Abstract
Plasmid encoded toxin (Pet) is a serine protease originally described in enteroaggregative Escherichia coli (EAEC) prototype strain 042 whose entire characterization was essentially obtained from studies performed with the purified toxin. Here we show that Pet is not exclusive to EAEC. Atypical enteropathogenic Escherichia coli (aEPEC) strains, isolated from diarrhea cases, express Pet and its detection in supernatants of infected HEp-2 cells coincides with the appearance of cell damage, which, in turn, were similar to those described with purified Pet. Pet secretion and the cytotoxic effects are time and culture medium dependent. In presence of DMEM supplemented with tryptone cell rounding and detachment were observed after just 5 h of incubation with the bacteria. In the absence of tryptone, the cytotoxic effects were detected only after 24 h of infection. We also show that, in addition to the prototype EAEC, other pet+ EAEC strains, also isolated from diarrhea cases, induce cellular damage in the same degree as the aEPEC. The cytotoxic effects of EAEC and aEPEC strains were significantly reduced in the presence of a serine protease inhibitor or anti-Pet IgG serum. Our results show a common aspect between the aEPEC and EAEC and provide the first evidence pointing to a role of Pet in aEPEC pathogenesis.
Highlights
Most bacterial enterotoxins, with a few exceptions, are proteases known to catalyze the hydrolytic cleavage of proteins and peptides in order to improve nutrient uptake by the bacteria [1]
We show that enteroaggregative Escherichia coli (EAEC) strains, obtained from diarrhea cases [27], induce cytotoxic effects similar to those observed with the EAEC prototype 042 [28], suggesting that the expression of plasmid encoded toxin (Pet) correlates with pathogenesis
A 104 kDa band, the molecular weight of Pet, was detected in all four Atypical enteropathogenic Escherichia coli (aEPEC) strains, but not in E. coli strain HB101, used as a negative control (Figure 1), strongly suggesting that Pet is secreted by these strains
Summary
With a few exceptions, are proteases known to catalyze the hydrolytic cleavage of proteins and peptides in order to improve nutrient uptake by the bacteria [1]. Members of the family of the serine protease autotransporters of the Enterobacteriaceae (SPATE) have been associated with pathogenic strains and are among the virulence proteins predominantly secreted by the pathogens of this family [4]. The plasmid encoded toxin (Pet), so far described only in enteroaggregative Escherichia coli (EAEC), is certainly the best studied member of the SPATE family [5,6,7]. This toxin, an autotransporter prototype, is a 104 kDa protein that enters the cell via clathrin-coated vesicles. In addition cytokeratin 8 has recently been described as an important receptor for Pet in epithelial cells [10]
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