Type II Heat-Labile Enterotoxins from 50 Diverse Escherichia coli Isolates Belong Almost Exclusively to the LT-IIc Family and May Be Prophage Encoded

Some enterotoxigenic Escherichia coli (ETEC) produce a type II heat-labile enterotoxin (LT-II) that activates adenylate cyclase in susceptible cells but is not neutralized by antisera against cholera toxin or type I heat-labile enterotoxin (LT-I). LT-I variants encoded by plasmids in ETEC from humans and pigs have amino acid sequences that are ≥95% identical. In contrast, LT-II toxins are chromosomally encoded and are much more diverse. Early studies characterized LT-IIa and LT-IIb variants, but a novel LT-IIc was reported recently. Here we characterized the LT-II encoding loci from 48 additional ETEC isolates. Two encoded LT-IIa, none encoded LT-IIb, and 46 encoded highly related variants of LT-IIc. Phylogenetic analysis indicated that the predicted LT-IIc toxins encoded by these loci could be assigned to 6 subgroups. The loci corresponding to individual toxins within each subgroup had DNA sequences that were more than 99% identical. The LT-IIc subgroups appear to have arisen by multiple recombinational events between progenitor loci encoding LT-IIc1- and LT-IIc3-like variants. All loci from representative isolates encoding the LT-IIa, LT-IIb, and each subgroup of LT-IIc enterotoxins are preceded by highly-related genes that are between 80 and 93% identical to predicted phage lysozyme genes. DNA sequences immediately following the B genes differ considerably between toxin subgroups, but all are most closely related to genomic sequences found in predicted prophages. Together these data suggest that the LT-II loci are inserted into lambdoid type prophages that may or may not be infectious. These findings raise the possibility that production of LT-II enterotoxins by ETEC may be determined by phage conversion and may be activated by induction of prophage, in a manner similar to control of production of Shiga-like toxins by converting phages in isolates of enterohemmorhagic E. coli

Type II heat-labile enterotoxin-producing Escherichia coli isolated from animals and humans.

Heat-labile enterotoxin (LT)-producing Escherichia coli strains, as identified by the Y1 adrenal cell assay, were examined with a DNA probe coding for type I and type II LTs. Of 236 LT-producing E. coli isolates, 60% hybridized with LT-I, 17% hybridized with LT-II, and 23% did not hybridize with either probe and no longer produced LT as determined by the Y1 adrenal cell assay. These isolates presumably lost plasmids coding for LT-I during storage. A total of 75% of LT-producing E. coli isolates (27 of 36) from cows, 64% of LT-producing E. coli isolates (7 of 11) from buffalo, 31% of LT-producing E. coli isolates (4 of 13) from beef obtained in markets, and 2% of LT-producing E. coli isolates (3 of 168) from humans contained genes coding for LT-II. Genes coding for LT-II were not found in 50 LT-I-producing and heat-stable enterotoxin-producing E. coli isolates from 11 children with diarrhea and 44 LT-nonproducing and heat-stable enterotoxin-producing E. coli isolates from 12 other children with diarrhea. A total of 9% of LT-II-producing E. coli isolates (3 of 34) from cows and buffalo hybridized with DNA probes for genes coding for verocytotoxin 2 (VT2), and 18% (6 of 34) hybridized with a DNA probe coding for enterohemorrhagic E. coli (EHEC) adhesin fimbriae. E. coli SA-53, the original isolate in which LT-II was found, contained genes coding for VT2 and EHEC adhesin fimbriae. Five VT-producing, LT-II-producing E. coli isolates that hybridized with the EHEC probe did not contain DNA sequences coding for VT1 or VT2. LT-II-producing E. coli strains were frequently isolated from cattle and buffalo but were rarely isolated from humans

IDENTIFICAÇÃO DOS GENES QUE CODIFICAM PARA A ENTEROTOXINA TERMOLÁBIL LT-II EM AMOSTRAS DE ESCHERICHIA COLI ISOLADAS DE BEZERROS COM DIARRÉIA NA REGIÃO DE JABOTICABAL, SP, BRASIL IDENTIFICATION OF THE GENES THAT ENCODE FOR THE LT-II THERMOLABILE ENTEROTOXIN AMONG STRAINS OF ESCHERICHIA COLI ISOLATED FROM CALVES WITH DIARRHEA IN THE REGION OF JABOTICABAL, SP, BRAZIL)

Examinando 52 espécimes fecais de bezerros com diarréia de fazendas da região de Jaboticabal, SP, Brasil, uma das amostras de Escherichia coli isoladas, quando analisada pela Reação em Cadeia da Polimerase (PCR), demonstrou a presença dos genes que codificam para a enterotoxina termolábil do tipo LT-II. Este é o primeiro relato de amostra de E. coli enterotoxigênica, isolada de bezerros com diarréia no Brasil, contendo os genes para codificação da enterotoxina LT-II. Encontram-se apenas citações de isolamento no Brasil de amostras de E. coli LT-II+ de alimentos de origem animal.<br>Examining 52 samples of stools from calves with diarrhea from the region of Jaboticabal, SP, Brazil , one of the strains of Escherichia coli isolated, as examined by the Polymerase Chain Reaction (PCR), harbored the genes encoding for the LT-II thermolabile enterotoxin. This is the first report on the isolation of enterotoxigenic E. coli from calves with diarrhea in Brazil harboring the genes for LT-II enterotoxin. In our country, strains of LT-II + E. coli have been reported to occur only in foods of animal origin

Cholesterol Depletion Reduces Entry of Campylobacter jejuni Cytolethal Distending Toxin and Attenuates Intoxication of Host Cells ▿

Campylobacter jejuni is a common cause of pediatric diarrhea worldwide. Cytolethal distending toxin, produced by Campylobacter jejuni, is a putative virulence factor that induces cell cycle arrest and apoptosis in eukaryotic cells. Cellular cholesterol, a major component of lipid rafts, has a pivotal role in regulating signaling transduction and protein trafficking as well as pathogen internalization. In this study, we demonstrated that cell intoxication by Campylobacter jejuni cytolethal distending toxin is through the association of cytolethal distending toxin subunits and membrane cholesterol-rich microdomains. Cytolethal distending toxin subunits cofractionated with detergent-resistant membranes, while the distribution reduced upon the depletion of cholesterol, suggesting that cytolethal distending toxin subunits are associated with lipid rafts. The disruption of cholesterol using methyl-β-cyclodextrin not only reduced the binding activity of cytolethal distending toxin subunits on the cell membrane but also impaired their delivery and attenuated toxin-induced cell cycle arrest. Accordingly, cell intoxication by cytolethal distending toxin was restored by cholesterol replenishment. These findings suggest that membrane cholesterol plays a critical role in the Campylobacter jejuni cytolethal distending toxin-induced pathogenesis of host cells