Characterization of the roles of hemolysin and other toxins in enteropathy caused by alpha-hemolytic Escherichia coli linked to human diarrhea

Abstract

Escherichia coli strains producing alpha-hemolysin have been associated with diarrhea in several studies, but it has not been clearly demonstrated that these strains are enteropathogens or that alpha-hemolysin is an enteric virulence factor. Such strains are generally regarded as avirulent commensals. We examined a collection of diarrhea-associated hemolytic E. coli (DHEC) strains for virulence factors. No strain produced classic enterotoxins, but they all produced an alpha-hemolysin that was indistinguishable from that of uropathogenic E. coli strains. DHEC strains also produced other toxins including cytotoxic necrotizing factor 1 (CNF1) and novel toxins, including a cell-detaching cytotoxin and a toxin that causes HeLa cell elongation. DHEC strains were enteropathogenic in the RITARD (reversible intestinal tie adult rabbit diarrhea) model of diarrhea, causing characteristic enteropathies, including inflammation, necrosis, and colonic cell hyperplasia in both small and large intestines. Alpha-hemolysin appeared to be a major virulence factor in this model since it conferred virulence to nonpathogenic E. coli strains. Other virulence factors also appear to be contributing to virulence. These findings support the epidemiologic link to diarrhea and suggest that further research into the role of DHEC and alpha-hemolysin in enteric disease is warranted.

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; ░⃞, cnf.

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 (a and b) and GenBank accession no. M10133 and M12863 (a). B, BglII; Bm, BamHI; E, EcoRI; P, PstI.

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 DH5α(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.

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 DH5α(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.

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 DH5α(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.

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.

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.

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.