Translocon-independent intracellular replication by Pseudomonas aeruginosa requires the ADP-ribosylation domain of ExoS

Hritonenko, Victoria; Evans, David J.; Fleiszig, Suzanne M. J.

doi:10.1016/j.micinf.2012.08.007

Cited by 16 publications

(27 citation statements)

References 42 publications

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“…We previously reported that the ADPr domain of ExoS confers intracellular replication without the T3SS translocon or other known effectors [30]. Thus, we tested if this domain of ExoS impacts P.…”

Section: Resultsmentioning

confidence: 99%

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Pseudomonas aeruginosa Utilizes the Type III Secreted Toxin ExoS to Avoid Acidified Compartments within Epithelial Cells

et al. 2013

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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.

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Section: Resultsmentioning

confidence: 99%

“…Like wild-type P. aeruginosa , replication of popB mutants is dependent on the ADPr activity of ExoS [30]. …”

Section: Introductionmentioning

confidence: 99%

Pseudomonas aeruginosa Utilizes the Type III Secreted Toxin ExoS to Avoid Acidified Compartments within Epithelial Cells

et al. 2013

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“…Electron microscopy suggests that intracellular P. aeruginosa remains confined in vacuoles in THP-1 cells for at least 24 h and can actively replicate therein. A vacuolar localization of P. aeruginosa has also been generally observed in epithelial cells (7,13,33), but the nature of the infected vacuoles has not yet been determined (31).…”

Section: Discussionmentioning

confidence: 99%

“…This is important for P. aeruginosa because it produces a variety of virulence factors that can affect host cell viability. In strain PAO1, pili, flagella, a type III secretion system, exotoxin A, and lipopolysaccharide are all known to contribute to virulence (30,31). Because the expression of these factors is variable between strains and critically depends on their growth status, preliminary investigations such as those performed here will probably be necessary when expanding the model to other laboratory or clinical strains.…”

Section: Discussionmentioning

confidence: 99%

Pharmacodynamic Evaluation of the Intracellular Activity of Antibiotics towards Pseudomonas aeruginosa PAO1 in a Model of THP-1 Human Monocytes

Buyck

Tulkens

Bambeke

2013

Antimicrob Agents Chemother

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Pseudomonas aeruginosa invades epithelial and phagocytic cells, which may play an important role in the persistence of infection. We have developed a 24-h model of THP-1 monocyte infection with P. aeruginosa PAO1 in which bacteria are seen multiplying in vacuoles by electron microscopy. The model has been used to quantitatively assess antibiotic activity against intracellular and extracellular bacteria by using a pharmacodynamic approach (concentration-dependent experiments over a wide range of extracellular concentrations to calculate bacteriostatic concentrations [C s ] and maximal relative efficacies [E max ]; Hill-Langmuir equation). Using 16 antipseudomonal antibiotics (three aminoglycosides, nine ␤-lactams, three fluoroquinolones, and colistin), dose-response curves were found to be undistinguishable for antibiotics of the same pharmacological class if data were expressed as a function of the corresponding MICs. Extracellularly, all of the antibiotics reached a bacteriostatic effect at their MIC, and their E max exceeded the limit of detection (؊4.5 log 10 CFU compared to the initial inoculum). Intracellularly, C s values remained unchanged for ␤-lactams, fluoroquinolones, and colistin but were approximately 10 times higher for aminoglycosides, whereas E max values were markedly reduced (less negative), reaching ؊3 log 10 CFU for fluoroquinolones and only ؊1 to ؊1.5 log 10 CFU for all other antibiotics. The decrease in intracellular aminoglycoside potency (higher C s ) can be ascribed to the acid pH to which bacteria are exposed in vacuoles. The decrease in the E max may reflect a reversible alteration of bacterial responsiveness to antibiotics in the intracellular milieu. The model may prove useful for comparison of antipseudomonal antibiotics to reduce the risk of persistence or relapse of pseudomonal infections. P seudomonas aeruginosa is among the leading causes of nosocomial infections in humans, with a particular tropism for the respiratory tract (see references 1 and 2 for reviews). Beside causing acute infections, it also chronically colonizes the lungs of cystic fibrosis patients, causing protracted and relapsing infections that are a primary cause of increased morbidity and mortality (3, 4).P. aeruginosa is difficult to eradicate for at least two non-mutually exclusive reasons. First, P. aeruginosa is not only naturally resistant to many common antibiotics (because of the poor permeability of its outer membrane) but can also express several mechanisms of resistance to most, if not all, of the drugs that have been selected and clinically developed over the years for their antipseudomonal activity (2). Second, P. aeruginosa can adopt specific modes of life that afford protection from host defenses and antibiotic action. The first one consists of the production of biofilm, a physical barrier to the access of antimicrobial agents that contributes to the persistence of infection with P. aeruginosa (5). The second one stems from the capacity of P. aeruginosa to invade and survive within eukaryotic cell...

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“…Our research has shown that clinical and laboratory isolates of P. aeruginosa can be divided into invasive or cytotoxic strains based upon how they interact with host cells (7). Invasive strains can survive and replicate inside epithelial cells dependent on the type III secretion system (T3SS) effector ExoS (8)(9)(10), while cytotoxic strains instead encode ExoU, which can induce rapid death of intoxicated host cells (11,12). While details of how invasive and cytotoxic strains impact host cells vary, both types can cause human disease, and they can each be virulent in animal models of infection (13)(14)(15).…”

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confidence: 99%

The Importance of the Pseudomonas aeruginosa Type III Secretion System in Epithelium Traversal Depends upon Conditions of Host Susceptibility

et al. 2015

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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. P seudomonas aeruginosa remains a leading cause of respiratory infections, septicemia, and urinary tract and burn wound infections (1-4). It is also the most common cause of corneal infection associated with contact lens wear (5, 6). Our research has shown that clinical and laboratory isolates of P. aeruginosa can be divided into invasive or cytotoxic strains based upon how they interact with host cells (7). Invasive strains can survive and replicate inside epithelial cells dependent on the type III secretion system (T3SS) effector ExoS (8-10), while cytotoxic strains instead encode ExoU, which can induce rapid death of intoxicated host cells (11,12). While details of how invasive and cytotoxic strains impact host cells vary, both types can cause human disease, and they can each be virulent in animal models of infection (13-15).Several in vivo models exist for studying P. aeruginosa pathogenicity. They include Caenorhabditis elegans (16-18) and Drosophila melanogaster (19), in addition to mouse models of pneumonia with sepsis (20-22), burn wound infection with sepsis (23-25), and gastrointestinal colonization and sepsis (26,27) and corneal scarification models (28)(29)(30)...

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Translocon-independent intracellular replication by Pseudomonas aeruginosa requires the ADP-ribosylation domain of ExoS

Cited by 16 publications

References 42 publications

Pseudomonas aeruginosa Utilizes the Type III Secreted Toxin ExoS to Avoid Acidified Compartments within Epithelial Cells

Pseudomonas aeruginosa Utilizes the Type III Secreted Toxin ExoS to Avoid Acidified Compartments within Epithelial Cells

Pharmacodynamic Evaluation of the Intracellular Activity of Antibiotics towards Pseudomonas aeruginosa PAO1 in a Model of THP-1 Human Monocytes

The Importance of the Pseudomonas aeruginosa Type III Secretion System in Epithelium Traversal Depends upon Conditions of Host Susceptibility

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