Vol. 58, No. 12INFECTION AND IMMUNITY, Dec. 1990, p. 3959-39650019-9567/90/123959-07$02.00/0Copyright X 1990, American Society for Microbiology

Acute Renal Tubular Necrosis and Death of Mice Orally Infectedwith Escherichia coli Strains That Produce Shiga-Like Toxin Type II

ELIZABETH A. WADOLKOWSKI,l LAWRENCE M. SUNG,' JENNIFER A. BURRIS,2 JAMES E. SAMUEL,ltAND ALISON D. O'BRIEN1*

Department of Microbiology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road,Bethesda, Maryland 2081447991 and Comparative Pathology, National Center for Research

Resources, National Institutes of Health, Bethesda, Maryland 208922

Received 16 July 1990/Accepted 27 September 1990

Escherichia coli 0157:H7 strains have been implicated as etiologic agents in food-borne outbreaks ofhemorrhagic colitis and the hemolytic-uremic syndrome. A prototype E. coli 0157:H7 strain, designated 933,produces Shiga-like toxin I (SLT-I) and SLT-II and harbors a 60-MDa plasmid. In a previous study,streptomycin-treated mice were fed 933 together with a derivative cured of the 60-MDa plasmid (designated933cu). Strain 933cu colonized poorly, but in approximately one-third of the animals, an isolate of 933cu wasobtained from the feces that had regained the ability to colonize well. This isolate, designated 933cu-rev, killedall of the animals when fed alone to mice. In this investigation, two types of experiments were done to assesswhether SLT-I, SLT-II, or both contributed to the death of mice fed 933cu-rev. (i) Mice were pretreated withmonoclonal antibodies to SLT-I, SLT-II, SLT-I and SLT-II, or cholera toxin (as a control) before infection with933cu-rev. (ii) Mice were fed either an E. coli K-12 strain carrying cloned SLT-I genes or the same K-12 straincarrying cloned SLT-II genes. The results of both types of experiments indicated that the deaths of the orallyinfected mice were due solely to SLT-II. Extensive histological and selected electron microscopic examinationsof various tissues from the infected animals suggested that death was due to acute renal cortical tubular necrosisconsistent with toxic renal damage. These data indicate a critical role for SLT-II, but not SLT-I, in renaldamage associated with E. coli 0157:117 infection of streptomycin-treated mice.

Enterohemorrhagic Escherichia coli (EHEC) strains ofserotype 0157:H7 have been associated with hemorrhagiccolitis and the hemolytic-uremic syndrome (HUS) (17, 36,50). EHEC strains possess a 60-MDa plasmid (16) andproduce elevated levels of Shiga-like cytotoxin I (SLT-I),SLT-II, or both (29-31). Toxin production by E. coli 0157:H7 strains is the result of lysogenization with one or twotoxin-converting phages (31, 43). SLTs, like Shiga toxin, areall composed of an enzymatically active A subunit and amultimeric B or binding subunit (6, 8, 39). The structuralgenes for Shiga toxin and SLT-I share greater than 99%nucleotide sequence homology and are referred to synony-mously as Shiga toxin-SLT-I (41). By contrast, Shiga toxin-SLT-I and SLT-II structural genes are about 58% homolo-gous, and the proteins they encode are not cross-neutralizable(15). SLT-I and SLT-II are cytotoxic for both Vero andHeLa cells and specifically bind the globotriaosyl ceramide(Gb3) present on many mammalian cells (14, 21-23, 44, 45).

Studies with rabbits (33) and mice (3) have indicated thatShiga-like toxins may play a critical role in the pathogenesisof diarrhea caused by E. coli 0157:H7. Barrett et al. recentlydescribed an animal model for SLT-II-induced disease (2).They reported that highly purified SLT-II continuouslyinfused into rabbits produced diarrhea accompanied bylesions similar to those seen in the colons of humans withhemorrhagic colitis (2, 35). Some of the animals developedrenal failure. However, the pathological lesions did notresemble those seen in humans with HUS (2).

Recently, we showed that when streptomycin-treatedmice were fed E. coli 0157:H7 strain 933cu-rev, an isolate

* Corresponding author.t Present address: Biocarb Inc., Gaithersburg, MD 20879.

obtained in a simultaneous-feeding experiment, the animalsdied from bilateral renal cortical tubular damage (46). Thepurpose of this study was to determine the role, if any, ofSLTs in the renal lesions and death of mice fed E. coli0157:H7 strain 933cu-rev.

MATERIALS AND METHODSAbbreviations. The following abbreviations are used in this

report: Glc, glucose; Gal, galactose; GlcNAc, N-acetylglu-cosamine; GalNAc, N-acetylgalactosamine; Cer, ceramide;CMH, Gal1-lCer; CDH, Gal1-4Glc1-lCer; Gb2, Galal-4GalB1-lCer; Gb3(CTH), Gala1-4GalP1-4GlcP1-1Cer; Gb4(GL4), GalNAc,1-3Galal-4Galp1-4Glcp1-lCer; Gb5, GalB1-3GalNAcI1-3Galol-4Gal31-4GlcPl-lCer; NeuAc, N-acetyl-neuraminic acid; GM3, NeuAca2-3Gal,1-4Glc,B1-1Cer; GM2,GalNAcp1-4(NeuAcot2-3)Galp1-4Glcp1-lCer; GM,, Gal,1-3GalNAcp1-4(NeuAcot2-3)GalB1-4Glc,31-lCer.

Bacterial strains and plasmids. The bacterial strains andplasmids used in this study are listed in Table 1. A sponta-neous streptomycin-resistant mutant was selected for eachstrain. E. coli 0157:H7 strain 933cu-rev, resistant to bothstreptomycin (Strr) and nalidixic acid (Nalr), was isolatedfrom the feces of a mouse cocolonized with E. coli 0157:H7strains 933 and 933cu as described previously (46).Media, enzymes, and biochemicals. Luria broth or Luria

broth agar (25) was used for routine culturing of bacteria.MacConkey agar (Difco Laboratories, Detroit, Mich.) wasprepared as instructed by the manufacturer. Where indi-cated, medium was supplemented with antibiotics (SigmaChemical Co., St. Louis, Mo.) at the following concentra-tions: streptomycin, 100 ,ug/ml; ampicillin, 100 Fig/ml; nali-dixic acid, 50 Fg/ml. Agarose for DNA electrophoresis waspurchased from International Biotechnologies, Inc., NewHaven, Conn. Restriction enzymes, calf intestinal alkaline

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TABLE 1. Bacterial strains and plasmids used in this study

Strain or Description or Referenceplasmid genotypea or source

E. coli strainsDH5a F' 480dlacZ A(lacZYA-argF)U169 40

recAl endAl hsdRJ7(rK MK )supE44 X- thi-l gyrA relAl

JE5505 F- lpo ppo his proA argE thi gal 12lac xyl mtl tsx

H1618 thr lac serjhuA rpsL thi hsdR 11fiu::MudI(Ap lac) fur-31

PlasmidspBR329 Tcr Apr Cmr; replicon ColEl 5pBluescript KS Apr expression vector StratagenepNAS10 pBR329 with stx; Apr 41pNN103 pBR328 with slt-II; Apr 27pMJ100 pBluescribe with slt-II; Apr 48

a Tcr, Apr, and Cmr, Resistance to tetracycline, ampicillin, and chloram-phenicol, respectively; stx, Shiga toxin operon; slt, SLT operon; A, A subunitgene; B, B subunit gene (of stx or sit); 0, operon fusion.

phosphatase, and T4 ligase were purchased from BoehringerMannheim Biochemicals, Indianapolis, Ind.Cloning and DNA preparation. The methods used to pre-

pare and clone DNA were described by Maniatis et al. (25).DNA fragments used for cloning were separated by electro-phoresis in 0.8% preparative agarose gels and eluted fromthe gels with a model UEA electroeluter (InternationalBiotechnologies).

Cytotoxicity assays. Microcytotoxicity assays were donewith Vero and HeLa cells by previously described modifi-cations (26) of the methods of Gentry and Dalrymple (10).The amount of toxin contained in the last 10-fold dilution ofthe sample in which greater than or equal to 50% of the Veroor HeLa cells detached from the plastic, as assessed by A620,was considered to be the 50% cytotoxic dose.Mouse colonization experiments. The methods used to

colonize mice with one or more E. coli strains were previ-ously described (46). Mice to be fed streptomycin-resistantE. coli K-12 strains carrying Shiga toxin or SLT-II genescloned in vectors with ampicillin resistance markers weregiven drinking water that contained streptomycin sulfate (5g/liter) and ampicillin (5 g/liter), whereas mice to be fedstreptomycin-resistant E. coli 0157:H7 strains were givendrinking water that contained only streptomycin (5 g/liter).On the next morning, groups of three mice were fed 1010CFU of each of the E. coli strains to be tested in 1 ml ofsterile 20% (wt/vol) sucrose as described previously (46).The mice readily ingested the bacterial suspensions and werethen returned to a normal diet that included drinking waterwith streptomycin sulfate and ampicillin or streptomycinsulfate alone. To assess the colonizing capacity of a strain,fecal samples were collected at 24- or 48-h intervals, homog-enized, diluted, and plated on selective agar medium. Theplates were incubated at 37°C for 18 to 24 h. The degree towhich a strain colonized was determined by the number ofCFU that persisted in feces. Each experiment was per-formed at least twice with identical results. Experimentsdone with mice fed E. coli strains transformed with plasmidpMJ100 or pNAS10 were carried out under BL-3 contain-ment by National Institutes of Health guidelines for the useof clones that express levels of cytotoxicity greater than thatof Shigella dysenteriae type 1 strain 60R (9).

Passive antibody transfer. Mice were injected intraperito-neally with 1 ml of diluted monoclonal antibody (MAb) to the

B subunit of SLT-I (MAb 13C4; see reference 42), the Asubunit of SLT-II (MAb llFll; see reference 34), the B andA subunits of SLT-I and SLT-II, respectively (MAbs 13C4and llFll), the B subunit of SLT-II (MAb BC5BB12; seereference 7), or cholera toxin (MAb 32D3; see reference 13)as a control. The anti-SLT MAbs were diluted to a cytotoxinneutralization titer of about 1:1,000 and injected in 1-mlvolumes at 24 and 1 h before oral infection with E. coli0157:H7 strain 933cu-rev. The method used to colonizestreptomycin-treated mice is described in the previous sec-tion.SLT binding to glycolipids directly on thin-layer chromato-

grams. Purified CMH, Gb3(CTH), and Gb4(GL4) were ob-tained from Supelco, Inc., Bellefonte, Pa. GDH (lactocere-brosides) was purchased from Sigma. Standard gangliosideswere obtained and purified by conventional methods (20) orobtained from BaChem, Inc., Torrance, Calif. Neutral gly-colipids from streptomycin-treated mouse kidneys, sepa-rated into renal medulla and renal cortex fractions, wereprepared by the method of Kyogashima et al. (18).The glycolipids to which SLTs bound were detected on

thin-layer chromatograms as described previously (24, 38).The chromatograms were overlaid with crude SLT-II pre-pared by the method of Samuel et al. (38). After incubationwith toxin, the chromatograms were washed and then over-laid with anti-SLT-II MAb llElO (34). The plates werewashed again and then overlaid with 125I-labeled goat anti-mouse immunoglobulin G, followed by autoradiography asdescribed previously (38).

Histology. Mice were sacrificed and kidneys and otherorgans were harvested at the first signs of disease after oralinfection with E. coli K-12 strains carrying cloned SLT-IIgenes or E. coli 0157:H7 strain 933cu-rev. The tissues werefixed in 10% buffered neutral Formalin and processed bystandard procedures. Sections of paraffin-embedded tissuewere stained with hematoxylin and eosin and examined bylight microscopy. All histological sections were coded topermit assessment of histopathologic changes without bias.

Transmission electron microscopy. At 7 days postfeedingor when signs of disease were first evident, animals colo-nized with strain 933, 933cu, or 933cu-rev were sacrificed.Kidneys were excised and cut into 3-mm pieces. Specimenswere fixed in 2% glutaraldehyde and processed by standardprocedures. Tissues were embedded in EPOX 812 (Ernest F.Fullam, Latham, N.Y.) as instructed by the manufacturer.The thin sections were stained with uranyl acetate and leadcitrate by standard procedures.

RESULTS

Passive protection with MAbs to SLTs. Animals passivelytreated with MAb culture supernatant against the A subunitof SLT-II or the B subunit of SLT-I and the A subunit ofSLT-II survived infection with strain 933cu-rev, as did micepretreated with ascites fluid, BC5BB12, directed against theB subunit of SLT-II (Table 2). By contrast, mice pretreatedwith MAb to the B subunit of SLT-I did not surviveinfection. In a previous study, extensive histological exam-ination of animals fed 933cu-rev revealed that they died frombilateral renal cortical tubular necrosis (46). The passive-transfer data strongly suggest that death of these mice wassolely due to SLT-II.

In vivo colonization of mice with SLT-producing E. coliK-12 strains. Mice were fed E. coli K-12 strains transformedwith plasmids carrying the genes for Shiga toxin or SLT-II toassess the role of SLT-II in the acute tubular necrosis that

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TABLE 2. Protection against E. coli 0157:H7 strain933cu-rev infection by passive transfer of MAb'

MAbbNo. of surviving mice/

Designation Directed against: no. passively immunized(reference)

13C4 (42) SLT-I B 0/5llF1l (34) SLT-II A 6/6BC5BB12 (7) SLT-II B 6/613C4 + llF1l SLT-I B + SLT-II A 6/632D3 (13) Cholera toxin B 1/5

a Mice were inoculated intraperitoneally with 1 ml of antibody at 24 and 1 hbefore feeding with E. coli 933cu-rev.

b MAbs, whether culture supematants (13C4, llFil, and 32D3) or asciticfluid (BC5BB12), were diluted to a cytotoxin-neutralizing titer of 1:1,000.

led to the death of these animals. An E. coli K-12 strainrather than an SLT-negative E. coli 0157:H7 strain wasselected as the host for recombinant plasmids because,according to the recombinant DNA guidelines established bythe National Institutes of Health (9), plasmids that expressShiga toxin or SLTs at levels of cytotoxicity greater than orequal to that of S. dysenteriae type 1 strain 60R may not beintroduced into a wild-type E. coli background. Although E.coli K-12 laboratory strains colonize the bowels of normalanimals or humans poorly (about 106 CFU/g of feces) rela-tive to normal fecal E. coli isolates (about 108 CFU/g offeces), if selective pressure, such as antibiotic treatment, isapplied to favor antibiotic-resistant E. coli K-12 strains,these strains colonize the bowel well and persist indefinitely(19). When mice treated with streptomycin sulfate andampicillin were individually fed 1010 CFU of E. coli K-12strains containing a recombinant plasmid carrying genes foreither Shiga toxin (pNAS10) or SLT-II (pNN103), eachstrain colonized well at about 107 to 108 CFU/g of feces (datanot shown) but none of the mice died. As controls, animalswere orally challenged with E. coli DH5a(pBluescript) orDH5a(pBR329). These vector-containing strains colonizedstreptomycin-treated mouse bowels at a level of about 107 to108 CFU/g of feces, and as expected, the animals remainedhealthy. By contrast, when mice were fed 1010 CFU of E.coli DH5a(pMJ100), which carries the genes for SLT-II on ahigh-expression pBluescript vector, the animals died 4 to 5days postinfection (Table 3). The stx operon was also clonedinto the high-expression pBluescript vector, but we wereunable to obtain viable clones of this type. Taken together,these data indicate that SLT-II produced by an E. coli K-12strain can kill orally challenged mice if toxin is produced athigh levels.

Since Shiga toxin-SLT-I is almost completely cell associ-ated and SLT-II is found primarily in culture supernatants(26), it is conceivable that Shiga toxin is not released atconcentrations high enough in vivo to kill mice. Therefore,Shiga toxin plasmid pNAS10 was transformed into E. coliJE5505, a mutant K-12 strain with a leaky periplasmicphenotype (12). The culture supernatants of E. coliJE5505(pNAS10) were 10- to 100-fold more cytotoxic invitro than those of E. coli DH5a(pNAS10) (Table 3), whichsuggests that Shiga toxin was released in greater quantities inthe E. coli JE5505 background than in the E. coli DH5abackground. However, when mice were fed E. coliJE5505(pNAS10), the animals remained healthy. As a con-trol, E. coli JE5505 was transformed with pMJ100, whichcarries the SLT-II operon, and then this strain was fed tomice. As expected, all of the mice died 4 to 5 days postfeed-ing (Table 3).

TABLE 3. Effects of SLT-producing E. coli onstreptomycin-treated mice

CytotoxicitybHost Plasmida Toxin genes Cell Culture Deathcstrain" associ- super-

ated natant

DH5a pNN103 slt-IIA, slt-IIB 104 103 NoDH5a pMJ100 slt-IIA, slt-IIB 106 105 YesDH5a pNAS10 stxA, stxB 107 106 NoDH5a pBluescript None <102 <102 NoDH5a pBR329 None <102 <10l NoJE5505 pNAS10 stxA, stxB 107 107 NoJE5505 pMJ100 slt-IIA, slt-IIB 106 106 YesH1618 pMJ100 slt-IIA, slt-IIB 106 105 YesH1618 pNAS10 stxA, stxB 107 106 No

a As described in Table 1.b Cell associated: log1o 50% cytotoxic doses per milliliter of sonically

disrupted E. coli transformed with the plasmid construct (total volume, 2 ml).Culture supernatant: 50% cytotoxic doses per milliliter (total volume, 50 ml).All cultures grew to approximately the same A6w.

c Colonization studies were done with three mice on two or more separateoccasions. The results of separate experiments for each infecting strain wereidentical. Yes indicates that all of the infected animals died, and no indicatesthat none of the infected animals died.

Shiga toxin-SLT-I production is iron regulated by a Furprotein-iron corepressor complex at the transcriptionallevel. To address the possibility that Shiga toxin productionis repressed in vivo by iron, pNAS10 was transformed intoE. coli H1618, a K-12 mutant strain with a Fur null pheno-type (11). Cytotoxicity assays confirmed that Shiga toxinwas produced constitutively in vitro by E. coli H1618(pNAS10) irrespective of iron levels in the growth medium(107 50% cytotoxic doses per ml). Nonetheless, mice fed E.coli H1618(pNAS10) were not killed. As a control, pMJ100carrying the SLT-II operon was introduced into E. coliH1618 and then fed to mice. As expected, the Fur nullphenotype had no effect on SLT-II production in vitro andthese strains killed orally infected mice in 4 to 5 days (Table3). These results suggest that iron repression of SLT-Iproduction, as demonstrated in vitro, does not account forthe avirulence of SLT-I-producing strains in this animalmodel.

Binding of SLT-II to glycolipids present in mouse kidneys. IfSLT-I1 is indeed responsible for renal lesions in CD-1 mice,then the kidneys of these animals should contain Gb3, theglycolipid receptor for SLT-II (21, 22, 45). To test for thepresence of toxin-binding glycolipids in CD-1 mouse kid-neys, lipid extracts of the renal cortex and medulla wereprepared and separated by thin-layer chromatography. Thechromatograms were then overlaid with crude SLT-I1, andthe glycolipid-toxin complexes were identified by immuno-staining (Fig. 1). SLT-II bound to Galal-4Gal containingglycolipids, Gb3 and Gb4, present in the neutral fraction ofboth the renal cortex and renal medulla of the mouse kidney(Fig. 1B). These findings indicate that streptomycin-treatedmouse kidneys contain glycolipid receptors specific forSLTs.

Histological examination of kidneys from infected mice. In aprevious study, light microscopic examination of kidneysections from mice infected with strain 933cu-rev revealedsevere, widespread bilateral acute renal cortical tubularnecrosis (46). However, the glomeruli of mice colonized withstrain 933cu-rev appeared normal by light microscopy (46).In this study, kidneys from mice colonized with E. coli K-12strains carrying Shiga toxin or SLT-II genes were examined

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A

CMH -

CDH -

Gb3Gb4-

FORSSMAN -

GM3-UM 2

GM -

FIG. 1. Binding of SLT-II to glycolipids separated on thin-layerchromatograms. (A) Glycolipids visualized with orcinol reagent.Lanes: 1, 1 p.g each of CMH, CDH, Gb3(CTH), Gb4(GL4), Forss-man antigen, GM3, GM2, and GM1; 2, neutral glycolipids from 10 mg(wet weight) of CD-1 mouse renal cortex; 3, neutral glycolipids from10 mg (wet weight) of CD-1 mouse renal medulla. (B) Glycolipids towhich SLT-II bound as visualized by immunostaining with MAb toSLT-II, 11E10, and 1251I-labeled goat anti-mouse immunoglobulin G,followed by autoradiography. Lanes 1 to 3 were the same as those inpanel A.

by light microscopy for histological damage. Only micecolonized with E. coli DH5ot(pMJ100) and H1618(pMJ100)showed bilateral renal cortical tubular necrosis (data notshown). The glomeruli of these mice appeared normal bylight microscopy. Kidneys from mice colonized with theremaining SLT-producing E. coli K-12 strains appearednormal (see reference 46).The tubules and glomeruli of kidneys from mice passively

protected with MAb to SLT-II or SLT-I and SLT-II beforefeeding with E. coli 0157:H7 strain 933cu-rev appearednormal by light microscopy (data not shown), while thekidneys from mice passively protected with MAb to SLT-Ior cholera toxin before feeding with strain 933cu-rev showedcortical tubular necrosis but no glomerular damage (data notshown). These observations indicate that SLT-II was re-

sponsible for the bilateral renal cortical tubular necrosis seen

in mice orally infected with strain 933cu-rev.Ultrastructural examination of kidneys from mice colonized

with E. coli 0157:H7 isolates. The characteristic renal histo-pathologic changes in humans with EHEC-mediated HUSinclude thrombotic microangiopathy in the glomeruli, throm-botic microangiopathy with arterial involvement, and/orcortical tubular necrosis (35). In our mouse model, there wasno light microscopic evidence of glomerular damage, butacute cortical necrosis was demonstrated (46). Ultrastruc-tural examination of the glomeruli and tubules of the kidneysof mice infected with strains 933, 933cu, and 933cu-rev was

done to determine the extent of ultrastructural damage, ifany, in these mice. None of the kidneys, including thosefrom mice fed 933cu-rev, exhibited glomerular damage (Fig.2). Moreover, the proximal convoluted tubules of micecolonized with strain 933 or 933cu also appeared normal(Fig. 3). However, proximal convoluted tubules from micecolonized with strain 933cu-rev showed severe bilateralacute renal cortical tubular necrosis with preservation oftubular basement membrane (Fig. 4). The tubular epithelial

FIG. 2. (A) Transmission electron photomicrograph of a glomer-ulus from a streptomycin-treated mouse colonized with strain 933.The glomerulus was normal. The glomeruli from mice colonizedwith strain 933cu also appeared normal. The arrow indicates a

normal endothelial cell. Magnification, x 1,700. (B) Transmissionelectron photomicrograph of a glomerulus from a streptomycin-treated mouse infected with strain 933cu-rev. No glomerularchanges were seen compared with glomeruli from mice infected withstrain 933 or 933cu. The glomeruli were normal. The arrows indicatenormal endothelial cells. Magnification, xl1360.

cells were degenerate, with loss of microvilli and mitochon-dria; tubular basement membranes were intact; and numer-

ous necrotic renal epithelial cells were present in the lumensof the damaged tubules (Fig. 4). Therefore, the ultrastruc-tural results support our previous conclusions that CD-1

1 2 3B

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riAt b ''

FIG. 3. Transmission electron photomicrograph of normal prox-imal convoluted tubules from a streptomycin-treated mouse colo-nized with E. coli 0157:H7 strain 933. Note the tall, cuboidepithelial cells, the long microvilli and thick brush border, and thenumerous mitochondria. Proximal convoluted tubules from micecolonized with strain 933cu were also normal. Magnification,x 1,088.

mice die of acute tubular necrosis in the absence of glomer-ular damage.

DISCUSSIONThe pathogenesis of the HUS that sometimes follows

infection with EHEC is not understood. One proposedmechanism for the generation of damage to glomerularendothelial cells of the kidneys is that the SLT(s) producedby EHEC strains are hematogenously disseminated from thegut and are targeted to the kidneys (17, 35). Since thishypothesis can never be tested in volunteers because of thepotential life-threatening nature of HUS, we recently orallyinfected streptomycin-treated mice with an EHEC strain asa first step in the development of a small-animal model thatpotentially could be used to study HUS. In reporting thatstudy, we stated that streptomycin-treated mice fed E. coli0157:H7 strain 933cu-rev, which produces both SLT-I andSLT-II, were killed within 10 days of infection and that deathwas due to acute bilateral cortical tubular necrosis (46).Although the damage to the kidneys was tubular, not glo-merular, and hence, not characteristic of the HUS seen inmost patients (35), we were interested in whether the SLTswere responsible for the renal damage. Therefore, in thisstudy, we examined the role, if any, of SLT-I and/or SLT-IIin renal tubular damage in streptomycin-treated mice fedSLT-producing E. coli. The results of several types ofexperiments strongly suggested that SLT-II, but not SLT-I,was responsible for the death of the mice from acute corticaltubular necrosis. Furthermore, the level of SLT-II producedby an E. coli strain appeared to be critical to the virulence ofa strain as demonstrated by the finding that E. coli DH5ot(pNN103), which produced 100-fold less SLT-II in vitro thandid E. coli DH5ot(pMJ100), did not kill mice.

FIG. 4. Transmission electron photomicrograph of a damagedproximal convoluted tubule from a streptomycin-treated mouseinfected with E. coli 0157:H7 strain 933cu-rev. Note the dilatedlumen and sloughed necrotic intraluminal epithelial cell. The re-maining epithelial cells were degenerated. Note the loss of micro-villi, loss of mitochondria, and squamous (flattened) appearance ofthe epithelial cells. Note the intact tubular basement membrane.Magnification, x 1,700.

The finding that SLT-I did not contribute to the virulenceof orally administered E. coli 933cu-rev, coupled with theavirulence of E. coli DH5ot(pNAS10), which expresses highlevels of Shiga toxin-SLT-I in vitro, was surprising becauseboth crude and purified Shiga toxin can kill mice wheninoculated parenterally (28). One possible explanation forthe failure of the Shiga toxin-SLT-I-producing E. coli to killorally infected mice when high-level SLT-II-producing E.coli was able to do so is that expression of Shiga toxin-SLT-Iis repressed by iron whereas expression of SLT-II is not (47,49). To test this possibility, the Shiga toxin genes wereintroduced into E. coli H1618, a K-12 mutant strain whichhas a Fur null phenotype. When mice were fed E. coliH1618(pNAS10), they remained healthy, which suggestedthat iron regulation was not responsible for the avirulence ofShiga toxin-producing E. coli. A second possible explanationfor the inability of Shiga toxin-producing E. coli DH5a to killmice was that release of Shiga toxin by E. coli DH5at wasminimal compared with release of SLT-II by E. coli DH5a.Shiga toxin-SLT-I is primarily cell associated, whereasSLT-II is found predominantly in culture supernatants. Toexamine the possibility that Shiga toxin is not released invivo in quantities large enough to kill mice, the Shiga toxinplasmid was transformed into E. coli JE5505, a mutant K-12strain with a leaky periplasmic phenotype. Mice fed E. coliJE5505(pNAS10) were not killed, which suggested that thecell-associated nature of Shiga toxin did not contribute to theavirulence of E. coli DH5o(dpNAS10).

It remains unclear why SLT-II-producing E. coli, but notShiga toxin-producing E. coli, is virulent in the streptomy-cin-treated mouse model. It should be noted that renalfailure and death in our mouse model were not restricted to

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3964 WADOLKOWSKI ET AL.

oral infection with strain 933cu-rev. Recently, we found thatmice fed certain human EHEC strains that contain SLT-II orSLT-II-related genes died after challenge and the death ofthese mice was due to acute renal cortical tubular necrosis.In contrast, mice fed human EHEC isolates that contain onlythe genes for SLT-I remained healthy (E. A. Wadolkowski,S. W. Lindgren, and A. D. O'Brien, unpublished data).These data correlate well with epidemiological studies whichindicate that E. coli 0157:H7 strains with only SLT-II genesare more likely to cause diarrheal disease that laterprogresses to HUS and thrombotic-thrombocytopenic pur-pura than are organisms containing genes for SLT-I or bothSLT-I and SLT-II (32). Perhaps isolates with only SLT-IIgenes produce greater quantities of SLT-II in vivo than doisolates with both toxin genes. On the other hand, theimmunological response of the host to the infecting organismmay be important. It has been reported that commerciallyavailable pooled human sera contain antibody to SLT-I butnot antibody to SLT-II (1). We know, however, that pooledsera from normal adult CD-1 mice do not contain antibody toeither Shiga toxin or SLT-II (data not shown). Lastly,SLT-II production may be linked to, or be a marker for, anundetermined EHEC virulence factor. This possibility ap-pears to be unlikely, since E. coli DH5a containing the genesfor SLT-II with little flanking EHEC DNA caused death inorally infected mice.

It has been reported that rabbits continuously infused withSLT-II by miniosmotic pumps developed diarrhea and cecallesions on day 3 of infusion (2). The gut lesions resembledthose seen in humans with hemorrhagic colitis (36) and infantrabbits orally fed SLT-producing E. coli 0157:H7 (33). Thissimilarity suggests that SLT-II is responsible for the histo-pathological changes seen in hemorrhagic colitis associatedwith SLT-II-producing E. coli. The rabbits failed to developclear HUS-like lesions in the glomeruli of the kidneys (2),perhaps because of absence of the functional SLT receptor,Gb3, in the kidneys (4).Death of streptomycin-treated mice fed either strain

933cu-rev or strains expressing SLT-II was due to severe,widespread, acute necrosis of proximal convoluted tubuleswith preservation of tubular basement membrane (46). Thislesion is characteristic of toxic insult to renal tissue ratherthan ischemia due to dehydration (37). Passive-protectionstudies strongly indicate that the tubular necrosis of thekidneys of mice infected with strain 933cu-rev was due toSLT-II. Although there was no light microscopic evidence ofthrombotic microangiopathy in the glomeruli of the kidneysof the mice colonized with strain 933cu-rev, we were able todemonstrate toxic acute cortical necrosis (46). Ultrastruc-tural examination confirmed that the glomeruli of the kid-neys of 933cu-rev-infected mice were normal but acutecortical tubular damage was seen. The fact that the miceshowed no glomerular damage may be due to the absence ofa functional receptor for SLT in that compartment of CD-1mouse kidneys.

ACKNOWLEDGMENTS

This work was funded by a Public Health Service grant from theNational Institutes of Health (AI 20148-07).We thank Suzanne F. Pletcher, Diagnostic Services, Uniformed

Services University of the Health Sciences, for excellent technicalassistance with the transmission electron microscopy.

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