PEPA, A SECRETED PROTEIN OF PSEUDOMONAS-AERUGINOSA, IS NECESSARY FOR CYTOTOXICITY AND VIRULENCE

Pseudomonas aeruginosa is an opportunistic pathogen and a leading caus e of hospital-acquired pneumonia, We identified a 73 kDa protein, desi gnated Pseudomonas exoprotein A (PepA), that was secreted by P. aerugi nosa strain PA103, PepA was necessary for in vitro killing of epitheli al cells as well as virulence in a mouse model of acute pneumonia. Sev eral properties of PepA suggested that it was secreted by a type III s ystem, Secretion occurred without cleavage of a signal peptide and in low-calcium environments in the presence of a divalent cation chelator , as is the case for characterized P, aeruginosa type III secreted pro teins. Secretion of PepA was absent from isogenic mutants with defecti ve type III pathways. Finally, amino-terminal peptide sequence analysi s indicated that the amino-terminal five residues of PepA were identic al to those of ExoS and ExoT, two type III secreted proteins of P, aer uginosa, After secretion, PepA underwent cleavage at two sites, each w ith the sequence A-X-K-S, suggesting that the cleavage may be caused b y a protease, The gene encoding PepA, designated pepA, was cloned and sequenced, and comparisons with the genetic database using BLAST align ments indicated that the nucleotide sequence of pepA and the inferred protein sequence of PepA had no homology to known sequences, A nucleot ide sequence identical to the consensus element for binding of ExsA, a transcriptional activator of P, aeruginosa type III secretion genes, was located 84 bp 5' of the translational start codon. Analysis of tra nsposon insertion mutants indicated that the carboxy terminus was requ ired for cytotoxicity, Examination of respiratory clinical isolates de monstrated that pepA was a variable trait and probably acquired by hor izontal transmission. Consistent with this hypothesis was the identifi cation of a putative insertion element 94 bp 5' of the PepA translatio nal start site, Analysis of G+C content of the PepA coding sequence an d the adjacent insertion element suggested that they were acquired tog ether from a different species, In summary, PepA is a secreted protein of P, aeruginosa that is necessary for epithelial cell cytotoxicity i n vitro and virulence in a mouse model of pneumonia.