characterization of the roles of hemolysin and other ...iai.asm.org/content/66/5/2040.full.pdflogic...

INFECTION AND IMMUNITY,0019-9567/98/$04.0010

May 1998, p. 2040–2051 Vol. 66, No. 5

Copyright © 1998, American Society for Microbiology

Characterization of the Roles of Hemolysin and Other Toxins inEnteropathy Caused by Alpha-Hemolytic Escherichia coli

Linked to Human DiarrheaSIMON J. ELLIOTT,1,2* S. SRINIVAS,3 M. JOHN ALBERT,4 KHORSHED ALAM,4 ROY M. ROBINS-BROWNE,5

STUART T. GUNZBURG,2 BRIAN J. MEE,2 AND BARBARA J. CHANG2

Center for Vaccine Development1 and Department of Comparative Medicine and Pathology,3 University of MarylandSchool of Medicine, Baltimore, Maryland 21201; International Center for Diarrheal Disease Research, Dhaka,

Bangladesh4; and Department of Microbiology, The University of Melbourne, Parkville, Victoria 3052,5

and Department of Microbiology, The University of Western Australia, Nedlands,Western Australia 6009,2 Australia

Received 5 September 1997/Returned for modification 23 October 1997/Accepted 29 January 1998

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

Escherichia coli is one of the major causes of human infec-tious diseases, partly because of the wide variety of virulencemechanisms and pathotypes (15), and new pathotypes con-tinue to be described. A new pathotype was proposed by Gunz-burg et al. after examining diarrheal pathogens in a prospectivecommunity-based study among Australian Aboriginal children(22). One group of isolates was significantly (P , 0.05) asso-ciated with diarrhea, and these isolates were particularly com-mon among children younger than 18 months. The isolates didnot produce any recognized enterotoxin or classic enteric vir-ulence factor, although they exhibited diffuse or aggregativeadhesion in a modified adhesion assay (15). All isolates wereable to detach HEp-2 cell monolayers and were termed “cell-detaching E. coli.” This property was shown to be mediated byalpha-hemolysin, and we demonstrate below that all cell-de-taching E. coli strains produce alpha-hemolysin and that somemay also produce cytotoxic necrotizing factor 1 (CNF1) andother toxins. However, neither alpha-hemolysin nor CNF1 hasbeen clearly demonstrated to be an enteric virulence factor,and the role of hemolysin in particular is controversial. We willrefer to these isolates as diarrhea-associated hemolytic E. coli(DHEC) isolates.

Alpha-hemolytic E. coli strains have been associated withhuman 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 epidemio-

logic study of the association of alpha-hemolysin with humandiarrhea. Alpha-hemolytic bacteria are also associated withenteric disease and diarrhea in pigs, cattle, and dogs (9, 13, 33,36, 44, 45). Porcine diarrheal strains are almost universallyhemolytic (23a), and alpha-hemolysin in these isolates en-hanced virulence and colonization (37) but was not itself diar-rheagenic. More recent studies have found that Hly1 CNF11

strains caused fluid accumulation in piglets (33) and that neo-natal pigs were susceptible to challenge with Hly1 CNF1

strains, which caused bloody diarrhea, enterocolitis, and sys-temic disease (45).

In contrast, some earlier studies were unable to demonstratea role for hemolysin in enteric disease, since neither hemolyticbacteria nor their supernatants caused fluid accumulation inileal loops (10, 14, 37). Hemolytic strains may be isolated fromthe feces of asymptomatic people (26), and, among humans,hemolysin is more commonly associated with strains causingextraintestinal infections (5, 26).

The genetics and in vitro mechanisms of alpha-hemolysinare well known. The hlyCABD operon encodes the structural110-kDa hemolysin protein (HlyA) and proteins involved inprocessing and export (42). Once secreted, hemolytic activity isshort-lived, and this has complicated studies of hemolysin tox-igenicity (42). Hemolysin does not require a receptor to bind totarget cells, inserting instead into the target cell membrane toform a pore that allows the free flow of cations, sugars, andwater. This leads to leakage of intracellular contents and af-fects the cytoskeleton and metabolism (4, 9, 42, 43). In ex-traintestinal infections, hemolysin has multiple effects androles, including resistance to host defense, tissue damage, andlethality, either by direct action or by stimulation of inflamma-tory mediators and signal transduction pathways (7, 9, 16, 42).

* Corresponding author. Mailing address: Center for Vaccine De-velopment, University of Maryland School of Medicine, 685 W Balti-more St., Baltimore, MD 21201. Phone: (410) 706 2493. Fax: (410) 7066205. E-mail: [email protected].

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CNF is a 114-kDa protein with homology to a family ofdermonecrotic toxins (18) and is encoded by the monocistroniccnf gene, which lies just downstream of hly. The CNF1 toxincauses HeLa cells to become large and multinucleated as aresult of actin disassembly, which results from activation ofRho (10, 19, 31). Similar to alpha-hemolysin, the role of CNF1in diarrhea remains unclear. CNF1-producing strains havebeen isolated from diarrheal stools and have been associatedwith several outbreaks in humans (8, 10) and animals (13, 33,44). Unfortunately, no large, prospective, case-controlled stud-ies have been performed, and the best evidence for the patho-genicity of CNF1-toxigenic isolates is the marked virulence inpiglet challenge experiments (45), outlined above. PurifiedCNF1 did not show enterotoxic potential in the suckling mouseor induce fluid accumulation in the rabbit ileal loop (10, 14), incontrast to the related CNF2, which is linked to enteric diseasein animals (13, 14, 30). Both CNF toxins are extremely lethal,and have a variety of in vivo effects including tissue necrosisand edema (12–14).

In this paper, we characterize DHEC isolates that wereobtained from a study where alpha-hemolysin was significantlyassociated with disease (22) and show that they are able tocause disease in rabbits. Using molecular genetics, we attemptto analyze the role of each gene in pathogenesis.

MATERIALS AND METHODS

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

Assays for cell detachment, alpha-hemolysin, and CNF. Cell detachment wasassayed as described by Gunzburg et al. (22). A semiconfluent monolayer ofHEp-2 cells in a 24-well tissue culture tray was washed three times with phos-phate-buffered saline (PBS) supplemented with CaCl2 and MgCl2 (each at0.01%) (PBS-CM), and 1 ml of PBS-CM was added to each well. A 10-ml volumeof log-phase bacterial culture was added, and the plate was incubated at 37°C for90 min under 5% CO2. Monolayers were washed three times with PBS-CM, fixedwith 70% methanol for 10 min, stained with 0.13% crystal violet for 10 min, andthen briefly destained in water. Significant destruction of the monolayer was

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Relevant properties Reference

Wild typeA70.1 Hly, CNF1, DA, MSHA, pap, detaching-cytotoxin positive, spindle factor positive 22A98.1 Hly, CNF1, pap 2255.3 Hly, CNF1 22J96 Hly, CNF1, pap 29

ControlC600 Avirulent K-12 34

Mutant variants of 55.3SE368 55.3 CNF12 This paperSE371 55.3 Hly2 This paperSE372 55.3 Hly2, CNF12 This paper

Plasmidsp3E1 hlyI cnf cosmid clone This paperp3E3 hlyI cnf cosmid clone This paperp4D3 hlyII cosmid clone This paper

pSE376a Hly1 CNF1 28-kb SalI fragment from p3E1 This paperpSE377a Hly1 15.3-kb partial PstI fragment from p3E1 This paperpSE378a CNF11 4.3-kb EcoRI fragment This paper

pSE379 13-kb EcoRI fragment, containing cnf This paperpJP5603 oriR6K, lacZ Km 32pJP5608 oriR6K, lacZ Tet 32

pSE297 1.4-kb PstI-BglII fragment from pSE379 in pJP5603 This paperpSE298 1.4-kb PstI-BglII fragment from pSE379 in pJP5608 This paperpSE345 0.55-kb EcoRI fragment from pSE377 in pJP5608 This paperpSE346 0.55-kb EcoRI fragment from pSE377 in pJP5603 This paperpSE266 0.95-kb Sau3AI fragment from pSE377 cloned into pUC18 This paper

pBGS18 Kmr pUC18 derivative 38

pHC79 Apr Tcr cos

Miscellaneous cloning strainsDH5a supE44 DlacU169 (f80 lacZDM15) hsdR17 recA1 endA1 gyrA96 hi-1 relA1 34JM109lpir recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi-1D(lac-proAB) F9 [traD36 proAB1

lacIq lacZDM15] lpirR6K32

S17-1lpir thi pro hsdR hsdM1 recA::(RP4-2-Tc::Mu-Km::Tn7, Tpr Smr)lpir 32

a These plasmids were transformed into E. coli C600 for RITARD model studies.

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recorded as cell detachment and was quantified by eluting crystal violet with asolution of 50% ethanol, 49% water, and 1% sodium dodecyl sulfate (SDS) andmeasuring the absorbance of the eluate at 590 nm. Alpha-hemolysin was de-tected by the presence of characteristic zones of lysis on Columbia agar (Oxoid)containing 5% washed sheep erythrocytes, with observation at 4 h and again afterovernight incubation, as described by Beutin (5).

CNF was detected by the method of Oswald et al. (30) by assaying the abilityof freeze-thawed bacterial lysates to cause characteristic multinucleation, cyto-toxicity, and morphological changes to HeLa cells. CNF activity was quantified asthe 50% multinucleation titer (MN50), the maximal dilution able to causemultinucleation in 50% of HeLa cells. Antibody neutralization of CNF activitywas demonstrated by preincubation of the lysate overnight at 37°C with neutral-izing antiserum before addition to HeLa cells. Antisera to CNF1 and CNF2 werekindly provided by A. Caprioli, Instituto Superiore di Sanita, Rome, Italy.

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

To clone virulence factors from A70.1, chromosomal DNA of A70.1 waspartially digested with Sau3AI and ligated into the BamHI site of pHC79.Concatamers were packaged into phage heads and transfected into E. coli HB101as specified by the manufacturer (Amersham lDNA in vitro packaging kit). Atotal of 412 colonies were screened for cell detachment and alpha-hemolysin.

A 4.3-kb fragment containing cnf was sequenced by the method of Bankier etal. (3), and a library of contiguous, randomly sheared fragments was constructed.Double-stranded DNA sequencing was performed with the Advanced Biosys-tems Inc. (ABI) Prism dye terminator sequencing kit, as specified by the man-ufacturer, and an ABI automatic sequencer. The sequence was complied andanalyzed with software packages MacVector for the Macintosh and tfasta, fasta,and align written for Unix.

Chromosomal mutagenesis by marker exchange. Mutagenesis of chromo-somal genes was performed by the method of Penfold and Pemberton (32). ADNA fragment internal to the gene of interest was cloned into pJP5603 orpJP5608, which contain a lacZ with a multiple-cloning site from pUC18 and apir-dependent R6K origin of replication. Transformants containing the insert ofinterest were first screened in JM109lpir to enable blue-white selection and thentransformed into S17-1lpir for conjugation into a rifampin-resistant (Rifr) vari-ant of the DHEC strain. Mutations were selected by loss of the correspondingphenotype and confirmed by Southern blotting. All Rifr variants of DHECstrains used as recipients for conjugations were otherwise indistinguishable fromthe parent.

Animal challenge. The RITARD (reversible intestinal tie adult rabbit diar-rhea) assay was performed by the method of Albert et al. (1). Rabbits werechallenged 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 ofchallenge organisms. Shedding was monitored by performing daily rectal swab-bing. Animals that developed frank diarrhea were sacrificed, and all the remain-ing animals were sacrificed on day eight. Following sacrifice, the rabbits wereexamined for gross pathological changes, and sections were taken from themidjejunum, the proximal and distal ileum, the proximal and distal colon, thececum (excluding the blind segment that was surgically manipulated), the ap-pendix, the rectum, and the mesenteric lymph nodes. These were preserved inbuffered formal saline, sectioned, stained with hematoxylin-eosin, and examinedby light microscopy (see above). Pathological changes including inflammation,polymorphonuclear leukocyte (PMN) infiltration, and peritonitis were notedsemiquantitatively, ranging from normal (0) or mild (1) to severe (3).

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

For production of polyclonal antiserum to A70.1 hemolysin, alpha-hemolysinwas precipitated from broth by the method of Bhakdi et al. (6) and subjected toSDS-polyacrylamide gel electrophoresis. The band of interest was excised fromthe acrylamide and macerated, and a 50% suspension in saline was injectedintraperitoneally into male Swiss mice on a schedule of 1, 14, 21, and 28 days; themice were bled on day 35.

RESULTS

Cloning and characterization of alpha-hemolysin fromDHEC. A cosmid library of A70.1 DNA was screened forproduction of alpha-hemolysin, and three clones were isolated.Restriction analysis (Fig. 1) revealed that two clones, p3E1 andp3E3, were identical and differed from a third clone, p4D3.From p3E1, hemolytic activity was subcloned on a 28-kb SalIfragment (giving pSE376 [Table 1]) and a 15.3-kb partial PstIfragment (generating pSE377). pSE377 was further mappedwith probes, restriction enzymes, and transposon Tn1725 (41),which also confirmed the region containing hly (Fig. 2). Geneorder was determined with the use of a series of DNA probesdirected to different regions of the operon.

The hly operon of DHEC was compared to those previouslydescribed. The restriction map and gene order were very sim-

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 ofhly are designated hlyI (in p3E1) and hlyII (in p4D3). See the text for further details. B, BglII; Bm, BamHI; E, EcoRI; S, SalI;3, 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 asobtained from reference 29 (a and b) and GenBank accession no. M10133 and M12863 (a). B, BglII; Bm, BamHI; E, EcoRI; P, PstI.

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ilar to those of the chromosomally located hly of uropatho-genic E. coli (UPEC) J96 and the plasmid-borne hly fromplasmid pHly512, isolated from an animal pathogen (Fig. 2).Four monoclonal antibodies raised against HlyA from UPECrecognized a 110-kDa supernatant protein produced by A70.1and hly clones. A polyclonal antiserum raised against A70.1HlyA recognized a similar 110-kDa band from A70.1, hlyclones, and UPEC J96 (results not shown).

The remaining collection of 176 DHEC strains was exam-ined by colony blotting, and they all recognized hly DNAprobes (see below) and the polyclonal antiserum. Most, but notall, recognized at least one monoclonal antibody, indicatingantigenic variation in HlyA among DHEC strains.

Cloning and analysis of CNF1 toxin production. DHECstrains were examined for production of CNFs and other toxinson HeLa cells. Of 177 DHEC, 54 (30.5%) (Table 2) werefound to produce CNF1 toxin based on characteristic morpho-logical alterations (Fig. 3a). There was no evidence for pro-duction of CNF2 or verotoxins. In strains A70.1, A98.1, and55.3, the identity of the toxin was confirmed by neutralizationwith specific antisera against CNF1, and in strain A70.1, it wasconfirmed by sequence analysis (see below).

CNF1 activity was encoded on cosmid clones p3E1 and p3E3but not p4D3 and was subcloned from p3E1 as a 28-kb SalIfragment (pSE376) and a 13-kb EcoRI fragment (pSE379).Figure 4 shows the map of pSE379, illustrating the positions ofcnf and a Tn1725 insert (described below). The restriction mapof the cnf region was similar to that in UPEC EB35 (17). UsingTn1725, which contains an EcoRI site in the terminal repeatregions, we were able to insert an EcoRI site that enabled us toisolate cnf on a 4.3-kb fragment. This subclone, pSE378, washighly active in toxin assays, with an MN50 of 1:1,280 comparedto 1:40 to 1:80 for A70.1 (Fig. 3c) and strains 55.3 and A98.1.This subclone was sequenced as a series of overlapping randomfragments. Analysis of the 4,295-bp sequence (GenBank acces-sion no. A42629) and the predicted translation product indi-cated significant DNA and amino acid homology (99.6%amino acid identity) to cnf from UPEC (accession no. X7670)(18). Differences occurred in the 59 region of cnf and upstreamof the gene.

A probe for cnf was made by cloning a 902-bp Sau3AIfragment (corresponding to nucleotides 1061 to 1963) frompSE378. This probe hybridized to 60 of the 177 DHEC strains,including all the strains producing assayable toxin (Table 2),and is 90% specific and 100% sensitive for predicting CNF1toxin production.

Identification of two chromosomal hly loci in A70.1. Therestriction map of cosmids p3E1, p3E3, and p4D3 was deter-mined with the assistance of hly and cnf probes. This demon-strated that the identical cosmids p3E1 and 3E3 carried cnf

while p4D3 lacked cnf and possessed different BglII andBamHI sites. Identically sized fragments were observed inSouthern blots of chromosomal DNA, which confirmed thatA70.1 contained one cnf gene and two hly operons. These werenamed hlyI, which is present on p3E1 and linked to cnf, andhlyII, which is present on p4D3. Probing of chromosomal DNAfrom strains 55.3 and A98.1, as well as mutagenesis, suggestedthat these two isolates also possess two hly genes and one cnfgene.

Mutagenesis of chromosomal genes in wild-type DHEC. Tounderstand the role of hly and cnf in intestinal diseases, chro-mosomal genes were inactivated by marker exchange tech-niques. The CNF1 gene was inactivated by cloning a 1.4-kbPstI-BglII fragment (corresponding to nucleotides 1044 to 2495of the cnf open reading frame) into pJP5603, an oriR6K-basedsuicide vector (Fig. 5). After this recombinant plasmid(pSE298) was conjugated into A70.1 Rifr, 80% (38 of 48) of thetransconjugants had lost the ability to produce CNF1 toxin.Insertion into the chromosome was demonstrated by disrup-tion of the 23-kb BamHI fragment. The same technique onRifr variants of A98.1 and 55.3 inactivated CNF1 production in100% of transconjugants.

To inactivate hly by this strategy, we proposed to use suicidevectors with different antibiotic resistance markers to inacti-vate the two hly genes. A 500-bp EcoRI fragment from withinhlyA from hlyI was cloned into pJP5603, generating pSE346,which inserted into and inactivated cloned hly genes on plas-mid pSE377 (from hlyI). When pSE346 was introduced intoA70.1, it inserted into the chromosome at hlyII in 20/20 at-tempts. When other fragments were cloned from hlyI intovectors pJP5603 or pJP5608, the resultant recombinant suicidevectors always (30 of 30) inserted into hlyII in A70.1, as seen inSouthern blots of chromosomal DNA. To inactivate hlyI, atetracycline-resistant analog of pSE346 was constructed bycloning the 500-bp EcoRI hlyA fragment from hlyI intopJP5608 (a Tcr derivative of pJP5603). In A70.1, this plasmid(pSE345) inserted into the chromosome at hlyII and was neverobserved to insert into hlyI. When pSE345 was introduced intoA70.1 derivatives containing the insertion of pSE346 into hlyII(i.e., A70.1 hlyI hlyII::pSE346), all 1,400 Tcr Kmr transconju-gants screened were hemolytic, indicating a failure to insertinto and inactivate hlyI. These data collectively indicate thathlyI in A70.1 is resistant to mutagenesis by this strategy, pos-sibly due to the tertiary structure of the DNA.

This strategy was then attempted on A98.1 and 55.3 withoutsuccess. The lone exception was isolated after pSE346 wasintroduced into 55.3, generating a nonhemolytic variant,SE371 (Table 1). SE371 produced CNF1 and was otherwiseindistinguishable from the parent, and Southern blotting dem-onstrated that hlyI had been insertionally inactivated but thatthe pathogenicity island containing hlyII had spontaneouslyexcised. Given the difficulty in constructing a nonhemolyticvariant by genetic techniques, this spontaneous mutant wasused in further manipulations and was used to construct adouble mutant defective in both alpha-hemolysin and CNF1.The CNF1-targeting pSE297 was introduced into SE371, andCNF1 production was lost in 1 (2%) of 42 strains examined.This strain, SE372 (Table 1), lacks both Hly and CNF1 pro-duction. This and the other variants were used in animal chal-lenge experiments.

Other toxins produced by DHEC. A number of potentiallynovel toxins appear to be produced by some DHEC strains, asobserved in the effect of freeze-thawed bacterial lysates onHeLa cells in the 72-h CNF assay. It was demonstrated that thefollowing phenotypes were not due to alpha-hemolysin, since

TABLE 2. Association of CNF1 toxin and probe amongDHEC strainsa

CNF1 toxinNo. of strains with cnf probe Total no.

of strainsPresent Absent

Present 54 0 54Absent 6 117 123

Total 60 117 177

a DHEC strains were examined for production of CNF1 toxin as outlined inMaterials and Methods. Reactivity to the Sau3AI probe for CNF1 (from plasmidpSE266) was determined by colony hybridization. Specificity, 90%; sensitivity,100%; positive predictive value, 90%; negative predictive value, 100%.



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they were not mediated by the cloned hly from A70.1 or ob-served in most other DHEC strains.

The first toxin caused detachment of HeLa cells in the CNF1assay. While CNF1 can cause limited death and detachment,1:10- and 1:20-diluted lysates from several CNF11 strains, in-cluding A70.1, exhibited a pronounced cell-detaching activitythat was not observed in other CNF11 strains. This phenom-enon was not due to CNF1, since 55.3, A98.1, and A70.1exhibited a similar MN50 of approximately 1:40, implying sim-ilar levels of CNF1 production, yet only A70.1 caused a com-plete loss of the HeLa cell monolayer. Further, lysates fromDH5a containing the cloned cnf of A70.1(pSE378) exhibited

potent multinucleating activity (MN50, 1:1,250) but did notcause detachment. Finally, mutation of cnf in A70.1 abolishedmultinucleation but not detaching activity. In addition, twoDHEC isolates that did not produce CNF1 (as determined bymultinucleation of HeLa cells and DNA probe) were cytotoxic.These results demonstrate that cell-detaching cytotoxicity ex-ists separately from CNF1.

The second potentially novel toxin caused unusual morpho-logical alterations to HeLa cells. This phenotype was first iden-tified in lysates from A70.1, which caused (in addition to thecell enlargement, loss of border definition, and multinucleationcharacteristic of CNF1) some HeLa cells to become thin and

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, twotypes of abnormal cells are observed: (i) large, multinucleated cells with diffusely staining borders, characteristic of CNF1; and (ii) densely staining, mononuclear, highlyelongated cells, referred to as spindle cells. Magnification, 31,000. (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, 31,000. (c) Dilution (1/20) of lysate from DH5a(pSE260), containing the cloned cnf gene from A70.1 on ahigh-copy number plasmid. All cells are multinucleated. Spindle cells are absent. Magnification, 31,000.

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 aresubclones 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 probefor cnf. B, BglII; E, EcoRI; P, PstI; S, SalI; Sm, SmaI.



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elongated, a phenomenon referred to as spindle cells (Fig. 3).Dilution of A70.1 lysates led to reduced multinucleating andenlarging activity but had less effect on spindle-forming activ-ity, and at a 1:80 dilution, spindle cells dominated some fields(Fig. 3b). This activity was not observed in the lysates from thecnf clone (Fig. 3c). This demonstrates that CNF1 activity isdistinct from spindle-forming activity. However, insertional in-activation of cnf in A70.1 caused a marked reduction in thespindle-forming activity of lysates. Although it is possible thatCNF1 potentiates the spindle-forming factor, it appears un-usual that two toxins with completely opposite toxic effectscould act synergistically. CNF1 leads to large, rounded cellswith diffusely staining cytoplasm, while spindle cells are ex-tremely elongated and mononuclear, with a normally stainingcytoplasm.

Animal challenge experiments. Animal models were used toevaluate DHEC enteropathogenicity. Oral inoculation ofstreptomycin-treated specific-pathogen-free mice and rabbitileal loops were attempted. While some colonization andpathological changes were observed, virulence was difficult toreproduce and was often mild or moderate, and so these mod-els were abandoned (results not shown).

In the RITARD model, DHEC strains exhibited virulencewith a set of clear, unique pathologic findings. Rabbits infectedwith DHEC exhibited depression, cramping, and diarrheawhich ranged from mild to frank (Table 3). All rabbits thatdeveloped frank diarrhea produced mucoid diarrhea with analstaining. Some rabbits died. At sacrifice, there was evidence offluid accumulation in the small and large intestines, sometimeswith mucus and blood. Infected animals showed a number ofmarked histological changes in both the small and large intes-tines (Fig. 6). These included multifocal areas of mucosal ero-sion and necrosis, hyperemia, and colonic goblet cell hyperpla-sia. The level of hyperplasia was remarkable, and the mucosain rabbits infected with strain A70.1 was observed to be 2 to 3times the normal thickness. Other inflammatory changes in-cluded activation of the Peyer’s patches, PMN infiltration intothe lamina propria and lumen, edema, and lymphocytic hyper-plasia. However, the pathologic findings varied with each rab-bit and infecting organism. In contrast, rabbits infected withthe negative control E. coli C600 did not develop diarrhea andtheir intestines were entirely normal. It is clear that DHECstrains are more virulent in RITARD than is C600 and, overall,that DHEC strains are significantly (x2, P , 0.05) associatedwith diarrhea. To compare histopathological changes, out-

comes were scored semiquantitatively (Table 4). By using thisapproach, it was evident that there was considerable variationbetween DHEC strains but that, overall, DHEC strains weresignificantly (t test, P , 0.05) associated with inflammation andthat more inflammatory changes were observed in the largeintestine.

To examine the role of different virulence factors in DHEC-mediated enteric disease, we compared rabbits infected witheither wild-type DHEC strains, isogenic mutants, C600 con-taining cloned DHEC virulence genes, or C600. All rabbitsinfected with a C600 strain containing hlyI cloned on a high-copy-number vector developed frank diarrhea and showedmarked inflammatory reactions in both small and large intes-tine. This clone was significantly more (x2, P , 0.05) likely tocause diarrhea than was the plasmid-free variant, suggestingthat cloned hly is able to confer virulence upon nonpathogenicE. coli strains. Rabbits infected with the CNF11 clone devel-oped diarrhea yet were not statistically more likely to developdiarrhea than were controls (x2, P , 0.05). They also exhibitedintestinal inflammation, although less than that observed inrabbits infected with the hemolytic clone.

Paradoxically, the loss of either hly or cnf from DHEC 55.3did not lead to a measurable reduction in disease, as measuredby diarrheagenicity or histopathological changes, and thesestrains remained more virulent than C600. However, loss ofboth CNF1 and hemolysin production was associated with lowintestinal inflammation (Tables 3 and 4). These results suggestthat other virulence factors are present in DHEC strains andcomplicate our understanding of the role of hly in DHEC-associated diarrhea.

DISCUSSION

DHEC strains were initially described as a class of E. colithat were significantly more common in children with diarrheathan in controls (22). To examine the diarrheagenic ability ofDHEC in vivo, we used the RITARD model, in which theeffect of each strain on the entire gut of a rabbit is examinedfor up to a week. In this model, DHEC strains caused frankmucoid diarrhea and a set of clear, unique, and marked intes-tinal pathologic findings in both the small and large intestines,including necrosis, hyperplasia, and multiple indicators of in-flammation. In contrast, avirulent E. coli C600 caused nopathologic effects or diarrhea in rabbits.

The mechanism by which DHEC strains caused enteric dis-

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.



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TABLE 3. Development of diarrhea, death, and shedding of organisms in a RITARD model challenge with DHEC

Challenge strain Rabbitno.

Diarrhea Shedding(day stopped) Comments

Day started Day stopped Maximum severity

Wild typec

A70.1 1 —a — — — Dead on day 22 1 1 Mild 43 2 2*b Frank 2*4 — — — 65 1 2* Frank 2*

A98.1 6 — — — — Dead on day 17 — — — 68 1 2* Frank 2*9 1 1 Mild 210 1 1 Mild 5

55.3 11 — — — —12 — — — —13 3 3* Frank 3*14 3 3* Frank 3*

Mutant variantsSE368 (55.3 CNF2) 15 — — — 3 Dead on day 5

16 1 1* Frank 1*17 — — — 4 Dead on day 618 1 1* Frank 1*

SE371 (55.3 Hly2) 19 1 1* Frank 1*20 1 3* Frank 3* Moderate on day 1, frank on day 321 — — — 422 1 1* Frank 1*

SE372 (55.3 Hly2 CNF2) 23 — — — 724 2 2* Frank 2*25 3 3* Frank 3*26 3 4* Frank 3* Slight on day 3, frank on day 427 1 2* Frank 2* Slight on day 1, frank on day 2

Recombinant plasmids in C600pSE376 (C600 Hly1 CNF1)c 28 — — — 2

29 — — — —30 1 3* Frank 3* Slight on day 1, frank on day 331 3 3* Frank 3*

pSE377 (C600 Hly1)c 32 3 3* Frank 3*33 4 5* Frank 5* Slight on day 4, frank on day 534 6 7* Frank 7* Slight on day 6, frank on day 735 4 6* Frank 6* Slight on day 4, frank on day 6

pSE378 (C600 CNF1) 36 — — — 237 1 2* Slight 2* Died in lab accident38 — — — —39 2 2* Frank 2*40 — — — 4

Negative control C60041 — — — —42 — — — —43 — — — —44 — — — —

a —, no disease was observed.b Asterisk indicates that rabbit was sacrificed on day indicated.c Significant associations with diarrhea (x2 test, P # 0.05); CDEC wild type versus C600, Hly1 clones in C600 versus C600, C600(pSE377) versus C600.



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FIG. 6. Histopathological changes to rabbit intestines after challenge with A70.1. (a) Multifocal areas of mucosal erosion with submucosal accumulation ofneutrophils and lymphocytes, capillary congestion, and lacteal dilatation. Rabbit ileum stained with hemotoxylin and eosin. Magnification, 3400. (b) Diffuse presenceof 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-bodymacrophages. Rabbit ileum, stained with hemotoxylin and eosin. Magnification, 3400.



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ease was unknown as they lacked traditional diarrheal viru-lence factors (22). All DHEC strains produced alpha-hemoly-sin, and we have demonstrated that DHEC hemolysin couldnot be significantly distinguished from UPEC hemolysin on thebasis of the restriction map, DNA hybridization, protein size,or antibody reactivity. Since the role of hemolysin in entericdisease is unresolved, we sought other toxins among DHECstrains that may explain their apparent diarrheagenicity. Ap-proximately one-third of isolates produced CNF1 toxin, asdemonstrated by the toxin assay and DNA hybridization. Theanalysis of cnf in A70.1 indicates that it is very similar to cnffrom UPEC. There is evidence in a few DHEC isolates for twonovel toxins which are phenotypically and genotypically dis-tinct from alpha-hemolysin and CNF1. The first toxin is aHeLa cell cytotoxin found in A70.1 and some other DHECstrains. The second toxin observed in A70.1 possessed spindle-forming activity, since bacterial lysates caused a subset of HeLacells to become densely staining, thin and elongated. While thiswas not mediated by cnf, mutation of cnf in the wild typemarkedly reduced spindle-forming activity. It is possible thatCNF1 is necessary to potentiate toxin activity or, more likely,that mutation has had polar effects on downstream genes. It isnot known what is encoded downstream of cnf.

Since the majority of DHEC strains do not appear to pro-duce toxins other than hemolysin and CNF1, we examined therole of these factors in DHEC-induced disease with wild-typestrains, clones, and mutant variants. The evidence from clonedhly supports its role as a virulence factor. When hly was clonedon a high-copy-number vector, it conferred on avirulent C600the ability to cause diarrhea in rabbits and other pathologicchanges. Notably, hemolysin appeared to be associated withinflammation, especially in the colon. By using statistical tests,the presence of both plasmid pSE377 (hly) and the hly genewas significantly associated with diarrhea. In contrast, thecloned cnf gene could not be demonstrated statistically tocause diarrhea, and so its role, if any, in this model of diseaseappears to be minor.

Complicating this analysis, however, is the data obtainedfrom isogenic mutants of DHEC 55.3. Mutagenesis of cnf, hly,

or both did not significantly affect the onset, duration, or se-verity of diarrhea, although loss of both was associated with adecrease in intestinal inflammation. This indicates that factorsin strain 55.3 other than cnf or hly may also mediate diarrheain the RITARD model, suggesting multiple virulence mecha-nisms.

In summary, the data suggests that DHEC strains are viru-lent and that alpha-hemolysin, the factor shared among allDHEC strains, is a virulence factor. This supports our initialepidemiologic observation linking hemolysin to diarrhea andagrees with other studies that found statistically significantassociations and/or linkage to a particular outbreak (8–13,20–22, 33, 36, 43, 44). Further, it supports the findings of Wrayet al. (45), who demonstrated that Hly1 CNF11 strains frompigs were virulent in piglets and caused pathologic changesgenerally similar to those observed by us, including diarrhea,death, and effects on both small and large intestines such asedema, inflammation, and necrosis. Rather than followingclassic secretory diarrhea, the disease was closer to that due toproinflammatory and invasive pathogens. The strains studiedby Wray et al. (45) appeared to be more virulent in the pigletmodel, possibly reflecting their challenge of piglets with pigpathogens compared to our challenging of rabbits with humanpathogens.

We propose several modes by which alpha-hemolysin couldact as a diarrheal toxin. The first involves pore formation in theenterocytes, allowing the free flow of ions into the lumen. Thismay be enhanced by a Ca21 flux into the cell, stimulating thearachidonic acid pathway and upregulation of secretion, or bygeneralized cytotoxicity, perturbing both secretion and absorb-tion. Other pore-forming hemolysins shown to cause diarrheainclude Vibrio cholerae El Tor hemolysin (39), Vibrio parahae-molyticus TDH (28), Serpulina hyodysenteriae hemolysin (40),and delta-hemolysin of Staphylococcus aureus (27). BecauseDHEC diarrhea appears to be associated with marked inflam-mation, this suggests that the inflammatory effects of hemoly-sin seen in extraintestinal infections and in vitro (7, 9, 16, 42,43) are present in intestinal infection and may be important indiarrhea.

TABLE 4. Summary of findings in RITARD model challenges with DHECa

Strain Virulencefactor(s)

No. ofrabbits

No.dead

No. withdiarrhea

% withdiarrhea

Diarrhoealseverity(mean)b

Mean durationof diarrhea

(days)c

Inflammationin small

intestined

Inflammationin colond

Ratio ofinflammation(large:smallintestine)

A70.1 CNF1 Hly1 5 1 3 75 62.5 6.8 38 150 4A98.1 CNF1 Hly1 5 1 3 75 50 4 262 187 0.755.3 CNF1 Hly1 4 0 2 50 50 4 32.4 38 1.2SE368 55.3 CNF2 4 2* 2 50 50 7 75 125 1.7SE371 55.3 Hly2 4 0 3 75 75 10 125 50 0.4SE372 55.3 CNF2 Hly2 5 0 4 80 80 8.8 20 20 1pSE376 CNF1 Hly1 4 0 2 50 50 5.5 25 50 2pSE377 Hly1 4 0 4 100 100 6.5 100 125 1.25pSE379 CNF1 5 0* 2 40 30 3.8 0 40 NDe

C600 CNF2 Hly2 5 0 0 0 0 0 4 7 1.75

a Dead rabbits excluded, except when marked with an asterisk. In those cases, two rabbits infected with 55.3 CNF2 died late in the experiment but had not developeddiarrhea earlier. These rabbits were regarded for calculations as two nondiarrheal rabbits and were not excluded from calculations as for other deaths. One rabbitinfected with pSE260 died from noninfectious causes after developing mild diarrhea. This rabbit was included and was assumed to have suffered mild diarrhea for 7days, for purposes of calculation.

b The maximal severity of diarrhea in each rabbit, where mild 5 50 and frank 5 100, was added, and the total score was divided by the number of rabbits. A scoreof 100 indicates that all rabbits developed frank diarrhea.

c The number of days (in 7 days) of diarrhea 3 the severity of diarrhea (mild 5 1, frank 5 2) for each rabbit was added and divided by the number of rabbits.d Inflammation was recorded if inflammation, PMN infiltration, or peritonitis was observed. Histopathological descriptions of inflammatory reactions were graded

0, 1, 2, and 3, and the total score was divided by the number observed and adjusted for the number of rabbits observed. The small intestine score consists of scoresfrom the jejunum, proximal and distal ileum; while the large intestine included proximal and distal colon.

e ND, not done.



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If we are to consider alpha-hemolysin to be an enteric vir-ulence factor and DHEC strains to be diarrheal pathogens, itis possible that UPEC strains are also enterovirulent. Cer-tainly, the virulence factors observed in DHEC strains did notdistinguish them from uropathogens, and it is unclear if DHECstrains are uropathogenic organisms functioning as entericpathogens or are specialized enteric pathogens. The commondogma that divides intestinal from extraintestinal E. coli patho-gens may be an oversimplification that needs to be reexamined.However, it is possible that specific factors such as a specializedHlyA type are present in DHEC strains and are functionallydifferent from those in UPEC strains.

It has been demonstrated that hly from different isolates maybe more than 95% homologous but can differ markedly in theregulation of expression, HlyA stability, and virulence (4, 23).This was most elegantly shown in an animal model of UPECpathogenesis (23). Deletion of a chromosomally encoded hlyfrom a human UPEC led to a significant reduction in toxicityfor mice, and reintroduction of hly cloned from the chromo-somes of various UPEC strains on a recombinant plasmidrestored toxicity. However, hly isolated from a plasmid of ananimal enteropathogen led to a very marginal increase in tox-icity despite being very highly related to hly of UPEC strains.Therefore, the marked similarities of DHEC hly to both UPEChly and animal enteropathogen hly may nonetheless mask realand significant differences that are relevant to pathogenesis inthis model. Indeed, we have observed with monoclonal anti-bodies that DHEC produce different HlyA subtypes that clus-ter with other virulence factors (unpublished data). This link-age suggests that there are DHEC subtypes that may causedifferent types of disease, with only certain categories respon-sible for diarrhea, and may explain why the epidemiologic linkto diarrhea is often not as strong as that for “classic” entero-pathogens.

Finally, we must address the findings of earlier workers, whowere unable to demonstrate a role for hemolysin in entericdisease. As outlined above, the type of hly used may be acrucial 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 diarrheagenicityin short-lived models appropriate for classic secretory toxins(e.g., cholera toxin) such as the rabbit ileal loop and have usedtoxin preparations despite HlyA being very labile. We wouldnot expect that the type of disease observed with DHEC wouldyield a positive result in these studies, and we were unable toshow virulence in the rabbit ileal loop. Marked disease wasobserved only several days after whole bacteria were inocu-lated into the RITARD model, and most pathologic findingswere observed in the large intestine.

In conclusion, we believe that DHEC strains are potentialenteropathogens and that the variant of alpha-hemolysin en-coded on the chromosome is an important but by no means theonly virulence factor in the RITARD model of infection. Webelieve that these preliminary results call for further experi-ments, including experiments with larger groups of animalsand those with more precisely defined measures of pathogen-esis. Finally, we believe that this result may eventually cause usto redefine the distinction between uropathogens and entero-pathogens and how a factor that promotes virulence in one sitemay function in another. Certainly, the way in which UPECinteracts with the intestine may determine its ability to subse-quently cause urinary tract infection.

ACKNOWLEDGMENTS

We thank M. M. Islam, ICDDR,B, for the main body of RITARDmodel studies; A. Joseph, M. Beach, and P. Kumar for assistance with

RITARD at the CVD; and J. Nataro and J. Kaper for advice andsupport.

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