During the next period of support we, will continue our studies on the coordinate regulation of virulence gene expression in three different bacterial species, Salmonella typhimurium, Vibrio cholerae and Bordetella pertussis. In order to facilitate the identification of virulence genes, we have devised a genetic strategy that allows the direct selection of genes that are expressed in animal tissues. We call this methodology """"""""in vivo expression technology (IVET)"""""""" and now propose to identify genes in each of the three target organisms that show a pattern of regulation that is consistent with a virulence gene (i.e., expressed poorly during grow on laboratory media but highly expressed in animal tissues). The gene fusions displaying this regulation will be cloned and the nucleotide sequence of the fusion junction determined. The wild-type target gene will also be cloned and used to construct corresponding null mutations in the target organism. These mutants will be tested for virulence defects in appropriate animal models. The particular regulatory response of selected fusions will be studied in vitro in an effort to ascertain the host environmental signals controlling virulence gene expression. The regulatory genes mediating the host specific regulatory responses will be identified by genetic methods (e.g., isolation of constitutive mutations that derepress the fusion in vitro). We will also isolate new gene fusions that are coordinately regulated by the same regulatory system. We further propose to construct a series of improved IVET vectors one of which will allow identification of genes encoding secreted or membrane proteins that are expressed selectively in host tissues, while another that will provide a sensitive measurement of tissue-specific gene expression by using site-specific recombination as a reporter. Ongoing studies that will also be continued will include studies in S. typhimurium that will address the regulation of O-antigen synthesis by an in vivo induced anti-sense RNA. In B. pertussis, we will study the induction of Vir-repressed genes by intracellular growth. Finally, in V. cholerae we will study the temporal phasing of gene expression during the infection cycle focusing on genes that are both activated as well as repressed by the ToxR regulatory system and the heat shock response. These experiments should provide several practical applications in vaccine and antimicrobial drug development.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI026289-10
Application #
2413535
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1988-05-01
Project End
1998-04-30
Budget Start
1997-05-01
Budget End
1998-04-30
Support Year
10
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Skurnik, David; Clermont, Olivier; Guillard, Thomas et al. (2016) Emergence of Antimicrobial-Resistant Escherichia coli of Animal Origin Spreading in Humans. Mol Biol Evol 33:898-914
Roux, Damien; Danilchanka, Olga; Guillard, Thomas et al. (2015) Fitness cost of antibiotic susceptibility during bacterial infection. Sci Transl Med 7:297ra114
Fu, Yang; Mekalanos, John J (2014) Infant Rabbit Colonization Competition Assays. Bio Protoc 4:
Ho, Brian T; Dong, Tao G; Mekalanos, John J (2014) A view to a kill: the bacterial type VI secretion system. Cell Host Microbe 15:9-21
Danilchanka, Olga; Mekalanos, John J (2013) Cyclic dinucleotides and the innate immune response. Cell 154:962-970
Shneider, Mikhail M; Buth, Sergey A; Ho, Brian T et al. (2013) PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature 500:350-353
Basler, Marek; Ho, Brian T; Mekalanos, John J (2013) Tit-for-tat: type VI secretion system counterattack during bacterial cell-cell interactions. Cell 152:884-94
Ho, Brian T; Basler, Marek; Mekalanos, John J (2013) Type 6 secretion system-mediated immunity to type 4 secretion system-mediated gene transfer. Science 342:250-3
Fu, Yang; Waldor, Matthew K; Mekalanos, John J (2013) Tn-Seq analysis of Vibrio cholerae intestinal colonization reveals a role for T6SS-mediated antibacterial activity in the host. Cell Host Microbe 14:652-63
Skurnik, David; Roux, Damien; Cattoir, Vincent et al. (2013) Enhanced in vivo fitness of carbapenem-resistant oprD mutants of Pseudomonas aeruginosa revealed through high-throughput sequencing. Proc Natl Acad Sci U S A 110:20747-52

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