Insights Into The Contribution Of Hfq In Salmonella Pathogenesis : Possible Role In Immune Evasion And Vaccine Development

Abstract

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.