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.
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