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Diversity of virulence phenotypes among type III secretion negative Pseudomonas aeruginosa clinical isolates.

ABSTRACT: Pseudomonas aeruginosa is a frequent cause of acute infections. The primary virulence factor that has been linked to clinical disease is the type III secretion system, a molecular syringe that delivers effector proteins directly into host cells. Despite the importance of type III secretion in dictating clinical outcomes and promoting disease in animal models of infections, clinical isolates often do not express the type III secretion system in vitro. Here we screened 81 clinical P. aeruginosa isolates for secretion of type III secretion system substrates by western blot. Non-expressing strains were also subjected to a functional test assaying the ability to intoxicate epithelial cells in vitro, and to survive and cause disease in a murine model of corneal infection. 26 of 81 clinical isolates were found to be type III secretion negative by western blot. 17 of these 26 non-expressing strains were tested for their ability to cause epithelial cell rounding. Of these, three isolates caused epithelial cell rounding in a type III secretion system dependent manner, and one strain was cytotoxic in a T3SS-independent manner. Five T3SS-negative isolates were also tested for their ability to cause disease in a murine model of corneal infection. Of these isolates, two strains caused severe corneal disease in a T3SS-independent manner. Interestingly, one of these strains caused significant disease (inflammation) despite being cleared. Our data therefore show that P. aeruginosa clinical isolates can cause disease in a T3SS-independent manner, demonstrating the existence of novel modifiers of clinical disease.

SUBMITTER: Toska J

PROVIDER: S-EPMC3900666 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Diversity of virulence phenotypes among type III secretion negative Pseudomonas aeruginosa clinical isolates.

Toska Jonida J Sun Yan Y Carbonell Dalina Alvarez DA Foster Altreisha N-S AN Jacobs Michael R MR Pearlman Eric E Rietsch Arne A

PloS one 20140123 1

Pseudomonas aeruginosa is a frequent cause of acute infections. The primary virulence factor that has been linked to clinical disease is the type III secretion system, a molecular syringe that delivers effector proteins directly into host cells. Despite the importance of type III secretion in dictating clinical outcomes and promoting disease in animal models of infections, clinical isolates often do not express the type III secretion system in vitro. Here we screened 81 clinical P. aeruginosa is ...[more]

PMID: 24466261

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Project description:The opportunistic pathogen Pseudomonas aeruginosa utilizes an injectisome-type III secretion system (injectisome-T3SS) to elicit cytotoxicity toward epithelial cells and macrophages. Macrophage killing results from the cytotoxic properties of the translocated effector proteins (ExoS, ExoT, ExoU, and ExoY) and inflammasome-mediated induction of pyroptosis. Inflammasome activation can occur following Nlrc4-mediated recognition of cytosolic translocated flagellin (FliC). In the present study, we demonstrate that FliC is a secretion substrate of both the injectisome- and flagellum-associated T3SSs. Molecular analyses indicate that the first 20 amino-terminal residues of FliC are sufficient for secretion by the injectisome-T3SS and that the first 100 residues are sufficient for translocation of FliC into host cells. Although maximal inflammasome activation requires FliC, activation can also occur in the absence of FliC. This prompted us to examine whether other flagellar components might also be translocated into cells to elicit inflammasome activation. Indeed, we find that the flagellar cap (FliD), hook-associated (FlgK and FlgL), hook (FlgE), and rod (FlgE) proteins are secretion substrates of the injectisome-T3SS. None of these proteins, however, result in increased inflammasome activation when they are overexpressed in a fliC mutant and appear to be translocated into host cells. While a role in inflammasome activation has been excluded, these data raise the possibility that flagellar components, which are highly conserved between different bacterial species, trigger other specific host responses from the extracellular milieu or contribute to the pathogenesis of P. aeruginosa.The inflammasome is a host defense mechanism that recognizes invading bacteria and triggers an inflammatory immune response. The opportunistic pathogen P. aeruginosa produces both inflammasome agonists and antagonists. In this study, we demonstrate that overexpression of an agonist suppresses the activity of an antagonist, thereby resulting in inflammasome activation. Since the relative expression levels of agonists and antagonists likely vary between strains, these differences could be important predictors of whether a particular P. aeruginosa strain elicits inflammasome activation.

Project description:UNLABELLED:The Pseudomonas aeruginosa type III secretion system has been associated with poor outcomes in both animal models and human patients. Despite a large number of studies exploring the regulation of type III secretion in vitro, little is known about the timing of secretion during mammalian infection. Here we demonstrate that the exoU gene, which encodes the highly cytotoxic type III effector ExoU, is induced early during acute P. aeruginosa pneumonia. Immunofluorescence microscopy indicated that the amount of ExoU protein in the lung also increased over time. The importance of early expression was examined using a strain of P. aeruginosa with inducible production of ExoU. Delays in expression as short as 3 h led to reduced bacterial burdens in the lungs of mice and improved survival. Our results demonstrate that early expression of exoU is critical to bacterial survival during pneumonia and suggest that therapeutic interventions that delay ExoU secretion for even short periods of time may be efficacious. IMPORTANCE:Pseudomonas aeruginosa is a major contributor to the large numbers of health care-associated infections occurring annually, particularly for immunocompromised patients. Although this organism possesses many virulence factors, the type III secretion system plays an especially important role in both animal models and humans. This system forms a needle-like apparatus that injects toxins directly into eukaryotic cells. The most toxic protein secreted by this molecular machine is ExoU, which causes rapid cell death. In this study, we demonstrated that exoU was expressed and ExoU was produced early during acute pneumonia in a mouse model. Delaying expression of exoU by as little as 3 h enhanced clearance of bacteria and survival of infected mice. Our findings highlight the importance of understanding the regulation of virulence factor expression during infection when designing therapeutic strategies to inhibit the toxic effects of these proteins.

Dataset Information

Diversity of virulence phenotypes among type III secretion negative Pseudomonas aeruginosa clinical isolates.

Publications

Diversity of virulence phenotypes among type III secretion negative Pseudomonas aeruginosa clinical isolates.

Similar Datasets

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