Pseudomonas aeruginosa is an opportunistic pathogen of immuno- compromised individuals. This organism produces a number of extracellular virulence factors including pili, exotoxins, hemolysins and proteases. I have previously shown that three accessory genes are required for the expression of the Type IV pili of this organism. With a recently developed in vitro assay for pilin cleavage, I have determined that one of the genes, pilD, encodes the prepilin leader peptidase. In addition to its role in pilin biogenesis, we have also demonstrated that PilD is required for the extracellular secretion of exotoxin A, phospholipase C (heat-labile hemolysin), elastase and alkaline phosphatase. None of these excreted proteins, however, share any homology to pilin or are cleaved by PilD. I propose that PilD prepilin peptidase is required for the processing of one or more """"""""Type IV pilin-like"""""""" proteins that comprise the structural components of a novel excretory apparatus. These pilin-like proteins would show recognizable similarities to pilin including physical characteristics and amino acid sequence at the cleavage site of pilin recognized by PilD. I will present evidence for this model and suggest that protein excretion pathways of which pilin-like proteins are components are widespread among pathogenic gram-negative bacteria. I propose to initiate the study of this novel excretion apparatus by isolating and characterizing the gene sequences encoding the pilin-like proteins, to demonstrate PilD-dependent cleavage of their precursors and to show that pilin-like proteins are required for the excretion of virulence factors by P. aeruginosa.
