IDENTIFICATION AND CHARACTERIZATION OF SPCU, A CHAPERONE REQUIRED FOREFFICIENT SECRETION OF THE EXOU CYTOTOXIN

In recent studies, we have shown that Pseudomonas aeruginosa strains t hat are acutely cytotoxic in vitro damage the lung epithelium in vivo. Genetic analysis indicated that the factor responsible for acute cyto toxicity was controlled by ExsA and therefore was part of the exoenzym e S regulon, The specific virulence determinant responsible for epithe lial damage in vivo and cytotoxicity in vitro was subsequently mapped to the exoU locus. The present studies are focused on a genetic charac terization of the exoU locus, Northern blot analyses and complementati on experiments indicated that a region downstream of exoU was expresse d and that the expression of this region corresponded to increased Exo U secretion. DNA sequence analysis of a region downstream of exoU iden tified several potential coding regions. One of these open reading fra mes, SpcU (specific Pseudomonas chaperone for ExoU), encoded a small 1 5-kDa acidic protein (137 amino acids [pI 4.4]) that possessed a leuci ne-rich motif associated with the Syc family of cytosolic chaperones f or the Yersinia Yops, T7 expression analysis and nickel chromatography of histidine-tagged proteins indicated that ExoU and SpcU associated as a noncovalent complex when coexpressed in Escherichia coli. The ass ociation of ExoU and SpcU required amino acids 3 to 123 of ExoU. In P. aeruginosa, ExoU and Spell are coordinately expressed as an operon th at is controlled at the transcriptional level by ExsA.