The objectives of this proposal are to develop and characterize an attenuated strain of the food-borne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). This work will define the role of global virulence gene regulator gidA in Salmonella virulence. We will examine the ability of Salmonella gidA mutant to cause infections in vitro cell culture system as well as in animal model of infection. These data will also provide information on the immuno-inflammatory responses in the host and the potential use of such mutant for delivering heterologous antigens. Further, the proposed work will determine the ability of the gidA mutant to immunize mice and provide protection against subsequent challenge with the lethal dose of WT Salmonella and establish the mechanistic base of such immunity. This information will be essential in determining the suitability of such mutant as a vaccine candidate.
NON-TECHNICAL SUMMARY: Salmonella causes large numbers of human infections due to consumption of contaminated food. The disease is characterized by abdominal cramps, diarrhea, nausea, vomiting, and high number of bacteria in the blood. The Center for Disease Control and prevention estimates that 1.5 million people per year are affected by Salmonella-related illnesses. Long term goals of this research are to develop strategies to protect animal from Salmonella infection and hence reduce human outbreaks. We created weakened (attenuated) type of Salmonella for use as vaccine to protect animals against Salmonella infection. The attenuation of Salmonella was made by deletion of. gidA gene (code for bacterial factors (virulence) enable them to cause diseases. Therefore, this deletion makes Salmonella less capable of causing infections in animals and humans. The purpose of this study is to test the ability of attenuated Salmonella to invade cultured intestinal cell and to induce the release inflammatory mediators. We will test the ability of such attenuated Salmonella to cause death in mice infected with different bacterial doses, compared to mice infected with the wild-type (non-attenuated) Salmonella. It is essential to examine whether injection of mice with the attenuated bacteria would provide protection against lethal infection of Salmonella. This study will also tell us about how these animals are protected from death when infected with Salmonella. In this experiment, mice will also be infected with non-lethal dose of bacteria and blood samples and tissues will be collected and examined for the toxic effect of the bacteria. Such information is critical to determine potential use of attenuated bacteria as a vaccine candidate.
APPROACH: In objective 1, we will test the ability of gidA mutant to bind and invade T84 intestinal epithelial cells. Cell death and cytotoxic effect of the mutant on macrophages and intestinal epithelial cells will be determined using microscopic examination and LDH release assay. Levels of inflammatory cytokines/chemokines in RAW264.7 macrophages and T84 cells infected with the wild type Salmonella and its mutant will be measured using BioPlex assay. Objective 2 will investigate the effect of GidA deletions in an in vivo mouse model of salmonellosis. We will determine LD50, and induction of inflammatory cytokines, systemic bacterial load and clearance, and histopathology will also be evaluated in Swiss Webster mice. The ability of each mutant to invade, survive and replicate in vivo will be determined. In addition, samples from liver and spleen will be saved in 10% buffered formalin for H&E staining and histopathological examination. To evaluate the level of cytokines in mice following inoculation with the mutant and the WT, blood samples will be taken at day 0, 1, 3, and 7 post-infection to determine TNF, IFN-, IL-1, IL-6, and IL-8 levels in serum using Bioplex method. In objective 3, we will determine if mice immunized with such mutant would protect animals against lethal dose challenge of the wild-type (WT) Salmonella. We believe that the GidA mutant will be avirulent/less virulent in mice. We will examine whether mice infected with such mutant will be protected when challenged with lethal challenge of WT Salmonella. The immunized mice will be challenged with the WT counterpart 8 weeks after the mutated bacteria are cleared from the animals so that the innate immunity is back to normal. It is important to examine the immunological basis of protection in animals that have been immunized with the gidA mutant. Such studies are crucial in determining the effectiveness of any attenuated bacterial strain to be used as a vaccine candidate. To determine the role of cell-mediated immunity in providing protection, lymphocyte proliferation assay will be performed by measuring [3H]-thymidine incorporation during the last 18 hr of culture. Culture supernatants also will be assessed for levels of IFN- versus IL-4 using ELISA to determine the contribution of Th1 and Th2 related immune responses in providing protection against lethal infection. Additionally, we will determine the distribution of leukocyte subpopulations CD4/CD8 in the draining lymph nodes and spleens at selected time-points after immunization with mutant strain before challenge with wild type Salmonella.