Examining the Role of Actin-Plasma Membrane Association in Pseudomonas aeruginosa Infection and Type III Secretion Translocation in Migratory T24 Epithelial Cells

Bridge, Dacie R.; Martin, Karen H.; Moore, Elizabeth R.; Lee, Wendy M.; Carroll, James A.; Rocha, Claudia L.; Olson, Joan C.

doi:10.1128/iai.00231-12

Cited by 9 publications

(12 citation statements)

References 69 publications

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“…RalA can be ADP-ribosylated by Pa ExoS-WT at two to three sites, with Arg52 functioning as a preferred site of ExoS ADP-ribosylation, and Arg135 and Arg161 functioning as secondary sites of ADP-ribosylation (Fraylick et al, 2002a). ADP-ribosylation by ExoS causes RalA to shift in mobility in a manner dependent on the number of sites of ADP-ribosylation (Bridge et al, 2012; Fraylick et al, 2002a), and the different shift in RalA mobility detected between MTC and MTLn3 cells (Fig. 1B, noted by asterisks) can be explained by RalA being ADP-ribosylated at more sites in MTC cells.…”

Section: Resultsmentioning

confidence: 96%

“…Detection of Pa -T3S translocation into eukaryotic cells requires a 1.5 to 3.5 hr co-culture period, depending on the cell line and the method of analysis (Bridge et al, 2012; Bridge et al, 2010). To adapt Pa co-culture conditions to studies of MTC and MTLn3 cells, indicated infections were performed in medium containing 5% FBS (steady state conditions) to allow cells to move randomly during the co-culture period.…”

Section: Resultsmentioning

confidence: 99%

“…Notably, tumor-derived cell lines are generally highly sensitive to ExoS toxicity (McGuffie et al, 1999). More recent studies of human HT-29 colon and T24 bladder carcinoma cell lines identified a relationship between the leading edge of migrating epithelial cells and sensitivity to Pa -T3S and infection (Bridge et al, 2012; Bridge et al, 2010). The finding that carcinoma derived cell lines are highly sensitive to Pa -T3S and ExoS toxicity, while healthy polarized epithelial monolayers are resistant to both, introduces the possibility that responsiveness to ExoS toxicity might provide insight into mechanistic properties linked to carcinogenesis.…”

Section: Introductionmentioning

confidence: 99%

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Metastatic MTLn3 and non-metastatic MTC adenocarcinoma cells can be differentiated by Pseudomonas aeruginosa

et al. 2013

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SummaryCancer 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.

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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Metastatic MTLn3 and non-metastatic MTC adenocarcinoma cells can be differentiated by Pseudomonas aeruginosa

et al. 2013

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“…Recent studies have shown that the ADPRT activity of ExoS disrupts the epithelial cell barrier [35]. The authors suggest this as the mechanism of PA invasion and dissemination.…”

Section: Discussionmentioning

confidence: 99%

The GAP Portion ofPseudomonas AeruginosaType III Secreted Toxin ExoS Upregulates Total and Surface Levels of Wild Type CFTR

Tukaye

Kwon

Guggino

2013

Cell Physiol Biochem

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Background: Pseudomonas aeruginosa (PA) infections account for a large percentage of fatal hospital acquired pneumonias. One of the PA Type III secreted toxin (TTST) ExoS, a bifunctional protein with N-terminal GTPase activating protein (GAP) and C-terminal ADP rybosyl transferase (ADPRT) activities, significantly contributes to PA virulence by targeting small molecular weight G-proteins (SMWGP). In this study, we have looked at one of the mechanisms by which the GAP portion of ExoS (ExoS-GAP) mediates cellular toxicity. Methods: The effects of ExoS-GAP on CFTR trafficking were studied in CFBE41o- Kir 2.2 and MDCK cell lines stably expressing CFTR using a transient transfection system. Results: Transient transfection of ExoS-GAP increased the total and surface protein levels of mature wild type CFTR in epithelial cells stably expressing wild type (WT) CFTR. The effect of ExoS-GAP was specific to CFTR in bronchial epithelial cells since it did not affect the total protein levels of Na⁺/K⁺ATPase, another membrane protein. A point mutation in the ExoS GAP domain (R146K), known to disrupt its catalytic GAP activity, abolished the effect of ExoS-GAP on WT CFTR. Lysosomal inhibition studies with Bafilomycin A1 indicate that ExoS-GAP decreased lysosomal degradation of the mature WT CFTR with concomitant increase in the total levels of mature WT CFTR. However, ExoS-GAP did not increase the total protein levels of ∆F508CFTR. Conclusion: The GAP portion of the PA TTST ExoS increases the total and surface levels of wild type CFTR in vitro mammalian cell system. The effect of ExoS-GAP on WT CFTR total protein levels provides new insight into understanding the virulent pathophysiology of PA infections.

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“…RhoGAP activity due to a R146A mutation, or 5) ExoS that lacks catalytic activity (RhoG-/ADPr-) due to E379A/E381A/R146A mutations, were previously described (Bridge, Martin et al 2012). Bacteria were grown at 37°C in Lauria Broth with appropriate antibiotics.…”

Section: Bacteria Strainsmentioning

confidence: 99%

Regulation of Rab5 GTPase activity during Pseudomonas aeruginosa-macrophage interaction

Mustafi¹

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Examining the Role of Actin-Plasma Membrane Association in Pseudomonas aeruginosa Infection and Type III Secretion Translocation in Migratory T24 Epithelial Cells

Abstract: ABSTRACTThe opportunistic pathogenPseudomonas aeruginosatargets wounded epithelial barriers, but the cellular alteration that increases susceptibility toP. aeruginosainfection remains unclear. This study examined how… Show more

Cited by 9 publications

References 69 publications

Metastatic MTLn3 and non-metastatic MTC adenocarcinoma cells can be differentiated by Pseudomonas aeruginosa

Metastatic MTLn3 and non-metastatic MTC adenocarcinoma cells can be differentiated by Pseudomonas aeruginosa

The GAP Portion ofPseudomonas AeruginosaType III Secreted Toxin ExoS Upregulates Total and Surface Levels of Wild Type CFTR

Regulation of Rab5 GTPase activity during Pseudomonas aeruginosa-macrophage interaction

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