The Importance of the Pseudomonas aeruginosa Type Three Secretion System in Epithelial Traversal Depends Upon Conditions of Host Susceptibility (original) (raw)
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Infection and Immunity, 2015
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 ϳ16-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.
Investigative Ophthalmology & Visual Science, 2003
PURPOSE. The exoenzyme S regulatory protein ExsA regulates a type III secretion system in Pseudomonas aeruginosa. In vitro, cytotoxic strains use this system to secrete exotoxin (Exo)U and ExoT causing cytotoxicity and inhibiting their phagocytosis by epithelial cells. Invasive P. aeruginosa secrete ExoT and ExoS, but exsA mutation has little impact on their short-term interactions with epithelia. In the present study, the contribution of these ExsA-regulated proteins toward corneal infections in vivo was investigated. METHODS. After anesthesia, the left cornea of C57BL/6 mice was scratch injured and then inoculated with cytotoxic (PA103) or invasive (PAK) P. aeruginosa or with isogenic mutants in exsA-related genes. Inocula of 10 3 to 10 6 bacteria/5 L were used, and at least five animals were assigned to each experimental group. Corneal disease was quantified at regular intervals for 14 days in masked fashion with two different scoring systems. RESULTS. For the cytotoxic strain, mutation of either exoU or exoT alone had little effect on virulence, whereas simultaneous mutation of both exoT and exoU or of exsA resulted in a significantly reduced capacity to cause corneal disease. Complementation of the double exoUexoT mutant with exoU alone restored bacterial colonization levels (Ͼ3-log increase) and disease severity to wild-type levels. Complementation with exoT alone increased colonization (ϳ3-log increase) and increased virulence to almost the same levels as wild-type or exoU-complemented infections. Virulence of the invasive strain was not reduced by mutation of exsA or of genes encoding the ExsA-regulated secreted proteins. CONCLUSIONS. ExsA contributed to corneal virulence of only cytotoxic P. aeruginosa, with contributions made by both ExoU and ExoT to bacterial survival and disease severity. This differs from cytotoxic P. aeruginosa virulence in the lung, which is ExoU-dependent.
ExoT of Cytotoxic Pseudomonas aeruginosa Prevents Uptake by Corneal Epithelial Cells
Infection and Immunity, 2000
The presence of invasion-inhibitory activity that is regulated by the transcriptional activator ExsA of cytotoxic Pseudomonas aeruginosa has previously been proposed. The results of this study show that both ExoT and ExoS, known type III secreted effector proteins of P. aeruginosa that are regulated by ExsA, possess this activity. Invasion was reduced 94.4% by ExoT and 96.0% by ExoS. Invasion-inhibitory activity is not linked to ADP-ribosylation activity, at least for ExoS, since a noncatalytic mutant also inhibits uptake by an epithelial cell line (invasion was reduced 96.0% by ExoSE381A).
Investigative Ophthalmology & Visual Science, 2003
PURPOSE. The scarified cornea keratitis model was modified to study Pseudomonas aeruginosa infection of healing corneal epithelium. The new model was then used to study the role of ExsA, a transcriptional activator of P. aeruginosa, in bacterial penetration through injured and healing corneal epithelia. METHODS. Scratch-injured corneas of C57BL/6 mice were allowed to heal for 0, 6, 9, or 12 hours before inoculation with a cytotoxic (6206) or invasive (PAO1) P. aeruginosa strain. Disease progression was monitored for 14 days. The integrity of the healing epithelium was studied in uninfected eyes by fluorescein staining and by histologic examination. In other experiments, the effect of bacterial exsA mutation was studied after 0, 6, or 12 hours of healing. Three hours after infection, these eyes were used to quantify early bacterial colonization levels by viable counts, or they were sectioned to study bacterial penetration through the epithelium by microscopy. RESULTS. Corneas remained susceptible to infection 6 but not 12 hours after scratch injury. By 6 hours, the previously exposed stroma was already completely covered by several layers of epithelial cells. Fluorescein staining unexpectedly occurred even after 12 hours of healing time, showing that resistance to infection preceded full restoration of epithelial barrier function. Mutation of exsA reduced both bacterial colonization levels and penetration through the epithelium 3 hours after bacterial inoculation, but only in the 6-hour healing situation, and only for the cytotoxic strain (PA103). Mutation of exsA in the invasive strain (PAO1) had no effect on 3-hour colonization or penetration levels under any circumstances. CONCLUSIONS. The 6-hour healing infection model showed a role for ExsA in early interactions with the corneal epithelium that was not detectable with the conventional (0-hour) scratch model. Comparison of the 6-and 12-hour healing models, which showed that factors additional to barrier function contribute to defense against infection, could be used to gain new insights into corneal defense mechanisms, and the methods used by bacteria to circumvent them.
Infection and Immunity, 2006
Mutation of retS (rtsM) of Pseudomonas aeruginosa strain PA103 reduces its virulence in both ocular and respiratory murine models of infection. In vitro, retS mutants exhibit loss of the ExsA-regulated type III secretion system (TTSS), reduced twitching motility, and a decrease in association with, invasion of, and survival within corneal epithelial cells. In addition, transcription of multiple other virulence genes is positively and negatively affected by retS mutation. Since our published data show that ExoU and ExoT, the two TTSS effectors encoded by strain PA103, each confer virulence in this corneal model, we hypothesized that loss of virulence of retS mutants follows loss of type III secretion. Corneal pathology, bacterial colonization, and phagocyte infiltration were compared for wild-type PA103, retS mutants, and various TTSS mutants after infection with ϳ10 6 CFU bacteria. Results showed that either a retS or an exsA (TTSS) mutation delayed disease progression, as illustrated by reduced severity scores and colonization levels during the first 48 h postinfection. Surprisingly, retS mutant infections then became more severe than those involving exsA mutants. By day 7, colonization levels of retS mutants even surpassed those of wild-type bacteria (more than twofold, P ؍ 0.028). Although retS mutants caused more severe opacification of central corneas than both the wild type and the exsA mutants, neither mutant caused the peripheral ring opacity commonly associated with wild-type infection, suggesting that the TTSS was involved. Histological experiments with retS and various TTSS mutants showed that ring opacification required ExoU but not ExoT and that it consisted of dense polymorphonuclear phagocyte infiltration at the corneal periphery and the absence of any cell type in the central cornea. These data suggest that these P. aeruginosa TTSS effectors have different effects on innate immunity and that RetS influences virulence beyond its effects on the TTSS.
Infection and immunity, 1998
Previous characterization of Pseudomonas aeruginosa clinical isolates has demonstrated an inverse correlation between cytotoxicity and internalization by epithelial cells. To further investigate this relationship, we tested PA103, a cytotoxic P. aeruginosa strain, and 33 isogenic noncytotoxic transposon mutants for internalization by Madin-Darby canine kidney cells. The majority of the mutants were not internalized, demonstrating that an inverse correlation between cytotoxicity and bacterial uptake by epithelial cells is not absolute. Six of the noncytotoxic mutants, however, demonstrated measurable levels of internalization by standard aminoglycoside exclusion assays even though internalization of wild-type strain PA103 was not detectable. All six had evidence of protein secretion defects involving two proteins, a 40-kDa protein and a 32-kDa protein. These proteins, designated PepB (for Pseudomonas exoprotein B) and PepD, respectively, each had characteristics of type III transport...
Investigative Ophthalmology & Visual Science, 2012
PURPOSE. Pseudomonas aeruginosa isolates from microbial keratitis are invasive or cytotoxic toward mammalian cells, depending on their type III secreted toxins. Cytotoxic strains express ExoU, a phospholipase that contributes to corneal virulence. This study determined whether the ExoU phospholipase domain is required for P. aeruginosa traversal of the human corneal epithelium. METHODS. P. aeruginosa traversal of airlifted, multilayered, human corneal epithelial cells was quantified in vitro up to 8 hours after apical inoculation with ϳ10 6 cfu of strain PA14, or an isogenic exoU mutant (PA14⌬exoU). In addition, PA14⌬exoU or its triple effector mutant PA14⌬exoU⌬exoT⌬exoY, were complemented with exoU (pUCPexoU), phospholipase-inactive exoU (pUCPexoUD344A), or control plasmid (pUCP18). Transepithelial resistance (TER) was measured (by epithelial volt ohmmeter), and cytotoxicity was determined by trypan blue staining. RESULTS. PA14 traversed more efficiently than its exoU mutant at 4, 6, and 8 hours after inoculation (100-, 20-, and 8-fold, respectively; P Ͻ 0.05), but not at 2 hours. Cells exposed to PA14 lost TER to baseline (P Ͻ 0.05). Controls confirmed PA14 cytotoxicity toward these corneal epithelial cells that was absent with exoU mutants. Epithelial traversal, cytotoxicity, and lost TER were restored for PA14⌬exoU, or PA14⌬exoU⌬exoT⌬exoY, by complementation with pUCPexoU, but not by complementation with pUCPexoUD344A. CONCLUSIONS. Traversal of multilayered corneal epithelia in vitro by cytotoxic P. aeruginosa requires ExoU with an active phospholipase domain. Correlative loss of TER with traversal by wild-type, or exoU-complemented, bacteria suggests involvement of epithelial cell death and/or lost tight junction integrity. However, traversal by exoU mutants without reduced TER suggests that additional mechanisms are also operative.
mBio, 2018
is internalized into multiple types of epithelial cell and and yet is often regarded as an exclusively extracellular pathogen. Paradoxically, ExoS, a type three secretion system (T3SS) effector, has antiphagocytic activities but is required for intracellular survival of and its occupation of bleb niches in epithelial cells. Here, we addressed mechanisms for this dichotomy using invasive (ExoS-expressing) and corresponding effector-null isogenic T3SS mutants, effector-null mutants of cytotoxic with and without ExoS transformation, antibiotic exclusion assays, and imaging using a T3SS-GFP reporter. Except for effector-null PA103, all strains were internalized while encoding ExoS. Intracellular bacteria showed T3SS activation that continued in replicating daughter cells. Correcting the mutation in effector-null PA103 promoted internalization by >10-fold with or without ExoS. Conversely, mutating in PAO1 reduced internalization by >10-fold, also with or without ExoS. Effector-null...
2018
Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that is a substantial threat, particularly in hospital settings, causing severe infections in immunocompromised patients that may lead to death. Pseudomonas aeruginosa harbors a multitude of virulence factors that enable this pathogen to establish both acute and chronic infections in humans. A key determinant of acute infections is a hollow molecular needle structure used for injecting toxins into a host cell, called the type three secretion system (T3SS). The secretion machinery itself is highly complex and, together with the specific secreted factors, requires expression of more than 30 genes. Due to the high energy cost of its synthesis to the organism this system is highly regulated to finely time gene expression to coincide with host contact. ExsA, a member of the AraC-type transcription factor family, is the main transcriptional activator of all the genes necessary for expression of the T3SS. Members of the AraC fam...
Alveolar Response to Pseudomonas aeruginosa: Role of the Type III Secretion System
Infection and Immunity, 2005
The type III secretion system (TTSS) is a specialized cytotoxin-translocating apparatus of gram-negative bacteria which is involved in lung injury, septic shock, and a poor patient outcome. Recent studies have attributed these effects mainly to the ExoU effector protein. However, few studies have focused on the ExoUindependent pathogenicity of the TTSS. For the present study, we compared the pathogenicities of two strains of Pseudomonas aeruginosa in a murine model of acute lung injury. We compared the CHA strain, which has a functional TTSS producing ExoS and ExoT but not ExoU, to an isogenic mutant with an inactivated exsA gene, CHA-D1, which does not express the TTSS at all. Rats challenged with CHA had significantly increased lung injury, as assessed by the wet/dry weight ratio for the lungs and the protein level in bronchoalveolar lavage fluid (BALF) at 12 h, compared to those challenged with CHA-D1. Consistent with these findings, the CHA strain was associated with increased in vitro cytotoxicity on A549 cells, as assessed by the release of lactate dehydrogenase. CHA was also associated at 12 h with a major decrease in polymorphonuclear neutrophils in BALF, with a proinflammatory response, as assessed by the amounts of tumor necrosis factor alpha and interleukin-1, and with decreased bacterial clearance from the lungs, ultimately leading to an increased mortality rate. These results demonstrate that the TTSS has a major role in P. aeruginosa pathogenicity independent of the role of ExoU. This report underscores the crucial roles of ExoS and ExoT or other TTSSrelated virulence factors in addition to ExoU.