Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 4 uppsatser.

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.

Foodborne diseases are one among the diseases with high morbidity and mortality rate. The concern is raised with the emergence of pathogenic strains that are resistant to the available set of antibiotics. Conventional regimens fail to treat the infections caused by these pathogens prolonging the sickness leading to increased morbidity and mortality. The situation can get further complicated with the dietary intake of the host. Of late it has been understood that the dietary flavonoids play an important role in regulating the immune system. Curcumin, a pigment from turmeric, is one among such bioflavonoid with an immunomodulatory potential. Curcumin has been a front-line topic of mainstream scientific research for a variety of diseases from cancer to Alzheimer’s to infectious diseases. Curcumin being considered as a spicy panacea is not a remedy for all diseases. Its ability to act differentially as an antioxidant or pro-oxidant can be either beneficial or harmful for the host. It exhibits antioxidant properties at concentrations achievable in the body; this can make the host vulnerable to infections due to the suppression of innate immune responses. Curcumin also suppresses the type 1 immune response, which might lead to alleviation of type 1 immune response disorders. However, the inhibition of type 1 immune response might invite infections with opportunistic pathogens. We have chosen curcumin to assess the effect of diet on the regulation of pathogenesis of Salmonella along with few medically important pathogens like Yersinia enterocolitica, Staphylococcus aureus, Shigella flexneri and Listeria monocytogenes. The thesis is divided into five chapters. As the main focus of the thesis is on Salmonella, in Chapter 1 we introduce diverse aspects of curcumin and the basic biology of Salmonella. Initially the properties of curcumin, the molecule of interest are introduced followed by brief overview to Salmonella biology and pathogenesis. Various activities of curcumin dealing with the variety of diseases are discussed. Further, the introduction to the intricate underlying mechanisms and the functional determinants of curcumin is given. The subsequent sections give an overview of different phases of Salmonella pathogenesis and the molecular mechanisms of Salmonella virulence and host defense. Towards the end of the chapter we discuss the strength, limitations and the distinctive characteristics of the murine model of typhoid fever. Curcumin has gained immense importance for its vast therapeutic and prophylactic applications. Its anti-bacterial effect has been demonstrated in bacteria, like B. subtilis, H. pylori and E. coli. Contrary to this, the results of the Chapter 2 reveals that curcumin at a nontoxic concentration to both host and pathogen, regulates the defense pathways of Salmonella enterica serovar Typhimurium (S. Typhimurium) to enhance its pathogenicity. In a murine model of typhoid fever, we observed higher bacterial load in reticuloendothelial organs when infected with curcumin-treated Salmonella. Curcumin increased the resistance of S. Typhimurium against antimicrobial agents like antimicrobial peptides, reactive oxygen and nitrogen species. It up-regulated the genes involved in resistance against antimicrobial peptides - pmrD and pmrHFIJKLM and genes with antioxidant function - mntH, sodA and sitA. We implicate that the iron chelation property of curcumin has a role in regulating mntH and sitA. Interestingly, we see that the curcumin-mediated modulation of pmr genes is through the PhoPQ two-component regulatory system (TCS). Curcumin downregulates SPI-1 genes required for entry into epithelial cells and upregulates SPI-2 genes required for intracellular survival, through PhoPQ TCS. Thus, this common regulator (PhoPQ) could explain curcumin's mode of action. Another important factor for the pathogen’s success is its ability to counteract the action of antibiotics. Almost all the bactericidal antibiotics act via production of reactive oxygen species in the bacteria. Curcumin has anti-oxidant property that might interfere with the action of antibiotics. Ciprofloxacin is a commonly used anti-typhoidal drug. It kills the bacteria by inhibiting DNA replication and increasing reactive oxygen species in bacterial cell. In Chapter 3 we present the results obtained after the investigation of the interference of curcumin with the anti-bacterial action of ciprofloxacin against Salmonella. We found that curcumin indeed increased the proliferation of Salmonella Typhi and Salmonella Typhimurium in ciprofloxacin treated macrophages by reducing the ciprofloxacin-induced reactive oxygen species. It also inhibited ciprofloxacin mediated DNA damage and the resultant SOS response and filamentation. However, curcumin was unable to rescue the ciprofloxacin induced gyrase inhibition. The reduced antibiotic (ciprofloxacin) efficacy against Salmonella by curcumin might aggravate the disease. Thus, the results of chapter 1 and 2 urge us to rethink the indiscriminate use of curcumin especially during Salmonella outbreaks. Bacteria modulate its virulence determinants in response to the environmental cues. Salmonella being a foodborne pathogen has a very likely chance of getting exposed to turmeric and hence curcumin. In Chapter 4 we have assessed the modulation of motility of S. Typhimurium, an important virulence determinant, by curcumin. We show that curcumin reduced the motility of the S. Typhimurium by decreasing the flagellar density around it. Surprisingly, this was achieved without affecting the expression of the flagellin gene and protein. Curcumin physically adhered to the flagella making it fragile and breaking it into fragments. This can hinder bacterial motility, chemotaxis, adherence and invasion into the host cells. However, aflagellate bacteria are hypervirulent as is the case with our experimental results with curcumin treated bacteria. Curcumin regulates myriad of bacterial (Salmonella) activities increasing its pathogenicity. Curcumin is known to regulate the host defenses in response to the disease. In Chapter 5 we have sought to address the effect of curcumin treatment of host cells on the outcome of infection by different pathogens. Pathogens have evolved different strategies to evade the host innate immune system, one of them being avoiding lysosome mediated degradation. Pathogens like Salmonella, Yersinia, Mycobacterium and Staphylococcus have acquired molecular machinery to inhibit the fusion of the pathogen containing vacuole with lysosomes and multiply within the vacuole whereas other pathogens like Shigella, Listeria and Rickettsia escape into and multiply in the cytosol. In our study we show that pretreatment of macrophage with curcumin increased the fold proliferation of S. Typhimurium, S. aureus and Y. enterocolitica whereas decreased that of S. flexneri and L. monocytogenes. From the results obtained, we can state that curcumin differentially regulates the pathogenesis of vacuolar and cytosolic pathogen. We hypothesized that curcumin pretreatment stabilizes the membrane of pathogen containing vacuole retarding the lysis of the phagolysosome harboring the cytosolic pathogen and hence facilitating its clearance. We indeed observed that the membrane stabilizing effect of curcumin led to increased fusion of cytosolic pathogen with the lysosome, decreasing its proliferation in the cells. As the vacuolar pathogens have an inherent ability to inhibit this fusion, they proliferate better in curcumin treated cells. In a nutshell curcumin can have multiple and sometimes unexpected effects not only on a pathogen’s potential to successfully cause infection but also on the host’s ability to counter it. A brief summary of the study that does not directly deal with the modulation of bacterial pathogenesis by curcumin is included in the Appendix. In this study a novel, simple, sensitive and efficient PCR based assay was devised to detect Salmonella contamination in milk, fruit juice and ice-cream without any pre-enrichment.

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, which belong to the Salmonella enterica species. 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. Salmonella infection occurs via the orofecal route following which it invades the intestinal mucosa through several ways, namely by antigen sampling M cells, CD18+ macrophages present in the intestinal lumen or via a forced entry in the non phagocytic enterocytes. Upon entry Salmonella resides in an intracellular phagosomal compartment called the Salmonella containing vacuole (SCV). The SCV only transiently acquires endocytic markers like TfnR, EEA1, Rab4, Rab5, Rab11 and Rab7. It eventually uncouples from the endocytic pathway to avoid lysosomal fusion and ultimately reaches the golgi apparatus achieving a perinuclear position. The mechanisms by which phagocytes kill the virulent Salmonella are not completely understood, however the role of nicotinamide-adenine dinucleotide phosphate (NADPH) phagocytic oxidase system has been strongly implicated. The generation of reactive oxygen species (ROS) occurs via a membrane-bound flavocytochrome b558, consisting of two phagocytic oxidase components (gp91phox and p22phox) and four cytosolic components, p40phox, p47phox, p67phox, and a GTP-binding Rac protein. Further, professional phagocytes like macrophages generate nitric oxide (NO) that acts as a potent agent to limit the growth of many intracellular pathogens including Salmonella. Chapter:2 Resistance to host Nitrosative stress in Salmonella by quenching L-arginine. Arginine is a common substrate for both inducible nitric oxide synthase (iNOS) and arginase. The competition between iNOS and arginase for arginine contributes to the outcome of several parasitic and bacterial infections. Salmonella infection in macrophage cell line RAW264.7 induces iNOS. Because the availability of L-arginine is a major determinant for nitric oxide (NO) synthesis, we hypothesize that in the Salmonella infected macrophages NO production may be regulated by arginase. Here we report for the first time that Salmonella up-regulates arginase II but not arginase I isoform in RAW264.7 macrophages. Blocking arginase increases the substrate L-arginine availability to iNOS for production of more nitric oxide and perhaps peroxynitrite molecules in the infected cells allowing better killing of virulent Salmonella in a NO dependent manner. RAW264.7 macrophages treated with iNOS inhibitor aminoguanidine reverts the attenuation in arginase blocked condition. Further, the NO block created by Salmonella was removed by increasing concentration of L-arginine. In the whole-mice system arginase I, although constitutive, is much more abundant than the inducible arginase II isoform. Inhibition of arginase activity in mice during the course of Salmonella infection reduces the bacterial burden and delays the disease outcome in a NO dependent manner. Chapter:3 Hrg (hydrogen peroxide resistant gene), a LysR type transcriptional regulator confers resistance to oxidative stress in Salmonella LysR type transcriptional regulators are one of the key players that help bacteria adapt to different environments. We have christened STM0952, a putative LysR type transcriptional regulator in Salmonella enterica serovar Typhimurium as the hydrogen peroxide resistance gene (hrg). By generating a knock out of the hrg gene, we demonstrate that the hrg mutant serovar Typhimurium is sensitive to oxidative products of the respiratory burst, specifically to hydrogen peroxide. The hrg mutant is profoundly attenuated in the murine model of infection and shows decreased intracellular proliferation in macrophages. It was also found to induce increased amount of reactive oxygen species and co-localization with gp91phox in the macrophage cell line, when compared to the wild type. An overproducing strain of this gene showed a survival advantage over the wild type Salmonella under hydrogen peroxide induced stress condition. Microarray analysis suggested the presence of a Hrg regulon, which is required for resistance to the toxic oxidative products of the reticulo-endothelial system. Chapter:4 Importance of the host oxidative stress in antigen presentation and its modulation by Salmonella: Role of TLR Synthetic CpG containing oligodeoxynucleotide TLR-9 agonist (CpG ODN) activates innate immunity and can stimulate antigen presentation against numerous intracellular pathogens. We report that Salmonella Typhimurium growth can be inhibited by the CpG ODN treatment in the murine dendritic cells. This inhibitory effect was shown to be mediated by an increased reactive oxygen species (ROS) production. We further show that the CpG ODN treatment of the dendritic cells during Salmonella infection leads to a ROS dependent increased antigen presentation. In addition, TLR-9 signaling inhibitor was able to inhibit the CpG ODN mediated increased antigen presentation, ROS production and pathogen killing. These data indicate that CpG ODN can improve the ability of the murine dendritic cells to contain the growth of the virulent Salmonella through ROS dependent killing and could as well be used as an effective adjuvant in vaccines against Salmonella infection.

Chapter I Introduction Salmonellae are facultative Gram-negative intracellular pathogens. Different serovars of it causes a variety of diseases in multiple hosts with different disease outcomes. Salmonella enterica serovar Enteritidis and Typhimurium (STM) can infect domestic animals causing gastroenteritis or typhoid like fever. Typhoid fever in humans which is actually caused by Salmonella enterica serovar Typhi still remains a significant health problem in many parts of the world with an estimated annual incidence of nearly 16 million cases and about 600,000 deaths. The infection begins via the orofecal route following which it invades the intestinal mucosa through several ways, namely by antigen sampling M cells, CD18 macrophages present in the intestinal lumen or via a forced entry in the non-phagocytic enterocytes. Upon entry, Salmonella resides in an intracellular phagosomal compartment called Salmonella containing vacuole (SCV) and has several strategies to counteract the host defense mechanisms. Following phagocytosis and its compartmentalization into Salmonella containing vacuole (SCV), a series of defense mechanisms are initiated. These include toxic reactive oxygen species or super oxide production, nitric oxide production, phagosomal acidification and release of hydrolases and defensins through fusion of phagosome with lysosomes generating highly bactericidal environment. The SCV transiently acquires endocytic markers like TfnR, EEA1, Rab4, Rab5, Rab11 and Rab7 and resist killing by avoiding phagosomal maturation and vesicular trafficking of iNOS and NADPH oxidase vesicles. Moreover, Salmonella also uses acidic pH of the SCV (~ pH 4.5) to assemble the Salmonella Pathogenecity Island 2 (SPI-2) type three secretion system (TTSS) which is essential for survival inside the macrophages. Salmonella uses these hostile conditions inside the host as cues for regulating their virulence factors using global regulatory factors. Hfq is one such global regulator playing an important role in many physiological processes and stress responses. Understanding the importance of Hfq regulated genes which impart Salmonella survival advantage under hostile conditions for successful infection will be of particular significance. The host too recognizes pathogen using innate immune receptors present either on the cell surface like TLRs (Toll Like Receptors) or inside the cells like NLRs (Nod Like Receptors). Innate immune receptors recognize pathogen associated molecular patterns (PAMPs) such as Lipopolysacharide (LPS), peptidoglycon (PGN), or hypomethylated DNA or RNA. Recognition of PAMPs by innate receptors leads, via activation of transcription factors (NF-κB and IRF3), to the generation of pro and anti-inflammatory cytokines, chemokines. Vaccination has been practiced for many years and it is one of the most effective methods of controlling infectious diseases like typhoid. At present two licensed vaccines against Salmonella are in use globally namely, Vi polysaccharide subunit vaccine (Typhim Vi™) and live attenuated typhoid vaccine (Vivotif Berma™). Lack of immunological memory, low efficacy (55-75 % protection) and requirement of higher number of doses are the important practical shortcomings associated with the currently used vaccines. So there is a need for a safer and immunogenic vaccine to combat Salmonella infection. Chapter II Salmonella Typhimurium lacking hfq gene induces long term memory response and confers protective immunity Currently available vaccines for typhoid have less-than-desired efficacy and certain unacceptable side effects, making it pertinent to search for new improved ones. Of the various strategies used for the generation of vaccine strains, focus is on manipulation of virulence regulator genes for bacterial attenuation. Hfq is a RNA chaperon which mediates the binding of small RNA to the mRNA and assists in post-transcriptional gene regulation in bacteria. Salmonella hfq deletion mutant is highly attenuated in vitro as well as in vivo implying its role in bacterial virulence. In this study, we have evaluated the efficacy of the Salmonella Typhimurium hfq deletion mutant as a candidate for live oral vaccine against Salmonella infection in murine salmonellosis model. The hfq deletion mutant is not only able to confer protection when administered orally to the mice against oral challenge with serovar Typhimurium virulent strain, but also elicits cross protective immune responses to other Salmonella serovars. The vaccine candidate appears to be safe for use in pregnant mice. This protection is partially mediated by the increase in the number of CD4+ T lymphocytes upon vaccination. STM hfq deletion mutant further exhibited significant increase in the lipopolysaccharide as well as outer membrane protein specific IgG in the serum as well as secretory S-IgA in the intestinal washes. In addition, vaccination led to an increased serum IFN-γ and IL-6. Taken together, our results suggest that the Salmonella Typhimurium hfq deletion mutant can be an excellent live oral vaccine candidate. Chapter III Acidic pH induced STM1485 gene governs intracellular replication and pathogenesis in Salmonella During the course of infection, Salmonella has to face several potentially lethal environmental conditions such as low pH both inside and outside the host. The ability to sense and respond to the acidic pH is crucial for survival and replication of Salmonella. Exposure to acidic pH results in the expression of large pool of virulence genes. One such gene highly up regulated inside the macrophage is STM 1485. In order to understand physiological role of STM 1485 in Salmonella pathogenesis, STM 1485 gene was deleted chromosomally and characterized in vitro and in vivo. In vitro the mutant did not show any growth defects at pH 4.5 and no difference in acid tolerance response. The 1485 deletion mutant was compromised in its capacity to proliferate inside the cells and is further lowered inside activated macrophages. We further showed that surface translocation of SPI-2 encoded translocon protein SseB was reduced at low pH in vitro in STM 1485 mutant and the mutant was found to colocalize with lysosomes higher than the wild type. In addition, the STM 1485 deletion mutant displayed decreased virulence in murine typhoid model when infected intragastrically. Based on our results, we hypothesize that the acid shock protein encoded by the STM 1485 might be involved in the formation of SPI-2 translocon at low pH and there by contributing to the virulence of Salmonella. Chapter IV Role of Nod1 in sensing vacuolar pathogen Salmonella in epithelial cells Nod1 and Nod2 are the archetypal members of the Nod like receptor family (NLR) and they recognize distinct peptidoglycan motifs of Gram-negative and Gram-positive bacteria respectively. Role of Nod1 and Nod2 in sensing bacterial pathogens have been elucidated. However, the role of Nod1 in sensing vacuolar pathogen Salmonella in epithelial cells is not understood. So in this study we investiged the role of Nod1 in the innate immune response against Salmonella in epithelial cells. We demonstrate that the recognition of Salmonella by Nod1 leads to NF-κB activation and this activation is diminished in epithelial cells expressing a dominant-negative Nod1 construct or Nod1 shRNA. Using a set of Salmonella mutants we show that the availability of ligand is higher when the bacteria were in cytosol rather than in vacuole. Further we also observed that the Nod1 mediated killing of Salmonella is mediated through the defensins. Based on our results we hypothesize that Salmonella uses its vacuolar niche to evade Nod1 mediated innate immune response.

Chapter-I Introduction The bacteria known as Salmonellae are gram-negative, rod-shaped intracellular pathogenic bacilli that belong to the family Enterobacteriacea and causes typhoid fever. Enteric fever or typhoid fever is a systemic infection caused by human specific enteric pathogen S.typhi. Another very similar but less severe disease, paratyphoid fever, is caused by another human pathogen S.paratyphi A, B and C and S.sendai. Typhoid fever is estimated to have caused 21.6 million illness and 1-4 % death worldwide in the year of 2000 effecting all ages and 90% of death occurs in Asia. In Asia, the incident of typhoid fever was highest with 274 cases per 100,000 persons worldwide, especially in Southeast Asian countries and the Indian subcontinent, followed by sub-Saharan Africa and Latin America with 50 cases per 100,000 persons. Transmission of the disease occurs through faecal-oral route upon ingestion of contaminated water and food. Salmonella can stay for long in ground and pond water. Typhoid fever can be fatal if left untreated and there are reports of 10-30 fatality in such cases and can persist for weeks. Prevention is better than cure. Same hold true even for typhoid fever also. The important and key preventive measures are clean and safe water, safe food, personal hygiene and appropriate sanitation. There are many antibiotics for typhoid fever but till now there are only two licensed vaccine recommended by the World Health Organization for the typhoid fever, one Vi polysaccharide subunit vaccine (sold as Typhim Vi by Sanofi Pasteur and Typherix by GlaxoSmithKline) which is administered through intramuscular route and another one is live oral attenuated vaccine Ty21a (sold as Vivotif Berna) for oral immunization. Both the vaccines are recommended to be used for the children above the age of 3-5 years. Both are between 50 to 80% protective and are recommended for travelers to areas where typhoid is endemic. Salmonella has evolved many strategies to survive inside host system especially during initial time of infection when bacteria counteract to host AMPs in intestine lumen. Salmonella has many pathogenesis island which help bacteria to invade the host system e.g. SPI-1(Salmonella pathogenicity island -1) and also help in intracellular survival as well proliferation e.g. SPI-2 (Salmonella pathogenicity island -2). Salmonella has many strategies to evade host immune system, one of them which is very important for bacteria is LPS modification. Salmonella is capable to modify its own LPS by increasing the +ve charge and increasing AMPs resistance. This modification and resistance is brought about by PhoP/Q and pmrA/B two different two-component system (TCS). These TCS regulate many genes like pmrD, pmrC, pmrG, pmrH-M operon, pmrE etc, which are important for LPS modification by adding 4-amino-arabinose and provide antimicrobial peptide resistance. Chapter-II Development of live attenuated Salmonella vaccine The superiority of live attenuated vaccines in systemic salmonellosis has been proven over killed and subunit vaccines, because of its ability to induce protective cell mediated immunity by CD8+ T cells. A live attenuated Salmonella enterica serovar Typhimurium vaccine has been developed by systematic site directed deletion of the pmrG-HM-D chromosomal genomic loci. This gene confers involved in antimicrobial peptide resistance and is involved in LPS modification, both of which are the major immune evasive mechanisms in Salmonella. The efficacy of the newly developed strain in inducing protection against mortality after challenge with the virulent wild type Salmonella typhimurium 12023 was evaluated in mice model of typhoid fever. Animals were immunized and then boosted on days 7 and 14. Following challenge with virulent S. typhimurium 12023, organ burden and mortality of vaccinated mice were less compared to non-immunized controls. The vaccine strain also induced elevated CD8+ T cells in the vaccinated mice. This multiple mutant vaccine candidate appears to be safe for use in pregnant mice and provides a model for the development of live vaccine candidates against naturally occurring salmonellosis and typhoid fever. Chapter -III A Safe and Efficient Vaccine against Salmonella Infection During Pregnancy Pregnancy is a transient immuno-compromised condition which has evolved to avoid the immune rejection of the fetus by the maternal immune system. The altered immune response of the pregnant female leads to increased susceptibility to invading pathogens, resulting in abortion and congenital defects of the fetus and a subnormal response to vaccination. Active vaccination during pregnancy may lead to abortion induced by heightened cell mediated immune response. In this study, we have administered the highly attenuated vaccine strain ΔpmrG-HM-D (DV-STM-07) in female mice before onset of pregnancy and followed the immune reaction against challenge with virulent S. typhimurium in pregnant mice. This vaccine strain gives protection against Salmonella in pregnant mice and also prevents Salmonella induced abortion. This protection is conferred by directing the immune response towards humoral immunity through Th2 activation and Th1 suppression. The low Th1 response prevents abortion. The use of live attenuated vaccine just before pregnancy carries the risk of transmission to the fetus. We have shown that this vaccine is safe as the vaccine strain is quickly eliminated from the mother and is not transmitted to the fetus. This vaccine also confers immunity to the new born mice of vaccinated mothers. Since there is no evidence of the vaccine candidate reaching the new born mice, we hypothesize that it may be due to trans-colostral transfer of protective anti-Salmonella antibodies. Chapter-IV Crosstalk between Salmonella genes involved in antimicrobial peptide resistance (pmrG, pmrD, pmr H-M) The pmr system of Salmonella consists of many genes and they are regulated by two component system (TCS), PmrA/B and PhoP/Q. These two component systems are activated at different Mg 2+and Fe3+ condition, low pH and the presence of antimicrobial peptides. Downstream genes like pmrD, pmrG, pmrH-M operon, pmrE, pmrC ect which are regulated by these TCS are involved in LPS modification and AMPs resistance. When these genes were deleted a highly attenuated strain with good vaccine potential was developed. The high degree of attenuation of the vaccine strain is a combined effect of the deletion of the all genes, when single mutation of the two single genes and the operon were created; the attenuation was not as good as the vaccine strain. When tried checking the cross-talk between these genes in vaccine strain and the single mutants of pmrD, pmrG and pmrH-M operon. In one of the previous report pmrH-M mutant was shown to be attenuated through oral route but not through intra-peritoneal route. However, pmrD-HM-G mutant (DV-STM-07) was attenuated when administered through both the routes of infection. To further explain the cross-talk and regulation of these genes, promoter analysis was done for all genes individually in different mutant background of pmrD, pmrG, pmrH-M and DV-STM-07. We hypothesize that the superior attenuation of the triple mutant is achieved because of transcriptional cross-regulation that exists between these genes which attenuates the bacteria when administered through the intra-peritoneal route.

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.

Chapter Introduction Salmonellae are gram negative bacteria that cause gastroenteritis and entericfever. S. enterica is divided into seven phylogenetic groups, subspecies 1, 2,3a, 3b, and 4, 6, 7. Subspecies1 includes 1,367 serovars, some of which are commonly isolated from infected birds and mammals. The other subspecies mainly colonize cold blooded animals. Salmonella typhimurium, Salmonella typhiandSalmonella enteritidis are some of the serovars, which belong to s.enterica species. S. typhimurium is one of the important causes for food poisoning in humans. It causes typhoid like fever in mice. In immuno compromised patients the infection is often fatal if it is not treated with antibiotics. Clinical features of food poisoning include abdominal pain, vomiting, nausea, abdominal cramps, dehydration etc. S. typhi causes typhoid fever in humans. No other host has been identified for this serovar. Main source of infection is contaminated food and water. No age is exempted but it is less common before2 years. Incubation period is 360 days. Clinical features include stepladder type fever, malaise, headache, hepato splenomegaly, coated tongue, Neutrogena etc. It may be fatal if untreated. Among the serovars of Salmonella infecting humans S. typhimurium and S. typhi are the most important. While S. typhimurium infects many host species including birds and mammals, S. typhi is single host adapted and infects only human. The single host adaptation of S. typhi presents it with the need for establishing are servoir of infection in the community which can serve as a source of fresh infection. Also the single host adaptation of S. typhi has made it a highly specialized pathogen which has evolved certain unique genes needed for human colonization at the same time has lost a set of genes which are needed for survival in other hosts and in the highly variable external environment. This has led to the accumulation of a vast number of pseudo genesin S. Typhi. A comparative study of the two serovars is useful in many ways. Due to varied host defense systems encountered by the two serovars owing to different niche of infection the bacterial counter defense mechanisms are also different. By focusing on the differences between genes involved in the bacterial defense of host immune response we can decipher the role played by various genes in combating the antibacterial host response. Chapter 2 The role of TolA and peptidoglycan modification in detergent resistance of pathogenic Salmonella The major Salmonella serovars that infect human are Salmonella enterica serovar Typhi (S.typhi) which cause systemic typhoid and Salmonella enterica serovar Typhimurium (S. typhimurium) which cause gastro enteritis. S. typhi resides in the gall bladder during chronic infection and S .typhimurium infects intestine .Thus both pathogens encounter high concentrations of bile and have developed mechanisms to counter it. The Tol Pal complex spanning the outermembrane and the inner cytoplasmic membrane plays an important role in maintaining the stability of the outer membrane and providing detergent resistance. The tolA gene of S. Typhi Is shorter by 27 aminoacid than S. typhimurium. The tolA gene knockout of S. typhimurium and S. typhi differed in their tritonX resistance behavoiur, morphology and low osmolality tolerance. S. typhi tolA was unable to complement the tolA defect in S. typhimurium which could probably due to the difference in the peptidoglycan layer. An analys is of the peptidoglycan modifying genes of both the serovars revealed that dacD, pbgP, ynhG are different. dacD, pbgP genes are pseudogenes in S. typhi and ynhG has a major deletion in S. typhi. Further studies reveal that a double knockout of dacD and pbpG in S. typhimurium makes it sensitive to low osmolality similar to S. typhi. Based on these results we propose a mechanism, where shortening of TolA increases detergent resistance by bringing the outer membrane into closer contact with the peptidoglycan layer, but this is achieved at the cost of reduced Lpp (Bruan’slipoprotein) peptidoglycan linkage which plays a major role in low osmolality tolerance. The pathogen S. typhi is highly adapted to the human host and cannot infect any other host. The single host adaptation and the need to survive in high concentrations of bile have made S. typhi to acquire higher bile resistance at the cost of lowered osmotic tolerance through shortening TolA and reduced Lpp and peptidoglycan binding. Chapter 3 Development of a DNA vaccine against Salmonella The immune response against Salmonella is multifaceted involving both the innate and the adaptive immune system. The characterization of specific Salmonella antigens inducing immune response could critically contribute to the development of epitope based vaccines for Salmonella. We have tried to identify aprotective Tcellepitope (s) of Salmonella, as cell mediated immunity conferred by CD8+T cells is the most crucial subset conferring protective immunity against Salmonella. It being a proven fact that secreted proteins are better in inducing cell mediated immunity than cell surface and cytosolic antigens, we have analyzed all the GenBank annotated Salmonella pathogenicity island 1 and 2 secreted proteins of S. typhimurium and S. typhi. They were subjected to BIMAS and SYFPEITHI analysis to map MHCI and MHC II binding epitopes. The huge profile of possible T cell epitopes obtained from the two classes of secreted proteins were tabulated and using a scoring system that considers the binding affinity and promiscuity of binding to more than one allele, SopB and SifB were chosen for experimental confirmation in murine immunization model. The entire Sop Band SifB genes were cloned into DNA vaccine vectors and were administered along with live attenuated Salmonella and it was found that SopB vaccination reduced the bacterial burden of organs by about 5fold on day4 and day8 after challenge with virulent Salmonella and proved to be a more efficient vaccination strategy than live attenuated bacteria alone. Chapter 4 PCR based diagnosis and Serovar Determination of Blood Borne Salmonella Typhoid fever is becoming an ever increasing threat in the developing countries. We have improved considerably upon the existing PCR based diagnosis method by designing primers against a region which is unique to S. typhiand S. paratyphiA, corresponding to the gene STY0312 in S. typhi and its homolog SPA2476 in S. paratyphiA. An additional set of primers amplify another region in S. typhi CT18 and S. typhiTy2 corresponding to the region between the genes STY0313 toSTY0316 but which is absent in S.paratyphi A. The threat of false negative result arising due to mutation in hypervariable genes has been reduced by targeting a gene unique to typhoidal Salmonella as a diagnostic marker. The amplified region has been tested for genomic stability by amplifying them from clinical is olates of patients from various geographical locations in India, there by showing that this region is potentially stable. These set of primers can also differentiate between S. typhiCT18, S. typhiTy2 and S. paratyphi A which have stable deletions in this specific locus. The PCR assay designed in this study has a sensitivityof95%ascompared to the Widal test which had only 63%. As observed, in certain cases the PCR assay was more sensitive than the blood culture test as the PCR based detection could also detect dead bacteria.

Chapter -I Introduction Salmonella is a pathogen well-known for its ability to infect a wide variety of hosts and causes disease ranging from mild gastroenteritis to typhoid fever. During infection, it is exposed to a myriad of conditions; from the aquatic environment, the gut lumen to the phagolysosome. The success of Salmonella as a pathogen lies in its ability to sense each of these environments and adapt itself for survival and proliferation accordingly. This is done mainly via the action of specific two-component systems (TCSs) which sense cues specific to each of these niches and trigger the appropriate transcriptional reprogramming. This reprogramming is best studied for the genes directly known to be involved in virulence. In the case of Salmonella, most of these genes are a part of specific clusters, acquired through horizontal gene transfer, known as Salmonella Pathogenicity Islands (SPIs). Of the various SPIs, the two most important are SPI-1 and SPI-2. SPI-1 is classically involved in orchestrating bacterial invasion of non-phagocytic cells in the gut, allowing the pathogen to invade the host. Furthermore, its role is well characterized in the classic inflammation associated with gastroenteritis. On the other hand, SPI-2 is specialized for survival within the harsh intracellular environment of host cells such as macrophages and epithelial cells. Other important virulence determinants include motility, chemotaxis as well as adhesins. The transcription of these virulence genes is under tight regulation and responsive to environmental conditions. Many small molecules such as short chain fatty acids, pp(p)Gpp, bile and acyl homoserine lactones among others are known to be potent regulators of virulence in Salmonella. Furthermore, the metabolic products of the normal flora in the gut also affect its virulence. Thus the metabolic status, of both the host as well as the pathogen, plays an important role in determining the outcome of the infection. Many metabolic enzymes and their products are now known to directly or indirectly affect virulence gene expression. In this study, we explore one such class of metabolic enzymes viz amino acid racemases. They catalyze the chiral conversion of L-amino acids to D-amino acids and vice versa. We have studied the biochemical properties of two such non-canonical racemases as well as their role in bacterial survival and pathogenesis. Chapter-II Identification and characterization of putative aspartate racemases in Salmonella Amino acid racemases, such as alanine and glutamate racemases, are ubiquitously found in all bacteria and they play an essential role in cell wall biosynthesis. Recently it has been found, that bacteria possess other amino acid racemases which produce non-canonical D-amino acids. These D-amino acids, upon secretion, further orchestrate various phenotypes such as cell wall remodeling and biofilm dispersal. In this study, we have explored the ability of Salmonella to produce such non-canonical D-amino acids. The genome of S. Typhimurium possesses genes encoding two putative aspartate racemases; ygeA and aspR. These genes were maximally expressed in mid-log phase of bacterial growth and their corresponding proteins ar localized in the outer membrane of the bacterium. The biochemical characterization of the proteins YgeA and AspR revealed that only the latter is catalytically active under in vitro conditions. AspR could catalyze the conversion of L-Aspartate to D-Aspartate and vice versa, however was unable to use any other amino acid as its substrate. With atleast one of the racemases showing catalytic activity, the profiling of the secreted D-amino acids in Salmonella conditioned medium was undertaken using LC-MS. It was observed that the bacterium actively secreted specific D-amino acids such as D-Ala and D-Met into the culture medium in a growth-phase dependent manner. Furthermore, analysis of the secreted D-amino acid profile of the strains lacking either one or both the racemases revealed that atleast a subset of the secreted D-amino acids were dependent on the activity of YgeA and AspR. Thus, D-amino acids secreted by S. Typhimurium might represent a novel class of signaling molecules. Chapter – III Role of aspartate racemases in growth and survival of S. Typhimurium In order to understand the role of ygeA and aspR in vivo, we created knockouts of these genes (both single as well as double knockout) in S. Typhimurium using λ Red recombinase strategy. These knockouts were then assessed for their growth and morphology. The aspartate racemase knockouts behave similar to the wild type during growth in LB as well as M9 minimal medium. While their gross morphology remained the same as the wild type, the size distribution of the racemase knockouts was slightly different in the stationary phase. Unlike the wild type bacteria, the mutants did not exhibit the characteristic reduction in cell size upon entry into stationary phase. In addition, the survival of the mutants in the presence of cell wall damaging agents such as bile and Triton-X 100 was compromised as compared to the wild type. This can be ascribed to changes in the cell wall of the bacterium, wherein the mutants accumulated peptidoglycan in the stationary phase of growth. This suggests that aspartate racemases might have an effect on cell wall biosynthesis in Salmonella in the stationary phase. Another important strategy employed by bacteria to survive in stress conditions is biofilm formation. It was seen that the mutants were compromised in their ability to form a biofilm at the liquid-air interface in vitro. This defect is due to a transcriptional downregulation of the genes required for biofilm formation. These results demonstrate that, contrary to the established inhibitory effects of D-amino acids on biofilms of various bacteria, the aspartate racemases appear to act as positive regulators of biofilm formation in Salmonella. Chapter – IV Involvement of aspartate racemases in the regulation of Salmonella pathogenesis Salmonella’s success as a pathogen can be broadly assessed by its ability to invade and replicate within two major cell types: epithelial cells and macrophage-like cells. We have studied the fate of the aspartate racemase knockout strains in both these cell types. While the mutants replicate as well as the wild type in macrophage cell lines, their ability to invade epithelial cell lines is highly compromised. This defect can be ascribed to the downregulation of the Salmonella Pathogenicity Island-1 (SPI-1) in the racemase knockouts at the transcriptional level. One of the major pathways that regulate SPI-1 activation is the flagellar pathway. It was observed that in addition to SPI-1, the motility of the racemase mutants was also highly compromised. The mutants did not possess any flagella and showed a high transcriptional downregulation of all the three classes of flagellar genes. Transcriptome analysis revealed a global reprogramming in the aspartate racemase mutants, resulting in the differential regulation of motility, adhesion, amino acid transport, cell wall biosynthesis and other pathways. Of the genes upregulated in the knockouts, FimZ is known for its negative effect on motility and might be responsible for the observed downregulation of the flagellar regulon. This suggests that ygeA and aspR might be repressors of fimbrial gene expression. In totality, the racemases affected the pathogenesis of Salmonella, where the double knockout was severely compromised in the colitis model of infection. Overall the study is the first to identify secretion of non-canonical D-amino acids by Salmonella and suggests that YgeA and AspR might be the source of the same. This is supported in part by in vitro studies with the purified proteins. Studies in vivo further highlight the possible substrates that might be utilized by these enzymes. Physiologically, the aspartate racemases appear to regulate cell wall remodeling and biofilm formation. In contrast to the established literature, aspartate racemases (and their possible D-amino acid products) seem to be essential for formation of biofilms and regulate this phenotype at the transcriptional level. Furthermore, our studies put forth aspartate racemases as novel positive regulators of Flagella and SPI-1, affecting the success of Salmonella in the colitis model of infection in mice. Transcriptome analysis hints at the pleiotropic effects of aspartate racemases in Salmonella, bringing forth hitherto unexplored roles for this class of enzymes in the biology of this pathogen.