The Hpm hemolysin secretion system of Proteus mirabilis has been implicated in the virulence of this common opportunistic gram-negative pathogen. HpmA is an extracellular, pore-forming hemolysin with an estimated molecular weight of 166kDa. HpmB is a 63kDa accessory protein required for activation and extracellular secretion of HpmA. Proteins related to HpmA and B have been identified as virulence factors among other species of Proteus, Serratia marcescens and a variety of other gram-negative bacterial pathogens. When characterized, the B proteins appear to be outer membrane proteins required for secretion of large A proteins (120-220kDa) beyond the outer membrane. The Proteus and Serratia proteins serve as a model for this group of protein. In these systems, the A and B proteins contain amino terminal signal sequences and are secreted beyond the inner membrane via the Sec protein secretion system. B proteins thus complement this common secretion pathway and may prove valuable as a simple system for extracellular secretion of recombinant proteins fused to A proteins. How the B proteins function in A protein secretion and activation, and what B protein domains are involved in these activities, is not understood. No mutations have been described which result in reduced B protein function, rather all have resulted in the complete loss of function. The goal of this proposal is to identify functional domains of HpmB necessary for secretion and activation of HpmA by characterization of hpmB mutants.
The specific aims are: 1. Random point mutations will be introduced throughout hpmB via hydroxylamine mutagenesis and low fidelity PCR amplification. 2. Escherichia coli expressing wild type HpmA will be transformed with plasmids containing mutagenized hpmB, and the resulting transformants will be screened for an altered hemolytic phenotype on sheep blood agar plates. 3. Selected mutants will be characterized and classified based on hemolytic activity and steady state levels of HpmA or B in various cell fractions. 4. DNA sequence analysis will be performed on selected clones representative of each mutant class to identify the specific mutations responsible for the phenotype. Analysis of these mutants will provide data necessary to identify the functional domains of HpmB. This information will serve as a model for similar proteins and permit optimization of this system for recombinant protein secretion from E. coli.
