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Role of the membrane localization domain of the Pseudomonas aeruginosa effector protein ExoU in cytotoxicity.

ABSTRACT: ExoU is a potent effector protein that causes rapid host cell death upon injection by the type III secretion system of Pseudomonas aeruginosa. The N-terminal half of ExoU contains a patatin-like phospholipase A(2) (PLA(2)) domain that requires the host cell cofactor superoxide dismutase 1 (SOD1) for activation, while the C-terminal 137 amino acids constitute a membrane localization domain (MLD). Previous studies had utilized insertion and deletion mutations to show that portions of the MLD are required for membrane localization and catalytic activity. Here we further characterize this domain by identifying six residues that are essential for ExoU activity. Substitutions at each of these positions resulted in abrogation of membrane targeting, decreased ExoU-mediated cytotoxicity, and reductions in PLA(2) activity. Likewise, each of the six MLD residues was necessary for full virulence in cell culture and murine models of acute pneumonia. Purified recombinant ExoU proteins with substitutions at five of the six residues were not activated by SOD1, suggesting that these five residues are critical for activation by this cofactor. Interestingly, these same five ExoU proteins were partially activated by HeLa cell extracts, suggesting that a host cell cofactor other than SOD1 is capable of modulating the activity of ExoU. These findings add to our understanding of the role of the MLD in ExoU-mediated virulence.

SUBMITTER: Veesenmeyer JL

PROVIDER: S-EPMC2916264 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Role of the membrane localization domain of the Pseudomonas aeruginosa effector protein ExoU in cytotoxicity.

Veesenmeyer Jeff L JL Howell Heather H Halavaty Andrei S AS Ahrens Sebastian S Anderson Wayne F WF Hauser Alan R AR

Infection and immunity 20100517 8

ExoU is a potent effector protein that causes rapid host cell death upon injection by the type III secretion system of Pseudomonas aeruginosa. The N-terminal half of ExoU contains a patatin-like phospholipase A(2) (PLA(2)) domain that requires the host cell cofactor superoxide dismutase 1 (SOD1) for activation, while the C-terminal 137 amino acids constitute a membrane localization domain (MLD). Previous studies had utilized insertion and deletion mutations to show that portions of the MLD are r ...[more]

PMID: 20479080

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Project description:UnlabelledPseudomonas aeruginosa is a Gram-negative pathogen commonly associated with nosocomial infections such as hospital-acquired pneumonia. It uses a type III secretion system to deliver effector proteins directly into the cytosol of host cells. Type III secretion in P. aeruginosa has been linked to severe disease and worse clinical outcomes in animal and human studies. The majority of P. aeruginosa strains secrete ExoS, a bifunctional toxin with GTPase-activating protein and ADP-ribosyltransferase activities. Numerous in vitro studies have investigated the targets and cellular effects of ExoS, linking both its enzymatic activities with inhibition of bacterial internalization. However, little is known about how this toxin facilitates the progression of infection in vivo. In this study, we used a mouse model to investigate the role of ExoS in inhibiting phagocytosis during pneumonia. We first confirmed previous findings that the ADP-ribosyltransferase activity of ExoS, but not the GTPase-activating protein activity, was responsible for bacterial persistence and decreased host survival in this model. We then used two distinct assays to demonstrate that ExoS inhibited phagocytosis during pneumonia. In contrast to the findings of several in vitro studies, this in vivo inhibition was also dependent on the ADP-ribosyltransferase activity, but not the GTPase-activating protein activity, of ExoS. These results demonstrate for the first time the antiphagocytic function of ExoS in the context of an actual infection and indicate that blocking the ADP-ribosyltransferase activity of ExoS may have potential therapeutic benefit.ImportancePseudomonas aeruginosa is a major cause of hospital-acquired infections. To cause severe disease, this bacterium uses a type III secretion system that delivers four effector proteins, ExoS, ExoT, ExoU, and ExoY, into host cells. The majority of P. aeruginosa strains secrete ExoS, a bifunctional toxin with GTPase-activating protein and ADP-ribosyltransferase activities. In cell culture models, both enzymatic activities have been associated with decreased bacterial internalization. However, our study is the first to examine a role for ExoS in blocking phagocytosis in an animal model. We report that ExoS does inhibit phagocytosis during pneumonia. The ADP-ribosyltransferase activity, but not the GTPase-activating protein activity, of ExoS is necessary for this effect. Our findings highlight the ability of P. aeruginosa to manipulate the inflammatory response during pneumonia to facilitate bacterial survival.

Dataset Information

Role of the membrane localization domain of the Pseudomonas aeruginosa effector protein ExoU in cytotoxicity.

Publications

Role of the membrane localization domain of the Pseudomonas aeruginosa effector protein ExoU in cytotoxicity.

Similar Datasets

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