The hemolysin (Hly) of uropathogenic E. coli is the prototype for the RTX family of hemolysins/leukotoxin produced by many different important human and animal pathogens. The putative role for RTX exotoxins in pathogenesis is the inhibition and killing of phagocytic cells. This occurs by formation of cation-selective pores in plasma membranes that leads to a metabolically disruptive Ca++ influx and eventual cell lysis at high toxin doses. The RTX exotoxin structure, mechanism, and sequence of events responsible for cytotoxicity are unknown. In contrast to the RTX leukotoxin, the E. coli hemolysin is cytotoxic to many cell types from different hosts. It contains a cysteineless, Ca++-bound, acylated, 1023 amino acid hemolysin polypeptide. The native size of hemolysin varies from 300 to > 1,000 kDa. There is conflicting evidence about the multimeric state of HlyA and if HlyA is associated with lipopolysaccharide (LPS). The proposal's specific aims are: 1) to derive models for the structure and action of hemolysin in soluble, host-membrane bound, and lytic-complex states based on different methods of composition and structural analysis. These methods involve CD spectroscopy, mass spectrometry, tryptophan fluorescence spectrometry, FACS analysis of hemolysin-MAb reactivities, and site-specific-fluorescein-labeled hemolysin. 2) to perform genetic and biochemical tests of hypothetical hemolysin structure and function models. The following types of mutants will be sought: gain of function by Hly A mutants affected in acylation and wild type Hly A expressed in LPS deep rough backgrounds, dominant negative Hly A mutants, altered pore-size Hly mutants and superhemolytic Hly A mutants. Mutants isolated from these screens will be tested for altered structural and functional changes by the appropriate methods covered in aims #1. Together the work in aims #1 and 2 will assess the critical residues and structures necessary for target cell binding, acylation, membrane insertion, multimerization, LPS interactions, and host-cell specificity of RTX toxins. 3) to produce mg quantities of Hly. Hly will be provided to a X-ray crystallographer with the long term, collaborative goal of the solution of a high resolution Hly structure. Increased knowledge of the structure and function of the prototype for this large family of exotoxins may lead the identification of novel RTX toxin inhibitors and prophylactic strategies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI020323-18
Application #
6373024
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Korpela, Jukka K
Project Start
1983-08-01
Project End
2003-03-31
Budget Start
2001-04-01
Budget End
2003-03-31
Support Year
18
Fiscal Year
2001
Total Cost
$313,612
Indirect Cost
Name
University of Wisconsin Madison
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Ravindran, Sriram; Grys, Thomas E; Welch, Rodney A et al. (2004) Inhibition of plasma kallikrein by C1-inhibitor: role of endothelial cells and the amino-terminal domain of C1-inhibitor. Thromb Haemost 92:1277-83
Lathem, Wyndham W; Bergsbaken, Tessa; Welch, Rodney A (2004) Potentiation of C1 esterase inhibitor by StcE, a metalloprotease secreted by Escherichia coli O157:H7. J Exp Med 199:1077-87
Lathem, Wyndham W; Bergsbaken, Tessa; Witowski, Sarah E et al. (2003) Acquisition of stcE, a C1 esterase inhibitor-specific metalloprotease, during the evolution of Escherichia coli O157:H7. J Infect Dis 187:1907-14
Lathem, Wyndham W; Grys, Thomas E; Witowski, Sarah E et al. (2002) StcE, a metalloprotease secreted by Escherichia coli O157:H7, specifically cleaves C1 esterase inhibitor. Mol Microbiol 45:277-88
Lim, K B; Walker, C R; Guo, L et al. (2000) Escherichia coli alpha-hemolysin (HlyA) is heterogeneously acylated in vivo with 14-, 15-, and 17-carbon fatty acids. J Biol Chem 275:36698-702
Serwecinska, L; Pytlos, M; Lukomski, S et al. (1997) The simultaneous production of both Hly- and Hpm-like hemolysins is characteristic of the Proteus penneri species. J Basic Microbiol 37:361-70
Leeds, J A; Welch, R A (1997) Enhancing transcription through the Escherichia coli hemolysin operon, hlyCABD: RfaH and upstream JUMPStart DNA sequences function together via a postinitiation mechanism. J Bacteriol 179:3519-27
Bauer, M E; Welch, R A (1997) Pleiotropic effects of a mutation in rfaC on Escherichia coli hemolysin. Infect Immun 65:2218-24
Moayeri, M; Welch, R A (1997) Prelytic and lytic conformations of erythrocyte-associated Escherichia coli hemolysin. Infect Immun 65:2233-9
Bauer, M E; Welch, R A (1996) Characterization of an RTX toxin from enterohemorrhagic Escherichia coli O157:H7. Infect Immun 64:167-75

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