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The long-term goal of this research is to develop attenuated strains of Salmonella that could be used as live attenuated vaccine to protect animals and ultimately humans against salmonellosis. Identification of key pathogenic mechanism of Salmonella would help design effective therapeutic and preventative measures against infections with this organism. <P> Our preliminary data indicated that GidA plays a principle role in modulation of Salmonella virulence. Based on our previous data and the urgent need to develop better non-antibiotic means to prevent animal and human morbidity and mortality to salmonellosis, we propose the following specific aims: 1) Define immunological basis of protection afforded by immunization with the gidA mutant. 2) Determine subcellular localization of GidA protein. GidA will be cloned and expressed, and the purified GidA will be used to develop polyclonal antibody which will be used for localization of GidA using fractional separation, immunoblotting and immunogold electron microscopy. 3) Investigate mechanistic and molecular characterization of morphological change in gidA mutant using fluorescence, immunofluorescence and transmission electron microscopy. <P>We expect that the gidA mutant would immunize mice and generate protective immunity against subsequent challenge with the WT S. Typhimurium. This project will determine the mechanistic base of such immunity including the role of humoral and cell-mediated immune responses in protection against lethal infection.<P> This information will be important for designing strategies for future animals and human vaccines. Data from this study will also determine the sub-cellular location of GidA (cytoplasmic or membrane-associated) on Salmonella and determine how deletion of gidA contributed to morphological changes in Salmonella.
NON-TECHNICAL SUMMARY: <BR> Salmonella is the most frequently reported cause of food-borne illness in the United States. Controlling Salmonella in food-producing animals is a key strategy for reducing food-borne outbreaks in humans. Our studies have indicated that deletion of virulence gene regulator glucose-inhibited division (gidA) altered morphology and infectivity of Salmonella. The gidA mutant was defective in invasion of host cells, induction of cytotoxicity and bacterial motility. Additionally, deletion of gidA resulted in filamentous morphology, compared to rod-shaped wild-type (WT). Most importantly, gidA mutant was attenuated in mice and animals immunized with the mutant were protected from lethal dose challenge of the WT, indicating such a mutant could be a candidate for live attenuated vaccine. The objectives of this proposal are to 1) investigate the mechanistic basis of protection afforded by the gidA mutant. 2) determine subcellular localization of GidA in Salmonella. 3) identify the method by which GidA contribute to Salmonella filamentous morphology. These studies would provide information on the mechanism of immunity provided by the gidA mutant and the potential use of such mutant as live attenuated vaccine against slamonellosis. Further, data from this study will determine the method by which GidA affects bacterial morphology.
<P>APPROACH: <BR> Methods Objective 1. Define the immunological basis of protection afforded by gidA mutant. Humoral immune response will be determined using solid phase ELISA in which bacteria will be immobilized to the surface of a microtiter well. Levels of IgM and the subclasses of IgG will also be assessed using peroxidase-conjugated anti-IgM, IgG1, IgG2a, IgG2b, and IgG3 antibodies. The role of cell-mediated immunity in providing protection will be examined by lymphocyte proliferation assay using the CellTiter-Glo Luminescent Cell Viability Assay. Splenocyte culture supernatants also will be assessed for levels of IFN-gamma versus IL-4 using ELISA to determine the contribution of Th1 and Th2 related immune responses in providing protection against lethal infection. Additionally, distribution of CD4/CD8 leukocyte subpopulations in lymph nodes and spleens will be determined using antibodies labeled with fluorescein isothiocyanate [FITC] and R-phycoerythrin (R-PE) using the FACSCalibur flow cytometer (BD Biosciences). Objective 2. Determine subcellular location of GidA protein. Cloning & expression of gidA gene will be performed using the pET15b vector and purified using BugBuster His-Bind purification kit (EMD Biosciences, San Diego Ca) following manufacturer's instructions. The purified GidA protein will be used as an antigen to generate polyclonal antibodies in rabbits. The gidA antibody will be used in immunofluorescence microscopy and transmission electron microscopic studies to determine cellular localization of GidA on Salmonella. Cellular fractions containing bacterial cell membranes, periplasmic and cytoplasmic contents will be separated by ultracentrifugation to determine the cellular location of GidA in Salmonella by immunoblotting. Immunogold electron microscopy will also be used for localization of GidA in the cytoplasmic and periplasmic regions Objective 3. Investigate mechanistic and molecular characterization of morphological change in gidA mutant. Fluorescence microscopy will be used to determine whether filamentous morphology observed in the gidA mutant was because of defects in chromosomal segregation or arrest in cell division. Bacteria will be stained with Sypro Orange stain, a fluorescent protein dye that does not stain nucleic acids, and diamidino-2-phenylindole (DAPI) nucleic acid stain (Molecular Probes). Slides will be observed under Zeiss 200M Axiovert fluorescence microscope to visualize bacterial chromosomes and membranes. Transmission electron microscopy and ultra thin-sections will be used to visualize chromosome structure and positioning, cell membrane and septa in the normal and filamentous (gidA mutant) bacterial cells.