| Summary: | Salmonella typhimurium is a facultative intracellular pathogen which is able to infect a variety of animal species and mammalian cells in culture. It has been estimated that there are in excess of 400,000 cases of Salmonellosis every year in the United States, but very little is known about the mechanisms of pathogenesis. Several bacterial proteins are synthesized upon encountering mammalian cells which may be involved in the infection process. Tn5-VB32 transposon mutagenesis was used to create transcriptional fusions in genes in the virulent S. typhimurium strain, SL1344. Subsequent screening identified mutants that contained the neomycin phosphotransferase II gene (from Tn5-VB32) under the control of a bacterial promoter which was induced in the presence of cultured epithelial cells. Induction was measured as an increase in the resistance of the bacteria to treatment with neomycin sulfate. Eight mutants that displayed increased resistance to neomycin upon contact with Madin-Darby canine kidney cells (MDCK) cells were identified during the initial screening process. In five of the mutants, the transposon was inserted in the same place, one mutant had two transposon insertions and the other two mutants (6-30 and 20-10) contained single insertions in unique sites. Mutant 1-12 was selected from the group of five mutants and was studied further, as were mutants 6-30 and 20-10, which contained unique single site insertions. Each of these three mutants displayed significantly higher survival rates when treated with neomycin in the presence of MDCK cells as compared to those treated with tissue culture fluid alone indicating that there were transcriptional fusions to induced genes. Mutant 1-12 survived neomycin treatment an average of 6 times better, 6-30 survived 23.7 times better and 20-10 survived 51.7 times better in the presence of MDCK cells than in tissue culture fluid. All of the mutants were motile, contained intact LPS, and grew at similar rates compared to the parental strain. These three mutants also adhered to fixed MDCK cells at parental levels and had a similar effect on the transepithelial
resistance of an MDCK cell monolayer. Invasion of MDCK cells by 1-12 was about 65% less than the wild type parent, mutant 6-30 invaded 95% less efficiently and 20-10 about 98% less. The growth kinetics of 1-12 and 6-30 inside MDCK cells were similar to the wild type parent. Mutant 20-10 replicated more slowly, culminating in about 50% fewer intracellular bacteria than wild type after 24 hours. Mutants 1-12 and 6-30 also showed enhanced survival to neomycin treatment in the presence of extracellular matrix components including collagen, entactin, laminin and heparin sulfate proteoglycan and in agarose, indicating that the transcriptional fusions were induced by the presence of a solid support. Mutant 20-10 did not display enhanced survival under these conditions. Since 20-10 had several altered phenotypic characteristics, it is possible that the gene affected in this mutant plays a more specific role in pathogenicity than the genes induced by the presence of a solid support. Wild type Salmonella were incubated in the presence of extracellular matrix components of tissue culture fluid before assaying their invasion into MDCK cells in an attempt to determine if pre-induction of the solid support genes enhanced invasiveness. After 1 hour, invasion by bacteria from the samples pre-incubated with the extracellular matrix components was considerably better than invasion by bacteria pre-incubated in tissue culture fluid. Thus, it appears that at least two of the bacterial genes induced in the presence of MDCK cells are induced by the presence of a solid support and that activation of these genes by pre-incubation with a solid support enhances invasion into MDCK cells. The nature of the gene affected in mutant 20-10 was not determined, but the the presence of several altered phenotypic characteristics caused by the transposon insertion indicate that the gene may be involved in regulation of additional virulence factors and may be regulated by contact with epithelial cells. === Science, Faculty of === Microbiology and Immunology, Department of === Graduate
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