The specific aims of this proposal are to identify and characterize some of the genetic determinants of pathogenicity in Campylobacter. The major goals we accomplished in the first proposal were to develop methods to permit genetic exchange that would allow us to clone putative virulence determinants and to construct isogenic mutants. We have successfully completed these goals by developing shuttle plasmids that can be introduced into Campylobacter via bacterial conjugation and by transformation via high voltage electroporation. Therefore, we can now proceed to establish genetic libraries in Campylobacter for the first time. Among the determinants of pathogenicity that we will examine are those genes encoding for Campylobacter flagellin, cytotoxin, and LPS in C. jejuni and surface proteins mediating serum resistance in C. fetus. While the primary focus will be on Campylobacter flagellin and cytotoxin, we will also examine the others, in part as a means to establish the utility of the two genetic exchange methods. Using shuttle vectors developed in the laboratory we will establish isogenic mutants deficient in these factors via gene exchange and transposon insertion. By examining the behavior of isogenic mutants in relevant models of infection, and by employing cloned pathogenic determinants as probes, we will be able to more precisely assess the contribution of each factor in the pathogenesis of gastrointestinal tract infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI023796-05
Application #
3136226
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1986-09-30
Project End
1994-08-31
Budget Start
1990-09-01
Budget End
1991-08-31
Support Year
5
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Stanford University
Department
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
Segal, E D; Cha, J; Lo, J et al. (1999) Altered states: involvement of phosphorylated CagA in the induction of host cellular growth changes by Helicobacter pylori. Proc Natl Acad Sci U S A 96:14559-64
Segal, E D; Lange, C; Covacci, A et al. (1997) Induction of host signal transduction pathways by Helicobacter pylori. Proc Natl Acad Sci U S A 94:7595-9
Segal, E D; Falkow, S; Tompkins, L S (1996) Helicobacter pylori attachment to gastric cells induces cytoskeletal rearrangements and tyrosine phosphorylation of host cell proteins. Proc Natl Acad Sci U S A 93:1259-64
Solnick, J V; Josenhans, C; Suerbaum, S et al. (1995) Construction and characterization of an isogenic urease-negative mutant of Helicobacter mustelae. Infect Immun 63:3718-21
Batterman, H J; Peek, J A; Loutit, J S et al. (1995) Bartonella henselae and Bartonella quintana adherence to and entry into cultured human epithelial cells. Infect Immun 63:4553-6
Solnick, J V; O'Rourke, J; Lee, A et al. (1994) Molecular analysis of urease genes from a newly identified uncultured species of Helicobacter. Infect Immun 62:1631-8
Solnick, J V; O'Rourke, J; Lee, A et al. (1993) An uncultured gastric spiral organism is a newly identified Helicobacter in humans. J Infect Dis 168:379-85
Segal, E D; Tompkins, L S (1993) Identification and characterization of a Helicobacter pylori hemolysin. Infect Agents Dis 2:178-82
Grant, C C; Konkel, M E; Cieplak Jr, W et al. (1993) Role of flagella in adherence, internalization, and translocation of Campylobacter jejuni in nonpolarized and polarized epithelial cell cultures. Infect Immun 61:1764-71
Segal, E D; Tompkins, L S (1993) Transformation of Helicobacter pylori by electroporation. Biotechniques 14:225-6

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