DHEC strains were initially described as a class of E. coli
that were significantly more common in children with diarrhea than in controls (22
). To examine the diarrheagenic ability of DHEC in vivo, we used the RITARD model, in which the effect of each strain on the entire gut of a rabbit is examined for up to a week. In this model, DHEC strains caused frank mucoid diarrhea and a set of clear, unique, and marked intestinal pathologic findings in both the small and large intestines, including necrosis, hyperplasia, and multiple indicators of inflammation. In contrast, avirulent E. coli
C600 caused no pathologic effects or diarrhea in rabbits.
The mechanism by which DHEC strains caused enteric disease was unknown as they lacked traditional diarrheal virulence factors (22
). All DHEC strains produced alpha-hemolysin, and we have demonstrated that DHEC hemolysin could not be significantly distinguished from UPEC hemolysin on the basis of the restriction map, DNA hybridization, protein size, or antibody reactivity. Since the role of hemolysin in enteric disease is unresolved, we sought other toxins among DHEC strains that may explain their apparent diarrheagenicity. Approximately one-third of isolates produced CNF1 toxin, as demonstrated by the toxin assay and DNA hybridization. The analysis of cnf
in A70.1 indicates that it is very similar to cnf
from UPEC. There is evidence in a few DHEC isolates for two novel toxins which are phenotypically and genotypically distinct from alpha-hemolysin and CNF1. The first toxin is a HeLa cell cytotoxin found in A70.1 and some other DHEC strains. The second toxin observed in A70.1 possessed spindle-forming activity, since bacterial lysates caused a subset of HeLa cells to become densely staining, thin and elongated. While this was not mediated by cnf
, mutation of cnf
in the wild type markedly reduced spindle-forming activity. It is possible that CNF1 is necessary to potentiate toxin activity or, more likely, that mutation has had polar effects on downstream genes. It is not known what is encoded downstream of cnf.
Since the majority of DHEC strains do not appear to produce toxins other than hemolysin and CNF1, we examined the role of these factors in DHEC-induced disease with wild-type strains, clones, and mutant variants. The evidence from cloned hly supports its role as a virulence factor. When hly was cloned on a high-copy-number vector, it conferred on avirulent C600 the ability to cause diarrhea in rabbits and other pathologic changes. Notably, hemolysin appeared to be associated with inflammation, especially in the colon. By using statistical tests, the presence of both plasmid pSE377 (hly) and the hly gene was significantly associated with diarrhea. In contrast, the cloned cnf gene could not be demonstrated statistically to cause diarrhea, and so its role, if any, in this model of disease appears to be minor.
Complicating this analysis, however, is the data obtained from isogenic mutants of DHEC 55.3. Mutagenesis of cnf, hly, or both did not significantly affect the onset, duration, or severity of diarrhea, although loss of both was associated with a decrease in intestinal inflammation. This indicates that factors in strain 55.3 other than cnf or hly may also mediate diarrhea in the RITARD model, suggesting multiple virulence mechanisms.
In summary, the data suggests that DHEC strains are virulent and that alpha-hemolysin, the factor shared among all DHEC strains, is a virulence factor. This supports our initial epidemiologic observation linking hemolysin to diarrhea and agrees with other studies that found statistically significant associations and/or linkage to a particular outbreak (8
). Further, it supports the findings of Wray et al. (45
), who demonstrated that Hly+
strains from pigs were virulent in piglets and caused pathologic changes generally similar to those observed by us, including diarrhea, death, and effects on both small and large intestines such as edema, inflammation, and necrosis. Rather than following classic secretory diarrhea, the disease was closer to that due to proinflammatory and invasive pathogens. The strains studied by Wray et al. (45
) appeared to be more virulent in the piglet model, possibly reflecting their challenge of piglets with pig pathogens compared to our challenging of rabbits with human pathogens.
We propose several modes by which alpha-hemolysin could act as a diarrheal toxin. The first involves pore formation in the enterocytes, allowing the free flow of ions into the lumen. This may be enhanced by a Ca2+
flux into the cell, stimulating the arachidonic acid pathway and upregulation of secretion, or by generalized cytotoxicity, perturbing both secretion and absorbtion. Other pore-forming hemolysins shown to cause diarrhea include Vibrio cholerae
El Tor hemolysin (39
), Vibrio parahaemolyticus
), Serpulina hyodysenteriae
), and delta-hemolysin of Staphylococcus aureus
). Because DHEC diarrhea appears to be associated with marked inflammation, this suggests that the inflammatory effects of hemolysin seen in extraintestinal infections and in vitro (7
) are present in intestinal infection and may be important in diarrhea.
If we are to consider alpha-hemolysin to be an enteric virulence factor and DHEC strains to be diarrheal pathogens, it is possible that UPEC strains are also enterovirulent. Certainly, the virulence factors observed in DHEC strains did not distinguish them from uropathogens, and it is unclear if DHEC strains are uropathogenic organisms functioning as enteric pathogens or are specialized enteric pathogens. The common dogma that divides intestinal from extraintestinal E. coli pathogens may be an oversimplification that needs to be reexamined. However, it is possible that specific factors such as a specialized HlyA type are present in DHEC strains and are functionally different from those in UPEC strains.
It has been demonstrated that hly
from different isolates may be more than 95% homologous but can differ markedly in the regulation of expression, HlyA stability, and virulence (4
). This was most elegantly shown in an animal model of UPEC pathogenesis (23
). Deletion of a chromosomally encoded hly
from a human UPEC led to a significant reduction in toxicity for mice, and reintroduction of hly
cloned from the chromosomes of various UPEC strains on a recombinant plasmid restored toxicity. However, hly
isolated from a plasmid of an animal enteropathogen led to a very marginal increase in toxicity despite being very highly related to hly
of UPEC strains. Therefore, the marked similarities of DHEC hly
to both UPEC hly
and animal enteropathogen hly
may nonetheless mask real and significant differences that are relevant to pathogenesis in this model. Indeed, we have observed with monoclonal antibodies that DHEC produce different HlyA subtypes that cluster with other virulence factors (unpublished data). This linkage suggests that there are DHEC subtypes that may cause different types of disease, with only certain categories responsible for diarrhea, and may explain why the epidemiologic link to diarrhea is often not as strong as that for “classic” enteropathogens.
Finally, we must address the findings of earlier workers, who were unable to demonstrate a role for hemolysin in enteric disease. As outlined above, the type of hly used may be a crucial factor. Thus, while DHEC hly may be a virulence factor, this is unlikely to apply to hly from all E. coli strains. Second, most studies have attempted to demonstrate diarrheagenicity in short-lived models appropriate for classic secretory toxins (e.g., cholera toxin) such as the rabbit ileal loop and have used toxin preparations despite HlyA being very labile. We would not expect that the type of disease observed with DHEC would yield a positive result in these studies, and we were unable to show virulence in the rabbit ileal loop. Marked disease was observed only several days after whole bacteria were inoculated into the RITARD model, and most pathologic findings were observed in the large intestine.
In conclusion, we believe that DHEC strains are potential enteropathogens and that the variant of alpha-hemolysin encoded on the chromosome is an important but by no means the only virulence factor in the RITARD model of infection. We believe that these preliminary results call for further experiments, including experiments with larger groups of animals and those with more precisely defined measures of pathogenesis. Finally, we believe that this result may eventually cause us to redefine the distinction between uropathogens and enteropathogens and how a factor that promotes virulence in one site may function in another. Certainly, the way in which UPEC interacts with the intestine may determine its ability to subsequently cause urinary tract infection.