Survival Strategies Of Salmonella Under Host Nitrosative Stress And Its Role In Pathogenesis
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.
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