Effects of the plasmid-encoded toxin of enteroaggregative Escherichia coli on focal adhesion complexes

Renato E. Cappello, Guadalupe Estrada-Gutierrez, Claudine Irles, Silvia Giono-Cerezo, Robert J. Bloch, James P. Nataro

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Abstract

Enteroaggregative Escherichia coli (EAEC) is an emerging diarrheal pathogen. Many EAEC strains produce the plasmid-encoded toxin (Pet), which exerts cytotoxic effects on human intestinal tissue. Pet-intoxicated HEp-2 cells exhibit rounding and detachment from the substratum, accompanied by loss of F-actin stress fibers and condensation of the spectrin-containing membrane cytoskeleton. Although studies suggest that Pet directly cleaves spectrin, it is not known whether this is the essential mode of action of the toxin. In addition, the effects of Pet on cytoskeletal elements other than actin and spectrin have not been reported. Here, we demonstrate by immunofluorescence that upon Pet intoxication, HEp-2 and HT29 cells lose focal adhesion complexes (FAC), a process that includes the redistribution of focal adhesion kinase (FAK), α-actinin, paxillin, vinculin, F-actin, and spectrin itself. This redistribution was coupled with the depletion of phosphotyrosine labeling at FACs. Immunoblotting and immunoprecipitation experiments revealed that FAK was tyrosine dephosphorylated, before the redistribution of FAK and spectrin. Moreover, phosphatase inhibition blocked cell retraction, suggesting that tyrosine dephosphorylation is an event that precedes FAK cleavage. Finally, we show that in vitro tyrosine-dephosphorylated FAK was susceptible to Pet cleavage. These data suggest that mechanisms other than spectrin redistribution occur during Pet intoxication. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.
Original languageAmerican English
Pages (from-to)301-314
Number of pages14
JournalFEMS Immunology and Medical Microbiology
DOIs
StatePublished - 1 Apr 2011

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Focal Adhesions
Spectrin
Focal Adhesion Protein-Tyrosine Kinases
Plasmids
Escherichia coli
Tyrosine
Actins
Paxillin
Vinculin
Actinin
HT29 Cells
Stress Fibers
Phosphotyrosine
Cytoskeleton
Phosphoric Monoester Hydrolases
Immunoprecipitation
Immunoblotting
Fluorescent Antibody Technique
Membranes

Cite this

Cappello, Renato E. ; Estrada-Gutierrez, Guadalupe ; Irles, Claudine ; Giono-Cerezo, Silvia ; Bloch, Robert J. ; Nataro, James P. / Effects of the plasmid-encoded toxin of enteroaggregative Escherichia coli on focal adhesion complexes. In: FEMS Immunology and Medical Microbiology. 2011 ; pp. 301-314.
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abstract = "Enteroaggregative Escherichia coli (EAEC) is an emerging diarrheal pathogen. Many EAEC strains produce the plasmid-encoded toxin (Pet), which exerts cytotoxic effects on human intestinal tissue. Pet-intoxicated HEp-2 cells exhibit rounding and detachment from the substratum, accompanied by loss of F-actin stress fibers and condensation of the spectrin-containing membrane cytoskeleton. Although studies suggest that Pet directly cleaves spectrin, it is not known whether this is the essential mode of action of the toxin. In addition, the effects of Pet on cytoskeletal elements other than actin and spectrin have not been reported. Here, we demonstrate by immunofluorescence that upon Pet intoxication, HEp-2 and HT29 cells lose focal adhesion complexes (FAC), a process that includes the redistribution of focal adhesion kinase (FAK), α-actinin, paxillin, vinculin, F-actin, and spectrin itself. This redistribution was coupled with the depletion of phosphotyrosine labeling at FACs. Immunoblotting and immunoprecipitation experiments revealed that FAK was tyrosine dephosphorylated, before the redistribution of FAK and spectrin. Moreover, phosphatase inhibition blocked cell retraction, suggesting that tyrosine dephosphorylation is an event that precedes FAK cleavage. Finally, we show that in vitro tyrosine-dephosphorylated FAK was susceptible to Pet cleavage. These data suggest that mechanisms other than spectrin redistribution occur during Pet intoxication. {\circledC} 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.",
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Effects of the plasmid-encoded toxin of enteroaggregative Escherichia coli on focal adhesion complexes. / Cappello, Renato E.; Estrada-Gutierrez, Guadalupe; Irles, Claudine; Giono-Cerezo, Silvia; Bloch, Robert J.; Nataro, James P.

In: FEMS Immunology and Medical Microbiology, 01.04.2011, p. 301-314.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Effects of the plasmid-encoded toxin of enteroaggregative Escherichia coli on focal adhesion complexes

AU - Cappello, Renato E.

AU - Estrada-Gutierrez, Guadalupe

AU - Irles, Claudine

AU - Giono-Cerezo, Silvia

AU - Bloch, Robert J.

AU - Nataro, James P.

PY - 2011/4/1

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N2 - Enteroaggregative Escherichia coli (EAEC) is an emerging diarrheal pathogen. Many EAEC strains produce the plasmid-encoded toxin (Pet), which exerts cytotoxic effects on human intestinal tissue. Pet-intoxicated HEp-2 cells exhibit rounding and detachment from the substratum, accompanied by loss of F-actin stress fibers and condensation of the spectrin-containing membrane cytoskeleton. Although studies suggest that Pet directly cleaves spectrin, it is not known whether this is the essential mode of action of the toxin. In addition, the effects of Pet on cytoskeletal elements other than actin and spectrin have not been reported. Here, we demonstrate by immunofluorescence that upon Pet intoxication, HEp-2 and HT29 cells lose focal adhesion complexes (FAC), a process that includes the redistribution of focal adhesion kinase (FAK), α-actinin, paxillin, vinculin, F-actin, and spectrin itself. This redistribution was coupled with the depletion of phosphotyrosine labeling at FACs. Immunoblotting and immunoprecipitation experiments revealed that FAK was tyrosine dephosphorylated, before the redistribution of FAK and spectrin. Moreover, phosphatase inhibition blocked cell retraction, suggesting that tyrosine dephosphorylation is an event that precedes FAK cleavage. Finally, we show that in vitro tyrosine-dephosphorylated FAK was susceptible to Pet cleavage. These data suggest that mechanisms other than spectrin redistribution occur during Pet intoxication. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.

AB - Enteroaggregative Escherichia coli (EAEC) is an emerging diarrheal pathogen. Many EAEC strains produce the plasmid-encoded toxin (Pet), which exerts cytotoxic effects on human intestinal tissue. Pet-intoxicated HEp-2 cells exhibit rounding and detachment from the substratum, accompanied by loss of F-actin stress fibers and condensation of the spectrin-containing membrane cytoskeleton. Although studies suggest that Pet directly cleaves spectrin, it is not known whether this is the essential mode of action of the toxin. In addition, the effects of Pet on cytoskeletal elements other than actin and spectrin have not been reported. Here, we demonstrate by immunofluorescence that upon Pet intoxication, HEp-2 and HT29 cells lose focal adhesion complexes (FAC), a process that includes the redistribution of focal adhesion kinase (FAK), α-actinin, paxillin, vinculin, F-actin, and spectrin itself. This redistribution was coupled with the depletion of phosphotyrosine labeling at FACs. Immunoblotting and immunoprecipitation experiments revealed that FAK was tyrosine dephosphorylated, before the redistribution of FAK and spectrin. Moreover, phosphatase inhibition blocked cell retraction, suggesting that tyrosine dephosphorylation is an event that precedes FAK cleavage. Finally, we show that in vitro tyrosine-dephosphorylated FAK was susceptible to Pet cleavage. These data suggest that mechanisms other than spectrin redistribution occur during Pet intoxication. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.

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