Secretion of heat-labile enterotoxin by porcine-origin enterotoxigenic Escherichia coli and relation to virulence

Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of porcine-origin enterotoxigenic Escherichia coli (pETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system (T2SS), but its presence or importance in pETEC has not been established. Exposure of pETEC to glucose has been shown to result in different secretion levels of LT. Furthermore, the relationship between the level of LT secreted and the virulence potential of the respective pETEC strain has not been established. To determine the relationship between the capacity to secrete LT and virulence in wild-type (WT) pETEC, 16 strains isolated from cases of severe diarrheal disease were analyzed by monosialoganglioside-ELISA to measure LT concentrations in culture supernatants. All strains had detectable LT in culture supernatants; however, 3030-2, which was particularly virulent, had the highest LT level. Collectively, in gnotobiotic piglets inoculated with one of three strains varying in LT secretion level, viz., 3030-2, 2534-86, or G58-1, LT secretion correlated with 92% of the variation in time-to-a-moribund-condition (R2 = 0.92, P<0.0001). Further examination of the 16 WT pETEC strains revealed the presence of genes for a T2SS. Bioinformatic analysis of 4 pETEC strains for which complete genomic sequences were available revealed a T2SS with a high degree of amino acid sequence identity to that of H10407. Protein modeling of the ATPase gene (gspE) suggested a direct relationship between the predicted ATP-binding capacities and LT secretion levels. When grown in casamino acid-yeast extract medium with varying glucose levels (0, 0.25, 0.5, 1.0 or 2.0%), only media containing 0.25% glucose resulted in increased adherence and cAMP levels. Furthermore, LT+ pETEC strains adhered to porcine epithelial IPEC-J2 cells more than LT- strains. These studies support the hypothesis that glucose, at a concentration optimal for LT expression, enhances bacterial adherence through the promotion of LT production, and demonstrate a direct relationship between the predicted ATP-binding capacity of GspE and LT secretion, and between the latter and virulence. 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.