Differences in levels of secreted locus of enterocyte effacement proteins between human disease-associated and bovine Escherichia coli O157

Ongoing extensive epidemiological studies of verotoxin-carrying Escherichia coli O157 (stx(+) eae(+)) have shown this bacterial pathogen to be common in cattle herds in the United States and the United Kingdom. However, the i ncidence of disease in humans due to this pathogen is still very low. This study set out to investigate if there is a difference between strains isola ted from human disease cases and those isolated from asymptomatic cattle wh ich would account for the low disease incidence of such a ubiquitous organi sm. The work presented here has compared human disease strains from both sp oradic and outbreak cases with a cross-section, as defined by pulsed-field gel electrophoresis, of E. coli O157 strains from cattle. Human (n = 22) an d bovine (n = 31) strains were genotyped for carriage of the genes for Shig a-like toxin types 1, 2, and 2c; E. coli secreted protein genes espA, espB, and espP; the enterohemolysin gene; eae (intimin); ast (enteroaggregative E. coli stable toxin [EAST]); and genes for common E. coli adhesins. Strain s were also phenotyped for hemolysin, EspP, Tir, and EspD expression as wel l as production of actin and cytoskeletal rearrangement associated with att aching and effacing (A/E) lesions on HeLa cells. The genotyping confirmed t hat there was little difference between the two groups, including carriage of stx(2) and stx(2c), which was similar in both sets. ast alleles were con firmed to all contain mutations that would prevent EAST expression. espP mu tations were found only in cattle strains (5 of 30). Clear differences were observed in the expression of locus of enterocyte effacement (LEE)-encoded factors between strains and in different media. EspD, as an indicator of L EE4 (espA, -B, and -D) expression, and Tir levels in supernatants were meas ured. Virtually all strains from both sources could produce EspD in Luria-B ertani broth, although at very different levels. Standard trichloroacetic a cid precipitation of secreted proteins from tissue culture medium produced detectable levels of EspD from the majority of strains of human origin (15 of 20) compared with only a few (4 of 20) bovine strains (P < 0.001), which is indicative of much higher levels of protein secretion from the human st rains. Addition of bovine serum albumin carrier protein before precipitatio n and enhanced detection techniques confirmed that EspD could be detected a fter growth in tissue culture medium for all strains, but levels from strai ns of human origin were on average 90-fold higher than those from strains o f bovine origin. In general, levels of secretion also correlated with abili ty to form A/E lesions on HeLa cells, with only the high-level protein secr etors in tissue culture medium exhibiting a localized adherence phenotype. This research shows significant differences between human- and bovine-deriv ed E. coli O157 (stx(+) eae(+)) strains and their production of certain LEE -encoded virulence factors. These data support the recent finding of Kim et al. (J. Kim, J. Nietfeldt, and A. K. Benson, Proc. Natl. Acad. Sci. USA 96 :13288-13293, 1999) proposing different E. coli O157 lineages in cattle and humans and extend the differential to the regulation of virulence factors. Potentially only a subset of E. coli O157 isolates (stx(+) eae(+)) in catt le may be capable of causing severe disease in humans.