ExoS Controls the Cell Contact-Mediated Switch to Effector Secretion in Pseudomonas aeruginosa

Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4960, USA.
Journal of bacteriology (Impact Factor: 2.81). 05/2008; 190(8):2726-38. DOI: 10.1128/JB.01553-07
Source: PubMed


Type III secretion is used by many gram-negative bacterial pathogens to directly deliver protein toxins (effectors) into targeted host cells. In all cases, secretion of effectors is triggered by host cell contact, although the mechanism is unclear. In Pseudomonas aeruginosa, expression of all type III secretion-related genes is up-regulated when secretion is triggered. We were able to visualize this process using a green fluorescent protein reporter system and to use it to monitor the ability of bacteria to trigger effector secretion on cell contact. Surprisingly, the action of one of the major type III secreted effectors, ExoS, prevented triggering of type III secretion by bacteria that subsequently attached to cells, suggesting that triggering of secretion is feedback regulated. Evidence is presented that translocation (secretion of effectors across the host cell plasma membrane) of ExoS is indeed self-regulated and that this inhibition of translocation can be achieved by either of its two enzymatic activities. The translocator proteins PopB, PopD, and PcrV are secreted via the type III secretion system and are required for pore formation and translocation of effectors across the host cell plasma membrane. Here we present data that secretion of translocators is in fact not controlled by calcium, implying that triggering of effector secretion on cell contact represents a switch in secretion specificity, rather than a triggering of secretion per se. The requirement for a host cell cofactor to control effector secretion may help explain the recently observed phenomenon of target cell specificity in both the Yersinia and P. aeruginosa type III secretion systems.

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Available from: Pei-Chung Lee, Apr 25, 2014
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    • "Protein pellets were washed 1x with acetone, dried and resuspended in 1x SDS sample buffer normalized for OD600. Samples were separated by SDS-PAGE and either stained for total protein using SYPRO Ruby (Invitrogen) according to the manufacturer’s instructions or transferred to PVDF membrane and probed for the presence of ExoS, ExoT, ExoU, PopB and PopD [30] by western blot. Primary antibodies were detected with a HRP-conjugated anti-rabbit secondary antibody (Sigma) and WesternBright Quantum detection reagent (Advansta) using a GE ImageQuant LAS 4000 digital imaging system. "
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    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.
    PLoS ONE 01/2014; 9(1):e86829. DOI:10.1371/journal.pone.0086829 · 3.23 Impact Factor
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    • "Expression of ExoS by P. aeruginosa is activated in a low calcium environment or by contact with host cells [35,36]. ExoS also regulates contact-dependent T3SS expression [37]. Since our data showed that ExoS ADPr activity reduces bacterial occupation of acidified compartments, we used a transcriptional reporter to study both relative levels and spatial patterns of exoS expression by intracellular P. aeruginosa. "
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    ABSTRACT: Invasive Pseudomonas aeruginosa (PA) can enter epithelial cells wherein they mediate formation of plasma membrane bleb-niches for intracellular compartmentalization. This phenotype, and capacity for intracellular replication, requires the ADP-ribosyltransferase (ADPr) activity of ExoS, a PA type III secretion system (T3SS) effector protein. Thus, PA T3SS mutants lack these capacities and instead traffic to perinuclear vacuoles. Here, we tested the hypothesis that the T3SS, via the ADPr activity of ExoS, allows PA to evade acidic vacuoles that otherwise suppress its intracellular viability. The acidification state of bacteria-occupied vacuoles within infected corneal epithelial cells was studied using LysoTracker to visualize acidic, lysosomal vacuoles. Steady state analysis showed that within cells wild-type PAO1 localized to both membrane bleb-niches and vacuoles, while both exsA (transcriptional activator) and popB (effector translocation) T3SS mutants were only found in vacuoles. The acidification state of occupied vacuoles suggested a relationship with ExoS expression, i.e. vacuoles occupied by the exsA mutant (unable to express ExoS) were more often acidified than either popB mutant or wild-type PAO1 occupied vacuoles (p < 0.001). An exoS-gfp reporter construct pJNE05 confirmed that high exoS transcriptional output coincided with low occupation of acidified vacuoles, and vice versa, for both popB mutants and wild-type bacteria. Complementation of a triple effector null mutant of PAO1 with exoS (pUCPexoS) reduced the number of acidified bacteria-occupied vacuoles per cell; pUCPexoSE381D which lacks ADPr activity did not. The H(+)-ATPase inhibitor bafilomycin rescued intracellular replication to wild-type levels for exsA mutants, showing its viability is suppressed by vacuolar acidification. Taken together, the data show that the mechanism by which ExoS ADPr activity allows intracellular replication by PA involves suppression of vacuolar acidification. They also show that variability in ExoS expression by wild-type PA inside cells can differentially influence the fate of individual intracellular bacteria, even within the same cell.
    PLoS ONE 09/2013; 8(9):e73111. DOI:10.1371/journal.pone.0073111 · 3.23 Impact Factor
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    • "Use of ExoS enzyme activity to characterize mechanisms underlying differences in T3S translocation in MTC and MTLn3 cells ExoS is a bi-functional T3S effector having N-terminal GAP activity, defined by arginine residue 146 (R146), and C-terminal ADPRT activity, defined by glutamic acid residues 379 and 381 (E379 and E381) (reviewed by Barbieri and Sun, 2004). Both activities can alter ExoS translocation into eukaryotic cells (Aili et al., 2008; Bridge et al., 2012; Cisz et al., 2008; Fraylick et al., 2001). In turn, inactivation of ExoS-GAP or ADPRT activity has proven useful in dissecting cellular mechanisms that underlie T3S translocation and Pa infection (Bridge et al., 2012). "
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    ABSTRACT: Cancer patients are known to be highly susceptible to Pseudomonas aeruginosa (Pa) infection, but it remains unknown whether alterations at the tumor cell level can contribute to infection. This study explored how cellular changes associated with tumor metastasis influence Pa infection using highly metastatic MTLn3 cells and non-metastatic MTC cells as cell culture models. MTLn3 cells were found to be more sensitive to Pa infection than MTC cells based on increased translocation of the type III secretion effector, ExoS, into MTLn3 cells. Subsequent studies found that higher levels of ExoS translocation into MTLn3 cells related to Pa entry and secretion of ExoS within MTLn3 cells, rather than conventional ExoS translocation by external Pa. ExoS includes both Rho GTPase activating protein (GAP) and ADP-ribosyltransferase (ADPRT) enzyme activities, and differences in MTLn3 and MTC cell responsiveness to ExoS were found to relate to the targeting of ExoS-GAP activity to Rho GTPases. MTLn3 cell migration is mediated by RhoA activation at the leading edge, and inhibition of RhoA activity decreased ExoS translocation into MTLn3 cells to levels similar to those of MTC cells. The ability of Pa to be internalized and transfer ExoS more efficiently in association with Rho activation during tumor metastasis confirms that alterations in cell migration that occur in conjunction with tumor metastasis contribute to Pa infection in cancer patients. This study also raises the possibility that Pa might serve as a biological tool for dissecting or detecting cellular alterations associated with tumor metastasis.
    Biology Open 09/2013; 2(9):891-900. DOI:10.1242/bio.20133632 · 2.42 Impact Factor
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