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The Pseudomonas aeruginosa type III secretion translocator PopB assists the insertion of the PopD translocator into host cell membranes.

ABSTRACT: Many Gram-negative bacterial pathogens use a type III secretion system to infect eukaryotic cells. The injection of bacterial toxins or protein effectors via this system is accomplished through a plasma membrane channel formed by two bacterial proteins, termed translocators, whose assembly and membrane-insertion mechanisms are currently unclear. Here, using purified proteins we demonstrate that the translocators PopB and PopD in Pseudomonas aeruginosa assemble heterodimers in membranes, leading to stably inserted hetero-complexes. Using site-directed fluorescence labeling with an environment-sensitive probe, we found that hydrophobic segments in PopD anchor the translocator to the membrane, but without adopting a typical transmembrane orientation. A fluorescence dual-quenching assay revealed that the presence of PopB changes the conformation adopted by PopD segments in membranes. Furthermore, analysis of PopD's interaction with human cell membranes revealed that PopD adopts a distinctive conformation when PopB is present. An N-terminal region of PopD is only exposed to the host cytosol when PopB is present. We conclude that PopB assists with the proper insertion of PopD in cell membranes, required for the formation of a functional translocon and host infection.

SUBMITTER: Tang Y

PROVIDER: S-EPMC5995524 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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The <i>Pseudomonas aeruginosa</i> type III secretion translocator PopB assists the insertion of the PopD translocator into host cell membranes.

Tang Yuzhou Y Romano Fabian B FB Breña Mariana M Heuck Alejandro P AP

The Journal of biological chemistry 20180423 23

Many Gram-negative bacterial pathogens use a type III secretion system to infect eukaryotic cells. The injection of bacterial toxins or protein effectors via this system is accomplished through a plasma membrane channel formed by two bacterial proteins, termed translocators, whose assembly and membrane-insertion mechanisms are currently unclear. Here, using purified proteins we demonstrate that the translocators PopB and PopD in <i>Pseudomonas aeruginosa</i> assemble heterodimers in membranes, l ...[more]

PMID: 29685888

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Project description:UnlabelledThe 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.ImportanceThe 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

The Pseudomonas aeruginosa type III secretion translocator PopB assists the insertion of the PopD translocator into host cell membranes.

Publications

The <i>Pseudomonas aeruginosa</i> type III secretion translocator PopB assists the insertion of the PopD translocator into host cell membranes.

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