Characterization of Pic, a Secreted Protease of Shigella flexneri and Enteroaggregative Escherichia coli

We have identified and characterized a secreted protein, designated Pic, which is encoded on the chromosomes of enteroaggregative Escherichia coli (EAEC) 042 and Shigella flexneri 2457T. The product of the pic gene is synthesized as a 146.5-kDa precursor molecule which is processed at the N and C termini during secretion, allowing the release of a mature protein (109.8 kDa) into the culture supernatant. The deduced amino acid sequence of Pic shows high homology to autotransporter proteins, particularly a subgroup termed the SPATEs (serine protease autotransporters of the Enterobacteriaceae). Present in all members of this subgroup is a motif similar to the active sites of certain serine proteases. Pic catalyzes gelatin degradation, which can be abolished by disruption of the predicted proteolytic active site. Functional analysis of the Pic protein implicates this factor in mucinase activity, serum resistance, and hemagglutination. Our data suggest that Pic may be a multifunctional protein involved in enteric pathogenesis

Phylogenetic Analysis of Enteroaggregative and Diffusely Adherent Escherichia coli

The phylogenetics of the various pathotypes of diarrheagenic Escherichia coli are not completely understood. In this study, we identified several plasmid and chromosomal genes in the pathogenic enteroaggregative E. coli (EAEC) prototype strain 042 and determined the prevalence of these loci among EAEC and diffusely adherent E. coli strains. The distribution of these genes is analyzed within an evolutionary framework provided by the characterization of allelic variation in housekeeping genes via multilocus enzyme electrophoresis. Our data reveal that EAEC strains are heterogeneous with respect to chromosomal and plasmid-borne genes but that the majority harbor a member of a conserved family of virulence plasmids. Comparison of plasmid and chromosomal relatedness of strains suggests clonality of chromosomal markers and a limited transfer model of plasmid distribution

Pet, an Autotransporter Enterotoxin from Enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) is an emerging cause of diarrheal illness. Clinical data suggest that diarrhea caused by EAEC is predominantly secretory in nature, but the responsible enterotoxin has not been described. Work from our laboratories has implicated a ca. 108-kDa protein as a heat-labile enterotoxin and cytotoxin, as evidenced by rises in short-circuit current and falls in tissue resistance in rat jejunal tissue mounted in an Ussing chamber. Here we report the genetic cloning, sequencing, and characterization of this high-molecular-weight heat-labile toxin. The toxin (designated the plasmid-encoded toxin [Pet]) is encoded on the 65-MDa adherence-related plasmid of EAEC strain 042. Nucleotide sequence analysis suggests that the toxin is a member of the autotransporter class of proteins, characterized by the presence of a conserved C-terminal domain which forms a β-barrel pore in the bacterial outer membrane and through which the mature protein is transported. The Pet toxin is highly homologous to the EspP protease of enterohemorrhagic E. coli and to EspC of enteropathogenic E. coli, an as yet cryptic protein. In addition to its potential role in EAEC infection, Pet represents the first enterotoxin within the autotransporter class of secreted proteins. We hypothesize that other closely related members of this class may also produce enterotoxic effects

A novel dispersin protein in enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) is a diarrheal pathogen defined by its characteristic aggregative adherence (AA) to HEp-2 cells in culture. We have previously shown that EAEC strains secrete a 10-kDa protein that is immunogenic in a human EAEC challenge model. We report here that this protein is encoded by a gene (called aap) lying immediately upstream of that encoding the AggR transcriptional activator, and that aap is under AggR control. The product of aap has a typical signal sequence and is secreted to the extracellular milieu, where it remains noncovalently attached to the surface of the bacterium. EAEC aap mutants aggregate more intensely than the wild-type parent in a number of assays, forming larger aggregates and fewer individual bacteria. Infection of colonic biopsies with wild-type EAEC strain 042 and its aap mutant revealed more dramatic autoagglutination of the mutant compared with the wild-type parent. Our data suggest that the aap gene product participates in formation of a surface coat that acts to disperse the bacteria, thus partially counteracting aggregation mediated by aggregative adherence fimbriae. We have therefore named the aap gene product “dispersin,” and we propose that it may be representative of a functional class of colonization factors. Since dispersin is expressed in vivo, is highly immunogenic, and is present in most EAEC strains, it holds considerable promise as an EAEC immunogen

Enteroaggregative Escherichia coli related to uropathogenic clonal group A

Enteroaggregative Escherichia coli (EAEC) are heterogeneous, diarrheagenic E. coli. Of EAEC strains from Nigeria, 10 independent antimicrobial-resistant isolates belonged to the multilocus sequence type 69 clonal complex, to which uropathogenic E. coli clonal group A belongs. This finding suggests a recent common ancestor for these distinct groups of pathogenic E. coli