Yersinia enterocolitica causes a variety of gastrointestinal syndromes; systemic spread of the organism frequently occurs in immunocompromised individuals and has a mortality rate of 50%. To date, most of the research efforts on this organism have focused on either the virulence plasmid, or on the ability of this bacterium to efficiently invade host cells. Because Y. enterocolitica is amenable to genetic manipulation and an animal model mimicking many aspects of infection in humans exist, it is an excellent model system for studying host-pathogen interaction and the genetic basis of virulence. Exploiting models such as this is more important than ever before as more genome sequence information becomes available. Without homologies to sequences already in the data banks, much of this information will be difficult to interpret, and many of the organisms the genome sequences are derived from are either not amenable to genetic analysis and/or good animal models are not available. The long term goal is to gain a full understanding of the Y. enterocolitica virulence factors and their contribution to the disease process. This will expand understanding and ability to intervene not only with Yersinia but with many other pathogens as well. The PI recently used two genetic approaches to identify new virulence genes. From one of these approaches, of the mutants screened to date for effects on virulence in the murine model of infection, 75% exhibited a significant alteration in pathogenesis. From the second approach the PI identified 10 chromosomal mutants (ivs genes) that significantly reduce the ability of Yersinia to survive in the mouse.
The specific aims of this proposal are to follow up on the new virulence factors identified in these screens and to further screen their pool for additional virulence genes.
Aim 1. Characterization of the protease HreP and how it contributes to pathogenesis. The hre-22 locus has a gene, hreP, that encodes a homologue of the subtilisin family of serine proteases. A mutation in hreP causes a 50-fold increase in the LD50. The PI proposes to further characterize hre22, and its role in pathogenesis.
Aim 2. What genes does HreR regulate and how do they contribute to pathogenesis? The hre-20 locus has a regulatory gene in the lysR family of regulators and a mutation in the gene (hreR) causes a significant increase in the rate at which Y. enterocolitica spreads systemically to the liver and spleen. The PI proposes to further characterize hre-20, its role in pathogenesis, and to identify and characterize genes regulated in hreR.
Aim 3. How do other hre and ivs loci affect pathogenesis? There is a strong probability that the pool of mutants contains many other virulence genes. The PI presents methods for further pre-screening of this pool to identify loci worth checking in mice for their effect on virulence. She will also further characterize the ivs genes with the strongest phenotypes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI042736-01A1
Application #
2748597
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1998-12-01
Project End
2003-11-30
Budget Start
1998-12-01
Budget End
1999-11-30
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Washington University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Witowski, Sarah E; Walker, Kimberly A; Miller, Virginia L (2008) YspM, a newly identified Ysa type III secreted protein of Yersinia enterocolitica. J Bacteriol 190:7315-25
Mildiner-Earley, Shirly; Miller, Virginia L (2006) Characterization of a novel porin involved in systemic Yersinia enterocolitica infection. Infect Immun 74:4361-5
Walker, Kimberly A; Miller, Virginia L (2004) Regulation of the Ysa type III secretion system of Yersinia enterocolitica by YsaE/SycB and YsrS/YsrR. J Bacteriol 186:4056-66
Heusipp, Gerhard; Nelson, Kristin M; Schmidt, M Alexander et al. (2004) Regulation of htrA expression in Yersinia enterocolitica. FEMS Microbiol Lett 231:227-35
Heusipp, Gerhard; Schmidt, M Alexander; Miller, Virginia L (2003) Identification of rpoE and nadB as host responsive elements of Yersinia enterocolitica. FEMS Microbiol Lett 226:291-8
Suo, Z; Tseng, C C; Walsh, C T (2001) Purification, priming, and catalytic acylation of carrier protein domains in the polyketide synthase and nonribosomal peptidyl synthetase modules of the HMWP1 subunit of yersiniabactin synthetase. Proc Natl Acad Sci U S A 98:99-104
Heusipp, G; Young, G M; Miller, V L (2001) HreP, an in vivo-expressed protease of Yersinia enterocolitica, is a new member of the family of subtilisin/kexin-like proteases. J Bacteriol 183:3556-63
Nelson, K M; Young, G M; Miller, V L (2001) Identification of a locus involved in systemic dissemination of Yersinia enterocolitica. Infect Immun 69:6201-8
Walsh, C T; Chen, H; Keating, T A et al. (2001) Tailoring enzymes that modify nonribosomal peptides during and after chain elongation on NRPS assembly lines. Curr Opin Chem Biol 5:525-34
Miller, D A; Walsh, C T (2001) Yersiniabactin synthetase: probing the recognition of carrier protein domains by the catalytic heterocyclization domains, Cy1 and Cy2, in the chain-initiating HWMP2 subunit. Biochemistry 40:5313-21

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