Relationship between heat-labile enterotoxin secretion capacity and virulence in wild type porcine-origin enterotoxigenic Escherichia coli strains.

Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of enterotoxigenic Escherichia coli (ETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system (T2SS). We sought to determine the relationship between the capacity to secrete LT and virulence in porcine-origin wild type (WT) ETEC strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme-linked immunosorbent assay to measure LT concentrations in culture supernatants. All strains had detectable LT in supernatants by 2 h of culture and 1 strain, which was particularly virulent in gnotobiotic piglets (3030-2), had the highest LT secretion level all porcine-origin WT strains tested (P<0.05). The level of LT secretion (concentration in supernatants at 6-h culture) explained 92% of the variation in time-to-a-moribund-condition (R2 = 0.92, P<0.0001) in gnotobiotic piglets inoculated with either strain 3030-2, or an ETEC strain of lesser virulence (2534-86), or a non-enterotoxigenic WT strain (G58-1). All 16 porcine ETEC strains were positive by PCR analysis for the T2SS genes, gspD and gspK, and bioinformatic analysis of 4 porcine-origin strains for which complete genomic sequences were available revealed a T2SS with a high degree of homology to that of H10407. Maximum Likelihood phylogenetic trees constructed using T2SS genes gspC, gspD, gspE and homologs showed that strains 2534-86 and 3030-2 clustered together in the same clade with other porcine-origin ETEC strains in the database, UMNK88 and UMN18. Protein modeling of the ATPase gene (gspE) further revealed a direct relationship between the predicted ATP-binding capacities and LT secretion levels as follows: H10407, -8.8 kcal/mol and 199 ng/ml; 3030-2, -8.6 kcal/mol and 133 ng/ml; and 2534-86, -8.5 kcal/mol and 80 ng/ml. This study demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and between the latter and virulence.

Glucose significantly enhances enterotoxigenic Escherichia coli adherence to intestinal epithelial cells through its effects on heat-labile enterotoxin production.

The present study tested whether exposure of enterotoxigenic Escherichia coli (ETEC) to glucose at different concentrations in the media results in increased bacterial adherence to host cells through increased heat-labile enterotoxin (LT) production, thereby suggesting the effects are physiological. Porcine-origin ETEC strains grown in Casamino acid yeast extract medium containing different concentrations of glucose were washed and inoculated onto IPEC-J2 porcine intestinal epithelial cells to test for effects on adherence and host cell cAMP concentrations. Consistent with previous studies, all LT+ strains had higher ETEC adherence to IPEC-J2 cells than did LT- strains. Adherence of the LT- but not the LT+ strains was increased by pre-incubating the IPEC-J2 cells with LT and decreased by co-incubation with GM1 ganglioside in a dose-dependent manner (P<0.05). To determine whether the glucose concentration of the cell culture media has an effect on adherence, IPEC-J2 cells were inoculated with LT+ or LT- strains in cell culture media containing a final glucose concentration of 0, 0.25, 0.5, 1.0 or 2.0%, and incubated for 4 h. Only media containing 0.25% glucose resulted in increased adherence and cAMP levels, and this was limited to IPEC-J2 cells inoculated with LT+ strains. This study supports the hypothesis that glucose, at a concentration optimal for LT expression, enhances bacterial adherence through the promotion of LT production. Hence, these results establish the physiological relevance of the effects of glucose on LT production and provide a basis for how glucose intake may influence the severity of ETEC infection.

Inverse relationship between heat stable enterotoxin-b induced fluid accumulation and adherence of F4ac-positive enterotoxigenic Escherichia coli in ligated jejunal loops of F4ab/ac fimbria receptor-positive swine.

Heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli (ETEC) increases bacterial adherence to porcine enterocytes in vitro and enhances small intestinal colonization in swine. Heat-stable enterotoxin-b (STb) is not known to affect colonization; however, through an induction of net fluid accumulation it might reduce bacterial adherence. The relationship between fluid accumulation and bacterial adherence in jejunal loops inoculated with ETEC strains that produce LT, STb, both, or neither toxin was studied. Ligated jejunal loops were constructed in weaned Yorkshire pigs in two independent experiments (Exp. 1, n=5, 8-week-old; Exp. 2, n=6, 6-8-week-old). Each pig was inoculated with six F4ac(+)E. coli strains: (1) LT(+), STb(+) parent (WAM2317); (2) STb(-) (ΔestB) mutant (MUN297); (3) MUN297 complemented with STb (MUN298); (4) LT(-) STb(-) (ΔeltAB ΔestB) mutant (MUN300); (5) MUN300 complemented with LT (MUN301); and (6) 1836-2 (non-enterotoxigenic, wild-type). Pigs were confirmed to be K88 (F4)ab/ac receptor-positive in Exp. 2 by testing for intestinal mucin-type glycoproteins and inferred to be receptor-positive in both Exp. 1 and 2 based on histopathologic evidence of bacterial adherence. Strains that produced STb induced marked fluid accumulation with the response (ml/cm) to WAM2317 and MUN298 significantly greater than that to the other strains (P<0.0001). Conversely, bacterial adherence scores based on immunohistochemistry and CFU/g of washed mucosa were both lowest in the strains that expressed STb and highest in those that did not. For the two experiments combined, the Pearson correlation coefficient (R) between fluid volume (ml/cm) and log CFU per gram was -0.57021 (P<0.0001); R(2)=0.3521 (n=197). These results support the hypothesis that enterotoxin-induced fluid accumulation flushes progeny organisms into the lumen of the bowel, thereby increasing the likelihood of fecal shedding and transmission of the pathogen to new hosts.