PepA, a secreted protein of Pseudomonas aeruginosa, is necessary for cytotoxicity and virulence

Abstract

SummaryPseudomonas aeruginosa is an opportunistic pathogen and a leading cause of hospital-acquired pneumonia. We identified a 73 kDa protein, designated Pseudomonas exoprotein A (PepA), that was secreted by P. aeruginosa strain PA103. PepA was necessary for in vitro killing of epithelial cells as well as virulence in a mouse model of acute pneumonia. Several properties of PepA suggested that it was secreted by a type III system. 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 proteins. Secretion of PepA was absent from isogenic mutants with defective type III pathways. Finally, amino-terminal peptide sequence analysis indicated that the amino-terminal five residues of PepA were identical to those of ExoS and ExoT, two type III secreted proteins of P. aeruginosa. After secretion, PepA underwent cleavage at two sites, each with the sequence A-X-K-S, suggesting that the cleavage may be caused by a protease. The gene encoding PepA, designated pepA, was cloned and sequenced, and comparisons with the genetic database using BLAST alignments indicated that the nucleotide sequence of pepA and the inferred protein sequence of PepA had no homology to known sequences. A nucleotide sequence identical to the consensus element for binding of ExsA, a transcriptional activator of P. aeruginosa type III secretion genes, was located 84 bp 5Ј of the translational start codon. Analysis of transposon insertion mutants indicated that the carboxy terminus was required for cytotoxicity. Examination of respiratory clinical isolates demonstrated that pepA was a variable trait and probably acquired by horizontal transmission. Consistent with this hypothesis was the identification of a putative insertion element 94 bp 5Ј of the PepA translational start site. Analysis of G þ C content of the PepA coding sequence and the adjacent insertion element suggested that they were acquired together from a different species. In summary, PepA is a secreted protein of P. aeruginosa that is necessary for epithelial cell cytotoxicity in vitro and virulence in a mouse model of pneumonia.

Cloning and Characterization of the Gene Encoding the Major Cell-Associated Phospholipase A of Legionella pneumophila , plaB , Exhibiting Hemolytic Activity

“…However, it is noteworthy that the L. pneumophila plaB mutants constructed in this study were defective in cell-associated phospholipase A activity although the Km r cassette disrupting the plaB gene was placed after the putative catalytic domains, suggesting that the C terminus might be important for activation, stability, or proper transport of PlaB. Indeed, the cytotoxic activity of the type III secreted P. aeruginosa cytotoxin ExoU, recently found to be a phospholipase A (45,50), has been suggested to depend on the C-terminal region adjacent to the catalytic domain, since a mutant containing a transposon insertion 88 nucleotides from the exoU stop codon secretes a stable protein but is defective in cell killing (29). Furthermore, several type I secreted bacterial proteins, such as the E. coli hemolysin HlyA or the Erwinia chrysanthemi metalloprotease PrtG, contain their signal for transport via an ABC transporter in their C termini (8).…”

Interactions between effector proteins of the Pseudomonas aeruginosa type III secretion system do not significantly affect several measures of disease severity in mammals

“…As a result, ExoT intoxication is associated with inhibition of bacterial internalization but not cytotoxicity (Cowell et al, 2000;Garrity-Ryan et al, 2000;Krall et al, 2000). ExoU possesses phospholipase A 2 and lysophospholipase activities (Phillips et al, 2003;Rabin & Hauser, 2005;Sato et al, 2003;Tamura et al, 2004) that lead to rapid lysis of mammalian cells (Coburn & Frank, 1999; Finck-Barbançon et al, 1997;Fleiszig et al, 1997;Hauser et al, 1998a;Vallis et al, 1999). ExoY is an adenylate cyclase that increases intracellular levels of cAMP (Yahr et al, 1998).…”

“…ExoY is an adenylate cyclase that increases intracellular levels of cAMP (Yahr et al, 1998). Several studies have clearly demonstrated that the enzymic activities of ExoS, ExoT and ExoU contribute to pathogenesis by affecting bacterial persistence in infected tissues, bacterial dissemination and host survival (FinckBarbançon et al, 1997;Garrity-Ryan et al, 2000;Hauser et al, 1998a;Pankhaniya et al, 2004;Shaver & Hauser, 2004). ExoY, in contrast, has not been shown to play an appreciable role in disease progression in animal models (Holder et al, 2001;Lee et al, 2005).…”

Pseudomonas aeruginosa mediated apoptosis requires the ADP-ribosylating activity of ExoS

“…Some virulence factors are produced and secreted directly into the host cell using the cell-contact-mediated type III secretion machinery. To date, four such factors have been characterized, including ADP-ribosylating enzymes ExoS and ExoT (Frank, 1997 ;Frithz-Lindsten et al, 1997 ;Yahr et al, 1996a), an acute cytolytic factor ExoU (Finck-Barbancon et al, 1997 ;Hauser et al, 1998) an adenylate cyclase ExoY (Yahr et al, 1998). Expression of these secreted effector molecules as well as components of the type III secretory apparatus are under the control of the transcriptional activator, ExsA (Hovey & Frank, 1995).…”

“…cytolytic) strains harbour exoU. The cytolytic phenotype associated with noninvasive strains results mainly from the action of the exoU gene product (Finck-Barbancon et al, 1997 ;Hauser et al, 1998). Although the factor(s) for the invasive phenotype is not clearly understood, Cowell et al (2000) have recently shown that both ExoS and ExoT have an invasioninhibitory effect on cytolytic P. aeruginosa strains.…”