ExoY, an adenylate cyclase secreted by thePseudomonas aeruginosatype III system

Abstract

The exoenzyme S regulon is a set of coordinately regulated virulence genes of Pseudomonas aeruginosa. Proteins encoded by the regulon include a type III secretion and translocation apparatus, regulators of gene expression, and effector proteins. The effector proteins include two enzymes with ADP-ribosyltransferase activity (ExoS and ExoT) and an acute cytotoxin (ExoU). In this study, we identified ExoY as a fourth effector protein of the regulon. ExoY is homologous to the extracellular adenylate cyclases of Bordetella pertussis (CyaA) and Bacillus anthracis (EF). The homology among the three adenylate cyclases is limited to two short regions, one of which possesses an ATP-binding motif. In assays for adenylate cyclase activity, recombinant ExoY (rExoY) catalyzed the formation of cAMP with a specific activity similar to the basal activity of CyaA. In contrast to CyaA and EF, rExoY activity was not stimulated or activated by calmodulin. A 500-fold stimulation of activity was detected following the addition of a cytosolic extract from Chinese hamster ovary (CHO) cells. These results indicate that a eukaryotic factor, distinct from calmodulin, enhances rExoY catalysis. Site-directed mutagenesis of residues within the putative active site of ExoY abolished adenylate cyclase activity. Infection of CHO cells with ExoY-producing strains of P. aeruginosa resulted in the intracellular accumulation of cAMP. cAMP accumulation within CHO cells depended on an intact type III translocation apparatus, demonstrating that ExoY is directly translocated into the eukaryotic cytosol.

Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen

“…Type III secretion systems are found in many plant and human pathogens and are responsible for contact-dependent delivery of proteins into the cytoplasm of host cells. P. aeruginosa contains a single type III secretion system (PA1690±PA1725) which secretes several proteins including exoenzymes S, T and Y 24 .…”

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

“…araD139 ∆(argF-lac)U169 rpsL150(Str R ) relA1 fibB 5301 deoC1 ptsF25 rbsR recA56 Gherardini et al (1990) P. aeruginosa strains PAK Laboratory strain (invasive) D. Bradley* PAKexsA ::Ω exsA knockout mutant derivative of PAK Yahr et al (1997) 1997 ;Hauser et al, 1998 ;Yahr et al, 1998 ;Vallis et al, 1999), the mechanisms by which these virulence factors alter mammalian cell physiology remain enigmatic.…”

“…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).…”

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

“…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). 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).…”