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The rationale for the proposed research is that pigs become colonized early in life by S. enterica and become life long carriers of this bacterial food borne pathogen. Carrier animals remain asymptomatic but the stresses of shipping, feed withdrawal, and lairage prior to slaughter increase fecal shedding of this microbe and leads to contamination of food products that then can cause disease in humans. We identified a process of phenotypic phase variation that allows S. enterica to exist in a virulent phenotype or a non-virulent phenotype. Our data shows that cells can shift from one phenotype to the other. Using the technique RNA-Seq we were able to determine all of the genes differentially expressed in each phenotype. <P>We hypothesize that the carrier state is mediated by modulation of the fraction of cells in a population that are in each phenotype. Thus, there are sufficient numbers to grow in the pig, but insufficient to cause disease. The proposal was developed to test this hypothesis. The most direct way to test this hypothesis is to first create appropriate mutations that lock cells in each phenotype.
Non-Technical Summary:<br/>
Salmonella is a common cause of food borne diarrhea. Most infections are directly or indirectly linked to animals. One such animal, the pig, is frequent carrier of Salmonella and fecal contamination of meats or produce are a likely source of infection. Our research is targeted toward understanding how Salmonella can infect food animals for long periods of time during their growth but not cause disease. This is important because infected aniamls appear to be healthy. We identified a mechanism whereby Salmonella can switch between two forms; one of which is infectious and the other is not. We believe that the mechanism responsible for this switching is responsible for its ability to be kept in pigs. The goal of this project is to create mutants that can't switch between these two forms. We will use modern techniques in molecular biology to identify the mechanism controlling this switching with the long term goal to identify a means to block this switching and reduce food borne illnesses cause by Salmonella.
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Approach:<br/>
The most direct way to test the hypothesis that phenotypic phase variation controls the ability of S. Typhimurium to cause persistent asymptomatic infections of pigs is to create mutants that are locked in each phenotypic phase. We developed a two color screen to aid in the selection of these mutants. LacZ and PhoA gene fusions will be made with two different genes that are in the repertoire of genes expressed during the virulent phenotype : namely SipC and ompF. We will then mutagenize the S. Typhimurium with an antibiotic resistance transposon and using the two color screen identify mutants that lose both gene activities. These mutants will be characterized by DNA sequencing and phenotype analysis to identified bone fide phase locked off mutants. The mutant locus will then be transduced to cells in the virulent phenotype creating a phase locked on mutant. The mechanism of phase variation will then be inferred based on the location of the mutations and the genes they are associated with.