DEFECTS IN TYPE-III SECRETION CORRELATE WITH INTERNALIZATION OF PSEUDOMONAS-AERUGINOSA BY EPITHELIAL-CELLS

Previous characterization of Pseudomonas aeruginosa clinical isolates has demonstrated an inverse correlation between cytotoxicity and inter nalization by epithelial cells. To further investigate this relationsh ip, we tested PA103, a cytotoxic P. aeruginosa strain, and 33 isogenic noncytotoxic transposon mutants for internalization by Madin-Darby ca nine kidney cells. The majority of the mutants were not internalized, demonstrating that an inverse correlation between cytotoxicity and bac terial uptake by epithelial cells is not absolute. Six of the noncytot oxic mutants, however, demonstrated measurable levels of internalizati on by standard aminoglycoside exclusion assays even though internaliza tion of wild-type strain PA103 was not detectable. All six had evidenc e of protein secretion defects involving two proteins, a 40-kDa protei n and a 32-kDa protein. These proteins, designated PepB (for Pseudomon as exoprotein B) and PepD, respectively, each had characteristics of t ype III transported proteins. In addition, nucleotide sequencing studi es demonstrated that PepB and PepD are homologs of YopB and YopD, resp ectively, type III secreted proteins of Yersinia spp. necessary for th e translocation of effector molecules into the cytoplasmic compartment of eukaryotic cells. Thus, while many mutations in PA103 result in lo ss of cytotoxicity without an appreciable increase in internalization, defects in transport of type III secretion proteins PepB and PepD cor relate with both loss of cytotoxicity and gain of internalization. The se results are consistent with type III secretion of an inhibitor of i nternalization that requires PepB and PepD for translocation into the host cell.