This proposal is based on a fundamentally new approach to the isolation of bacterial virulence genes involved in pathogenesis, termed IVET (in vivo expression technology), that allows for the positive selection of bacterial genes that are specifically induced in host tissues. This methodology will be used to characterize the genetic and environmental factors that regulate the expression of in vivo induced genes (ivi) in the human pathogen, Salmonella typhimurium. Such studies will provide insights into the molecular mechanisms governing the regulation of pathogenicity, which is fundamental to the understanding of infectious disease. Genetic analysis of genes that answer the IVET selection is facilitated because the genes are transcriptionally fused to the reporter gene, lacZ, allowing the recovery of regulatory mutants that constitutively express ivi genes in vitro (normally repressing conditions). Our efforts will be focused on those regulatory factors that coordinately regulate ivi genes, thus defining the regulatory components that ensure a multifaceted bacterial response to a multifaceted host challenge. Trans-acting regulatory mutations will be used to characterize the positive and negative ivi regulatory elements that activate ivi transcription in vivo and repress ivi transcription in vitro. These mutants will be screened for those that confer virulence defects in an animal model, suggesting that they, or some of the products they regulate, play an important role in pathogenesis. Cis-acting mutations will be used to define the ivi promoter region with an emphasis on establishing a consensus site for coordinate ivi gene expression. Defining the nature of coordinate regulation provides a means to under stand the sophisticated signal transduction mechanisms that enable bacteria to grow under such different environmental conditions (e.g., laboratory media vs. host tissues). Additionally, the IVET selection will be extended to screening for ivi fusions whose survival is limited to a particular host tissue and thus may contain tissue-specific ivi genes, whose restricted expression will be examined by genetic analysis. These studies have practical applications for both vaccine and antimicrobial development.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
1R29AI036373-01
Application #
2072622
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1994-08-01
Project End
1999-07-31
Budget Start
1994-08-01
Budget End
1995-07-31
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of California Santa Barbara
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Santa Barbara
State
CA
Country
United States
Zip Code
93106
Julio, S M; Heithoff, D M; Mahan, M J (2000) ssrA (tmRNA) plays a role in Salmonella enterica serovar Typhimurium pathogenesis. J Bacteriol 182:1558-63
Heithoff, D M; Sinsheimer, R L; Low, D A et al. (2000) In vivo gene expression and the adaptive response: from pathogenesis to vaccines and antimicrobials. Philos Trans R Soc Lond B Biol Sci 355:633-42
Handfield, M; Lehoux, D E; Sanschagrin, F et al. (2000) In vivo-induced genes in Pseudomonas aeruginosa. Infect Immun 68:2359-62
Heithoff, D M; Sinsheimer, R L; Low, D A et al. (1999) An essential role for DNA adenine methylation in bacterial virulence. Science 284:967-70
Heithoff, D M; Conner, C P; Hentschel, U et al. (1999) Coordinate intracellular expression of Salmonella genes induced during infection. J Bacteriol 181:799-807
Conner, C P; Heithoff, D M; Julio, S M et al. (1998) Differential patterns of acquired virulence genes distinguish Salmonella strains. Proc Natl Acad Sci U S A 95:4641-5
Julio, S M; Conner, C P; Heithoff, D M et al. (1998) Directed formation of chromosomal deletions in Salmonella typhimurium: targeting of specific genes induced during infection. Mol Gen Genet 258:178-81
Conner, C P; Heithoff, D M; Mahan, M J (1998) In vivo gene expression: contributions to infection, virulence, and pathogenesis. Curr Top Microbiol Immunol 225:1-12
Handfield, M; Schweizer, H P; Mahan, M J et al. (1998) ASD-GFP vectors for in vivo expression technology in Pseudomonas aeruginosa and other gram-negative bacteria. Biotechniques 24:261-4
Rainey, P B; Heithoff, D M; Mahan, M J (1997) Single-step conjugative cloning of bacterial gene fusions involved in microbe-host interactions. Mol Gen Genet 256:84-7

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