictly dependent upon peptides in growth media as a source of amino acids, showed intracellular growth similar to the wild-type strain in host cells, indicating intracellular access to peptides. We found that the Salmonella-containing vacuole (SCV) acquires the host cell proteins LAMP-2A and Hsc73, key components of CMA, but excludes lysosomal proteins such as LAMP-2B and LIMP-2. In contrast to wild-type Salmonella, the peptide-dependent mutant strain showed a severe reduction in growth when host cells were treated with inhibitors of CMA.. These results reveal a novel means whereby an intracellular pathogen can access the host cell cytosol to acquire nutrients from within its membrane-bound compartment. We suggest these results may provide an explanation for relapse infections resulting from persistent Salmonella infections, and suggest a possible means of targeting antibacterials against intracellular pathogens.

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.