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Title: Expression of Virulence Genes in Salmonella
Authors: Thomas, Gracie Ann
Advisors: Dr. Amy M. Grunden, Committee Member
Dr. Geraldine Luginbuhl, Committee Member
Dr. Stephen J. Libby, Committee Chair
Keywords: Salmonella
Issue Date: 4-Sep-2002
Degree: MS
Discipline: Microbiology
Abstract: A role for slyA in Salmonella pathogenesis has been clearly demonstrated. Originally, slyA was identified as a gene that encoded a cryptic hemolysin. When cloned into E. coli, slyA was shown to be required for the cytolytic death of nucleated cells and the lysis of red blood cells. However, upon further investigation, it was discovered that expression of the hemolysin is directly regulated by slyA and is, therefore, not a function of the gene itself. It has been previously reported that SlyA is required for the intracellular survival of the bacteria within host macrophages and that slyA mutant strains of S. typhimurium are severely attenuated for virulence in the murine model. In addition, Daniels et al. showed that SlyA is required for the destruction of murine M cells but not for invasion. Further studies show that slyA may be required for resistance to oxidative stress and, more recently, that slyA may act as a regulator of virulence genes involved in the systemic phase of infection, but not the enteric phase. Based on sequence homology with other transcriptional regulatory proteins of other Gram-negative bacteria, it was first suggested by Ludwig et al. that SlyA may act as a regulatory protein, affecting expression of both E. coli and Salmonella genes. In view of this, SlyA was initially grouped with MarR and EmrR (MprA) of E. coli, Hpr of Bacillus subtilis, and PecS of Erwinia chrysanthemi. However, it is now apparent that SlyA is more distantly related to these regulatory proteins than previously reported. SlyA is currently classified as a member of a unique family of low molecular weight transcriptional regulatory proteins that include RovA of Yersinia pestis and Yersinia tuberculosis, Rap of Serratia marcescens, and Hor of Erwinia carotovora. SPI-4 is an approximate 25 kb sequence insertion located at centisome 92 on the Salmonella chromosomal map. This pathogenicity island is flanked on either side by the ssb and yciB genes in both S. typhimurium and S. typhi. Originally, SPI-4 was identified via a transposon insertion as a chromosomal sequence required for survival inside of murine macrophages. Wong et al. originally claimed that SPI-4 contained 18 ORFs designated A-R. The recently completed annotation of both S. typhimurium LT2 and S.typhi genomes shows that only 6 open reading frames exist. Ahmer et al. demonstrated SPI-4 regulation by SirA, a transcriptional regulator of SPI-1 (invasion locus). Experiments in our laboratory have shown that the beta-galactosidase activity of a SPI4-K::MudJ insertion (SL3277) in a slyA mutant background is reduced 8-fold, suggesting that SPI-4 is regulated by slyA. We recently discovered that the predicted SPI4-K protein has significant homology to a class of autotransporter toxins found in pathogenic E.coli, Bordetella, Neisseria, and Shigella species, referred to as SPATE toxins. Members of this family are serine proteases possessing a characteristic GDSGS motif with a central catalytic Ser residue. We have constructed and purified a His-tagged SPI4-K protein fragment based on the earlier annotation, and used this protein to obtain a polyclonal antibody. In this study, we examined the protein expression characteristics of SPI4-K in Salmonella typhimurium and other Salmonellae. Here, we confirm through immunoblot analysis that SPI4-K is a large molecular weight protein that appears to be secreted from the intracellular compartment. In an attempt to further clarify the role of both SlyA and SPI4-K in Salmonella pathogenesis, polyclonal antibodies were purified to these proteins. Here, we show that the anti-His-tagged SlyA polyclonal antibody reacted with several low molecular weight proteins. In addition, we have found that SlyA may be expressed in early to mid logarithmic phase and not in stationary phase.
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