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The importance of the Pseudomonas aeruginosa type III secretion system in epithelium traversal depends upon conditions of host susceptibility.

ABSTRACT: Pseudomonas aeruginosa is invasive or cytotoxic to host cells, depending on the type III secretion system (T3SS) effectors encoded. While the T3SS is known to be involved in disease in vivo, how it participates remains to be clarified. Here, mouse models of superficial epithelial injury (tissue paper blotting with EGTA treatment) and immunocompromise (MyD88 deficiency) were used to study the contribution of the T3SS transcriptional activator ExsA to epithelial traversal. Corneas of excised eyeballs were inoculated with green fluorescent protein (GFP)-expressing PAO1 or isogenic exsA mutants for 6 h ex vivo before bacterial traversal and epithelial thickness were quantified by using imaging. In the blotting-EGTA model, exsA mutants were defective in capacity for traversal. Accordingly, an ?16-fold variability in exsA expression among PAO1 isolates from three sources correlated with epithelial loss. In contrast, MyD88-/- epithelia remained susceptible to P. aeruginosa traversal despite exsA mutation. Epithelial lysates from MyD88-/- mice had reduced antimicrobial activity compared to those from wild-type mice with and without prior antigen challenge, particularly 30- to 100-kDa fractions, for which mass spectrometry revealed multiple differences, including (i) lower baseline levels of histones, tubulin, and lumican and (ii) reduced glutathione S-transferase, annexin, and dermatopontin, after antigen challenge. Thus, the importance of ExsA in epithelial traversal by invasive P. aeruginosa depends on the compromise enabling susceptibility, suggesting that strategies for preventing infection will need to extend beyond targeting the T3SS. The data also highlight the importance of mimicking conditions allowing susceptibility in animal models and the need to monitor variability among bacterial isolates from different sources, even for the same strain.

SUBMITTER: Sullivan AB

PROVIDER: S-EPMC4363407 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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The importance of the Pseudomonas aeruginosa type III secretion system in epithelium traversal depends upon conditions of host susceptibility.

Sullivan Aaron B AB Tam K P Connie KP Metruccio Matteo M E MM Evans David J DJ Fleiszig Suzanne M J SM

Infection and immunity 20150209 4

Pseudomonas aeruginosa is invasive or cytotoxic to host cells, depending on the type III secretion system (T3SS) effectors encoded. While the T3SS is known to be involved in disease in vivo, how it participates remains to be clarified. Here, mouse models of superficial epithelial injury (tissue paper blotting with EGTA treatment) and immunocompromise (MyD88 deficiency) were used to study the contribution of the T3SS transcriptional activator ExsA to epithelial traversal. Corneas of excised eyeba ...[more]

PMID: 25667266

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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.

Project description:Pseudomonas aeruginosa is an important opportunistic pathogen that employs a type III secretion system (T3SS) to inject effector proteins into host cells. Using a protein depletion system, we show that the endoribonuclease RNase E positively regulates expression of the T3SS genes. We also present evidence that RNase E antagonizes the expression of genes of the type VI secretion system and limits biofilm production in P. aeruginosa Thus, RNase E, which is thought to be the principal endoribonuclease involved in the initiation of RNA degradation in P. aeruginosa, plays a key role in controlling the production of factors involved in both acute and chronic stages of infection. Although the posttranscriptional regulator RsmA is also known to positively regulate expression of the T3SS genes, we find that RNase E does not appreciably influence the abundance of RsmA in P. aeruginosa Moreover, we show that RNase E still exerts its effects on T3SS gene expression in cells lacking all four of the key small regulatory RNAs that function by sequestering RsmA.IMPORTANCE The type III secretion system (T3SS) is a protein complex produced by many Gram-negative pathogens. It is capable of injecting effector proteins into host cells that can manipulate cell metabolism and have toxic effects. Understanding how the T3SS is regulated is important in understanding the pathogenesis of bacteria with such systems. Here, we show that RNase E, which is typically thought of as a global regulator of RNA stability, plays a role in regulating the T3SS in Pseudomonas aeruginosa Depleting RNase E results in the loss of T3SS gene expression as well as a concomitant increase in biofilm formation. These observations are reminiscent of the phenotypes associated with the loss of activity of the posttranscriptional regulator RsmA. However, RNase E-mediated regulation of these systems does not involve changes in the abundance of RsmA and is independent of the known small regulatory RNAs that modulate RsmA activity.

Project description:Pseudomonas aeruginosa exhibits flagellum-mediated swimming in liquid and swarming on hydrated surfaces under diverse nutrient conditions. Prior studies have implicated a phosphodiesterase, DipA, in regulating these flagellum-mediated motilities, but collectively, the necessity for DipA was unclear. In this study, we find that the medium composition conditionally constrains the influence of DipA on flagellar motility. We show that DipA exhibits more influence on minimal medium supplemented with glutamate or glucose, where flagellar motility was negated for the dipA mutant. Conversely, a dipA-deficient mutant exhibits flagellar motility when growing with LB Lennox broth and minimal medium supplemented with Casamino Acids. Swarming under these rich medium conditions occurs under elevated levels of c-di-GMP. We also demonstrate that the influence of DipA upon swimming often differs from that upon swarming, and we conclude that a direct comparison of the motility phenotypes is generally valid only when characterizing motility assay results from the same medium composition. Our results are consistent with the explanation that DipA is one of several phosphodiesterases responding to the nutrient environment sensed by P. aeruginosa On minimal medium with glutamate or glucose, DipA is dominant; however, on rich medium, the necessity of DipA is fully negated.IMPORTANCE Motile and ubiquitous bacteria such as Pseudomonas aeruginosa can quickly colonize surfaces and form biofilms in numerous environments such as water distribution systems, soil, and the human lung. To effectively disrupt bacterial colonization, it is imperative to understand how bacteria regulate motility in these different growth environments. Here, we show that the phosphodiesterase DipA is not required for flagellar motility under all nutrient conditions. Thus, the maintenance of intracellular c-di-GMP levels to promote flagellar motility or biofilm development must be conditionally regulated by differing phosphodiesterases in variation with select nutrient cues.

Dataset Information

The importance of the Pseudomonas aeruginosa type III secretion system in epithelium traversal depends upon conditions of host susceptibility.

Publications

The importance of the Pseudomonas aeruginosa type III secretion system in epithelium traversal depends upon conditions of host susceptibility.

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