In vitro effects of a high-molecular-weight heat-labile enterotoxin from enteroaggregative Escherichia coli

Department of Public Health, Faculty of Medicine, UNAM, 04510 Mexico DF, Mexico.
Infection and Immunity (Impact Factor: 3.73). 07/1998; 66(7):3149-54.
Source: PubMed


The pathogenic mechanisms of enteroaggregative Escherichia coli (EAggEC) infection are not fully elucidated. In this work we show that an ammonium sulfate precipitate of culture supernatant of EAggEC strain 049766 increased the potential difference (PD) and the short-circuit current (Isc) in rat jejunal preparations mounted in Ussing chambers. The precipitate contained two major proteins of 108 and 116 kDa, which were partially copurified by chromatography in DEAE-cellulose. This chromatographic fraction (peak I) increased jejunal PD and Isc in a dose-dependent manner, accompanied by a decrease in tissue electrical resistance. These effects were inhibited by incubation of peak I at 75 degreesC for 15 min or for 1 h with proteinase K at 37 degreesC. Rabbit polyclonal antibodies against peak I containing both the 108- and 116-kDa proteins inhibited the enterotoxic effect. Specific polyclonal antibodies raised against the 108-kDa but not against the 116-kDa protein inhibited the enterotoxic effect, suggesting that the 108-kDa protein is the active toxic species. Moreover, another EAggEC strain (065126) producing the 116-kDa protein but not the 108-kDa protein had no effect on rat jejunal mucosa in the Ussing chamber. The >100-kDa fraction derived from prototype EAggEC strain 042, which also expressed both 108- and 116-kDa proteins, also produced an enterotoxic effect on rat jejunal preparations in Ussing chambers; however, the same strain cured of its 65-MDa adherence plasmid did not. A subclone derived from the 65-MDa plasmid expressing the 108-kDa toxin (and not the 116-kDa protein) elicited rises in Isc. Tissue exposed to any preparation containing the 108-kDa toxin exhibited similar histopathologic changes, characterized by increased mucus release, exfoliation of cells, and development of crypt abscesses. Our data suggest that some EAggEC strains produce a ca. 108-kDa enterotoxin/cytotoxin which is encoded on the large virulence plasmid.

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    • "This could lead to the loss of epithelial integrity and a potentially life-threatening diarrhea (53). Pet is a serine protease autotransporter that was originally identified in the culture supernatants of EAEC strains causing diarrheal outbreaks in Mexican hospitals (54). The cytotoxicity and enterotoxicity of this toxin depends on its protease activity and serine protease inhibitors such as phenylmethylsulfonyl fluoride abolished both activities (55). "
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    ABSTRACT: The main features of enteroaggregative Escherichia coli (EAEC) pathogenesis include attachment of bacteria to the intestinal mucosa, production of various toxins and cytotoxins, and stimulation of mucosal inflammation. 'Virulence' genes encode these features. Comparison of different EAEC isolates has shown that the virulence gene content of these isolates varies considerably. The heterogeneity of EAEC strains was concluded from the results obtained from the volunteer as well as other studies. Although the underlying mechanism behind the apparent increase in O104:H4 virulence is not known, several bacterial factors have been implicated. In this review, the known virulence factors involved in pathogenesis of EAEC pathotype are summarized.
    Gastroenterology and hepatology from bed to bench 03/2013; 6(2):71-79.
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    • "Many potential virulence factors are shared with p042 plasmid such as the AAF (agg3) operon and the serine protease pet. Pet is secreted by many EAEC strains and exhibits enterotoxic activity (Navarro-García et al. 1998). "
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    ABSTRACT: The genome sequences of two Escherichia coli O104:H4 strains derived from two different patients of the 2011 German E. coli outbreak were determined. The two analyzed strains were designated E. coli GOS1 and GOS2 (German outbreak strain). Both isolates comprise one chromosome of approximately 5.31 Mbp and two putative plasmids. Comparisons of the 5,217 (GOS1) and 5,224 (GOS2) predicted protein-encoding genes with various E. coli strains, and a multilocus sequence typing analysis revealed that the isolates were most similar to the entero-aggregative E. coli (EAEC) strain 55989. In addition, one of the putative plasmids of the outbreak strain is similar to pAA-type plasmids of EAEC strains, which contain aggregative adhesion fimbrial operons. The second putative plasmid harbors genes for extended-spectrum β-lactamases. This type of plasmid is widely distributed in pathogenic E. coli strains. A significant difference of the E. coli GOS1 and GOS2 genomes to those of EAEC strains is the presence of a prophage encoding the Shiga toxin, which is characteristic for enterohemorrhagic E. coli (EHEC) strains. The unique combination of genomic features of the German outbreak strain, containing characteristics from pathotypes EAEC and EHEC, suggested that it represents a new pathotype Entero-Aggregative-Haemorrhagic Escherichiacoli (EAHEC). Electronic supplementary material The online version of this article (doi:10.1007/s00203-011-0725-6) contains supplementary material, which is available to authorized users.
    Archives of Microbiology 06/2011; 193(12):883-91. DOI:10.1007/s00203-011-0725-6 · 1.67 Impact Factor
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    • "The mode of action of Pet has been investigated mostly in HEp-2 cells, a system that helped to delineate the following progression of morphological events during Pet cell intoxication: elongation, followed sequentially by cell shrinkage, rounding, and finally exfoliation from the substratum (Navarro-Garcia et al., 1998). Cell retraction has been correlated with reorganization of the spectrin membrane cytoskeleton (Villaseca et al., 2000) and loss of F-actin stress fibers (Navarro-Garcia et al., 1998). Additional studies suggest that Pet must be internalized into epithelial cells in order to induce toxicity and that its protease activity is "
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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.
    FEMS Immunology & Medical Microbiology 12/2010; 61(3):301-14. DOI:10.1111/j.1574-695X.2010.00776.x · 3.08 Impact Factor
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