INTRODUCTION

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Escherichia coli is one of the major causes of human infectious diseases, partly because of the wide variety of virulence mechanisms and pathotypes (15), and new pathotypes continue to be described. A new pathotype was proposed by Gunzburg et al. after examining diarrheal pathogens in a prospective community-based study among Australian Aboriginal children (22). One group of isolates was significantly (P < 0.05) associated with diarrhea, and these isolates were particularly common among children younger than 18 months. The isolates did not produce any recognized enterotoxin or classic enteric virulence factor, although they exhibited diffuse or aggregative adhesion in a modified adhesion assay (15). All isolates were able to detach HEp-2 cell monolayers and were termed "cell-detaching E. coli." This property was shown to be mediated by alpha-hemolysin, and we demonstrate below that all cell-detaching E. coli strains produce alpha-hemolysin and that some may also produce cytotoxic necrotizing factor 1 (CNF1) and other toxins. However, neither alpha-hemolysin nor CNF1 has been clearly demonstrated to be an enteric virulence factor, and the role of hemolysin in particular is controversial. We will refer to these isolates as diarrhea-associated hemolytic E. coli (DHEC) isolates.

Alpha-hemolytic E. coli strains have been associated with human enteric disease, especially among young children (8, 10-12, 20-22), and the related enterohemolysin of E. coli O157 (35) appears to be involved in enteric disease. There has, however, been no large prospective case-controlled epidemiologic study of the association of alpha-hemolysin with human diarrhea. Alpha-hemolytic bacteria are also associated with enteric disease and diarrhea in pigs, cattle, and dogs (9, 13, 33, 36, 44, 45). Porcine diarrheal strains are almost universally hemolytic (23a), and alpha-hemolysin in these isolates enhanced virulence and colonization (37) but was not itself diarrheagenic. More recent studies have found that Hly⁺ CNF1⁺ strains caused fluid accumulation in piglets (33) and that neonatal pigs were susceptible to challenge with Hly⁺ CNF⁺ strains, which caused bloody diarrhea, enterocolitis, and systemic disease (45).

In contrast, some earlier studies were unable to demonstrate a role for hemolysin in enteric disease, since neither hemolytic bacteria nor their supernatants caused fluid accumulation in ileal loops (10, 14, 37). Hemolytic strains may be isolated from the feces of asymptomatic people (26), and, among humans, hemolysin is more commonly associated with strains causing extraintestinal infections (5, 26).

The genetics and in vitro mechanisms of alpha-hemolysin are well known. The hlyCABD operon encodes the structural 110-kDa hemolysin protein (HlyA) and proteins involved in processing and export (42). Once secreted, hemolytic activity is short-lived, and this has complicated studies of hemolysin toxigenicity (42). Hemolysin does not require a receptor to bind to target cells, inserting instead into the target cell membrane to form a pore that allows the free flow of cations, sugars, and water. This leads to leakage of intracellular contents and affects the cytoskeleton and metabolism (4, 9, 42, 43). In extraintestinal infections, hemolysin has multiple effects and roles, including resistance to host defense, tissue damage, and lethality, either by direct action or by stimulation of inflammatory mediators and signal transduction pathways (7, 9, 16, 42).

CNF is a 114-kDa protein with homology to a family of dermonecrotic toxins (18) and is encoded by the monocistronic cnf gene, which lies just downstream of hly. The CNF1 toxin causes HeLa cells to become large and multinucleated as a result of actin disassembly, which results from activation of Rho (10, 19, 31). Similar to alpha-hemolysin, the role of CNF1 in diarrhea remains unclear. CNF1-producing strains have been isolated from diarrheal stools and have been associated with several outbreaks in humans (8, 10) and animals (13, 33, 44). Unfortunately, no large, prospective, case-controlled studies have been performed, and the best evidence for the pathogenicity of CNF1-toxigenic isolates is the marked virulence in piglet challenge experiments (45), outlined above. Purified CNF1 did not show enterotoxic potential in the suckling mouse or induce fluid accumulation in the rabbit ileal loop (10, 14), in contrast to the related CNF2, which is linked to enteric disease in animals (13, 14, 30). Both CNF toxins are extremely lethal, and have a variety of in vivo effects including tissue necrosis and edema (12-14).

In this paper, we characterize DHEC isolates that were obtained from a study where alpha-hemolysin was significantly associated with disease (22) and show that they are able to cause disease in rabbits. Using molecular genetics, we attempt to analyze the role of each gene in pathogenesis.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. A collection of 177 DHEC strains were obtained as part of a study of diarrheal virulence factors (22), and 3 of these isolates were selected for further study (Table 1). Also listed are mutant variants of wild-type DHEC and plasmids including cloned constructs of hly and cnf genes from strain A70.1 and strains used in mutagenesis.

Assays for cell detachment, alpha-hemolysin, and CNF. Cell detachment was assayed as described by Gunzburg et al. (22). A semiconfluent monolayer of HEp-2 cells in a 24-well tissue culture tray was washed three times with phosphate-buffered saline (PBS) supplemented with CaCl₂ and MgCl₂ (each at 0.01%) (PBS-CM), and 1 ml of PBS-CM was added to each well. A 10-µl volume of log-phase bacterial culture was added, and the plate was incubated at 37°C for 90 min under 5% CO₂. Monolayers were washed three times with PBS-CM, fixed with 70% methanol for 10 min, stained with 0.13% crystal violet for 10 min, and then briefly destained in water. Significant destruction of the monolayer was recorded as cell detachment and was quantified by eluting crystal violet with a solution of 50% ethanol, 49% water, and 1% sodium dodecyl sulfate (SDS) and measuring the absorbance of the eluate at 590 nm. Alpha-hemolysin was detected by the presence of characteristic zones of lysis on Columbia agar (Oxoid) containing 5% washed sheep erythrocytes, with observation at 4 h and again after overnight incubation, as described by Beutin (5).

Molecular genetic techniques. DNA preparation, cloning, manipulation, and Southern hybridizations were performed as described by Sambrook et al. (34) unless otherwise stated. Transposon mutagenesis with Tn1725 was performed by the method of Ubben and Schmitt (41). Chromosomal DNA was prepared by the method of Ausubel et al. (2).

Chromosomal mutagenesis by marker exchange. Mutagenesis of chromosomal genes was performed by the method of Penfold and Pemberton (32). A DNA fragment internal to the gene of interest was cloned into pJP5603 or pJP5608, which contain a lacZ with a multiple-cloning site from pUC18 and a pir-dependent R6K origin of replication. Transformants containing the insert of interest were first screened in JM109pir to enable blue-white selection and then transformed into S17-1pir for conjugation into a rifampin-resistant (Rif^r) variant of the DHEC strain. Mutations were selected by loss of the corresponding phenotype and confirmed by Southern blotting. All Rif^r variants of DHEC strains used as recipients for conjugations were otherwise indistinguishable from the parent.

Animal challenge. The RITARD (reversible intestinal tie adult rabbit diarrhea) assay was performed by the method of Albert et al. (1). Rabbits were challenged with 10¹⁰ bacteria grown on colonization factor antigen agar and were observed for up to 7 days for diarrhea and other symptoms and for shedding of challenge organisms. Shedding was monitored by performing daily rectal swabbing. Animals that developed frank diarrhea were sacrificed, and all the remaining animals were sacrificed on day eight. Following sacrifice, the rabbits were examined for gross pathological changes, and sections were taken from the midjejunum, the proximal and distal ileum, the proximal and distal colon, the cecum (excluding the blind segment that was surgically manipulated), the appendix, the rectum, and the mesenteric lymph nodes. These were preserved in buffered formal saline, sectioned, stained with hematoxylin-eosin, and examined by light microscopy (see above). Pathological changes including inflammation, polymorphonuclear leukocyte (PMN) infiltration, and peritonitis were noted semiquantitatively, ranging from normal (0) or mild (1) to severe (3).

Protein analysis. Standard protein analysis, SDS-polyacrylamide gel electrophoresis, immunization, Western blotting, and immunostaining techniques were performed as outlined by Harlow and Lane (24), unless otherwise stated. Monoclonal antibodies to alpha-hemolysin (h2A, h11A, f11F, I1C) (25) were kindly provided by F. Hugo, Institute of Medical Microbiology, University of Giessen, Giessen, Germany.

	RESULTS

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Cloning and characterization of alpha-hemolysin from DHEC. A cosmid library of A70.1 DNA was screened for production of alpha-hemolysin, and three clones were isolated. Restriction analysis (Fig. 1) revealed that two clones, p3E1 and p3E3, were identical and differed from a third clone, p4D3. From p3E1, hemolytic activity was subcloned on a 28-kb SalI fragment (giving pSE376 [Table 1]) and a 15.3-kb partial PstI fragment (generating pSE377). pSE377 was further mapped with probes, restriction enzymes, and transposon Tn1725 (41), which also confirmed the region containing hly (Fig. 2). Gene order was determined with the use of a series of DNA probes directed to different regions of the operon.

View larger version (7K): [in this window] [in a new window]   FIG. 1.   Map of the two chromosomal loci from A70.1 containing the hlyCABD operon as predicted from the maps of cosmids p3E1 and p4D3. The two copies of hly are designated hlyI (in p3E1) and hlyII (in p4D3). See the text for further details. B, BglII; Bm, BamHI; E, EcoRI; S, SalI; , hly; &atyp0220;, cnf.

View larger version (10K): [in this window] [in a new window]   FIG. 2.   Map of fragments in pSE377, which contains hly subcloned from cosmid p3E1 from A70.1 and is shown here aligned with previously mapped hly loci as obtained from reference 29 (a and b) and GenBank accession no. M10133 and M12863 (a). B, BglII; Bm, BamHI; E, EcoRI; P, PstI.

Cloning and analysis of CNF1 toxin production. DHEC strains were examined for production of CNFs and other toxins on HeLa cells. Of 177 DHEC, 54 (30.5%) (Table 2) were found to produce CNF1 toxin based on characteristic morphological alterations (Fig. 3a). There was no evidence for production of CNF2 or verotoxins. In strains A70.1, A98.1, and 55.3, the identity of the toxin was confirmed by neutralization with specific antisera against CNF1, and in strain A70.1, it was confirmed by sequence analysis (see below).

View larger version (90K): [in this window] [in a new window]   View larger version (80K): [in this window] [in a new window]   View larger version (102K): [in this window] [in a new window]   FIG. 3.   Toxin production by CDEC A70.1. (a) A70.1 bacterial cell lysate, diluted 1:20 and inoculated onto HeLa cells. In addition to generalized cell toxicity, two types of abnormal cells are observed: (i) large, multinucleated cells with diffusely staining borders, characteristic of CNF1; and (ii) densely staining, mononuclear, highly elongated cells, referred to as spindle cells. Magnification, ×1,000. (b) Dilution (1/80) of A70.1 lysate. Multinucleated cells are few, but more spindle cells are observed, and they predominate in some fields. Magnification, ×1,000. (c) Dilution (1/20) of lysate from DH5(pSE260), containing the cloned cnf gene from A70.1 on a high-copy number plasmid. All cells are multinucleated. Spindle cells are absent. Magnification, ×1,000.

View larger version (5K): [in this window] [in a new window]   FIG. 4.   Map of pSE379 containing a 13-kb gene fragment including cnf, showing the position of the Tn1725 insertion that added the EcoRI site. Also shown are subclones derived from pSE379. pSE378 is a 4.3-kb EcoRI fragment cloned into pUC18. pSE266 is a 0.95-kb Sau3AI fragment cloned into pUC18 and used as probe for cnf. B, BglII; E, EcoRI; P, PstI; S, SalI; Sm, SmaI.

Identification of two chromosomal hly loci in A70.1. The restriction map of cosmids p3E1, p3E3, and p4D3 was determined with the assistance of hly and cnf probes. This demonstrated that the identical cosmids p3E1 and 3E3 carried cnf while p4D3 lacked cnf and possessed different BglII and BamHI sites. Identically sized fragments were observed in Southern blots of chromosomal DNA, which confirmed that A70.1 contained one cnf gene and two hly operons. These were named hlyI, which is present on p3E1 and linked to cnf, and hlyII, which is present on p4D3. Probing of chromosomal DNA from strains 55.3 and A98.1, as well as mutagenesis, suggested that these two isolates also possess two hly genes and one cnf gene.

Mutagenesis of chromosomal genes in wild-type DHEC. To understand the role of hly and cnf in intestinal diseases, chromosomal genes were inactivated by marker exchange techniques. The CNF1 gene was inactivated by cloning a 1.4-kb PstI-BglII fragment (corresponding to nucleotides 1044 to 2495 of the cnf open reading frame) into pJP5603, an oriR6K-based suicide vector (Fig. 5). After this recombinant plasmid (pSE298) was conjugated into A70.1 Rif^r, 80% (38 of 48) of the transconjugants had lost the ability to produce CNF1 toxin. Insertion into the chromosome was demonstrated by disruption of the 23-kb BamHI fragment. The same technique on Rif^r variants of A98.1 and 55.3 inactivated CNF1 production in 100% of transconjugants.

View larger version (9K): [in this window] [in a new window]   FIG. 5.   Derivation of fragments for recombinant suicide vectors. A 1.4-kb BglII-PstI fragment was cloned from cnf into pJP5603, giving pSE297, or into pJP5608, giving pSE298. A 0.55-kb EcoRI fragment was cloned from within hlyA into pJP5603 or pJP5608, yielding pSE346 and pSE345, respectively. B, BglII; Bm, BamHI; E, EcoRI; S, SalI.

Other toxins produced by DHEC. A number of potentially novel toxins appear to be produced by some DHEC strains, as observed in the effect of freeze-thawed bacterial lysates on HeLa cells in the 72-h CNF assay. It was demonstrated that the following phenotypes were not due to alpha-hemolysin, since they were not mediated by the cloned hly from A70.1 or observed in most other DHEC strains.

Animal challenge experiments. Animal models were used to evaluate DHEC enteropathogenicity. Oral inoculation of streptomycin-treated specific-pathogen-free mice and rabbit ileal loops were attempted. While some colonization and pathological changes were observed, virulence was difficult to reproduce and was often mild or moderate, and so these models were abandoned (results not shown).

View larger version (143K): [in this window] [in a new window]   FIG. 6.   Histopathological changes to rabbit intestines after challenge with A70.1. (a) Multifocal areas of mucosal erosion with submucosal accumulation of neutrophils and lymphocytes, capillary congestion, and lacteal dilatation. Rabbit ileum stained with hemotoxylin and eosin. Magnification, ×400. (b) Diffuse presence of active germinal centers in Peyer's patches along the length of the ileum with a marked increase in the number of intraepithelial lymphocytes (arrow) and tingible-body macrophages. Rabbit ileum, stained with hemotoxylin and eosin. Magnification, ×400.

	DISCUSSION

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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 (8-13, 20-22, 33, 36, 43, 44). Further, it supports the findings of Wray et al. (45), who demonstrated that Hly⁺ CNF1⁺ 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 TDH (28), Serpulina hyodysenteriae hemolysin (40), and delta-hemolysin of Staphylococcus aureus (27). 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, 9, 16, 42, 43) 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, 23). 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.