INFECTION AND IMMUNITY, Oct. 1979, p. 362-368 Vol. 26, No. 10019-9567/79/10-0362/07$02.00/0

Attachment Pili from Enterotoxigenic Escherichia coliPathogenic for Humans

CARL F. DENEKE,* GRACE M. THORNE, AND SHERWOOD L. GORBACHInfectious Diseases Service, Department ofMedicine, Tufts-New England Medical Center, Boston,

Massachusetts 02111

Received for publication 12 July 1979

Pili from enterotoxigenic Escherichia coli pathogenic for humans have beenisolated by adsorption to the surface of erythrocytes followed by thermal elution.The pili are composed of two protein subunits with molecular weights of 13,100and 12,500 as determined by sodium dodecyl sulfate-gel electrophoresis. Thesepili also bind to human buccal cells under temperature conditions (370C) whichprevent the binding of these pili to the erythrocytes. Analogous temperatureeffects on binding have previously been observed with whole bacterial cells. Thisbinding can be inhibited by antiserum prepared against the isolated pili.

The adhesion of enterotoxigenic Escherichiacoli (ETEC) to the mucosal surface of the smallbowel and their subsequent resistance to clear-ance mechanisms such as peristalsis are impor-tant steps in the pathophysiology of diarrhealdisease. This adhesion is an initial step in the"colonization" of the small bowel (8-10, 12, 14,19, 25). We would reserve the term colonizationfor demonstration of mucosal attachment, aswell as growth and multiplication of bacteriawithin the small bowel. Because colonization isa complex event, we have chosen to separatethese components, studying each individuallyrather than approaching the entire process witha complex model.

Previous studies have established that K88and K99 pili are responsible for the host-specificattachment of ETEC to the small bowel mucosaof pigs and calves, respectively (1, 16, 18, 20, 26,28, 33, 34). These pili are heat-labile proteinsurface antigens which project out from the bac-terial cell like "fur" (35). Bacteria with thesesurface pili also hemagglutinate (HA) erythro-cytes (RBC) in the presence of the sugar man-nose (5, 9, 21, 27, 29). This reaction may betermed mannose-resistant hemagglutination(MR-HA). In contrast, the HA reaction me-diated by another kind of pili, termed type 1 pili,is inhibited by mannose (6, 30). Type 1 pili arepresent on a wide variety of pathogenic andnonpathogenic E. coli strains (2-4) and thereforeare not thought to be correlated with the abilityto cause human diarrheal disease. A furtherdistinction is that the MR-HA reaction occursat 0°C and is reversed at elevated temperatures,whereas the type 1 pili-mediated HA is nottemperature sensitive.

The K88 and K99 pili are also morphologicallyand chemically different from type 1 pili. K88pili have a reported diameter of 7 to 8.4 nm (35).K99 pili (17) have a diameter of 8.4 nm and arecomposed of protein subunits with molecularweights of 22,500 (major) and 29,500 (minor). Incontrast, the type 1 pili or common fimbriae (3,4, 6, 31, 32) have been reported to have a diam-eter of 7 to 13 nm and are composed of a subunitof 17,500 molecular weight (31, 32). Bacteria canalso possess sex pili (37) which function in con-jugation. The F and I-like sex pili have a diam-eter in the range of 4.5 to 10 nm, with the F pilihaving a subunit molecular weight of 11,800 (3,4, 24).Evans and co-workers have recently reported

the isolation of pili morphologically similar toK88 and K99 on ETEC isolated from humancases of diarrhea (10). These pili, which theyhave designated as the CFA/I antigen, havebeen associated with the attachment of E. colito the intestine of infant rabbits and have beenimplicated in the production of diarrheal diseasein volunteers (9-12). CFA/I pili, have been im-plicated in MR-HA of human A and bovineRBC. These investigators have also reported asecond antigen (CFA/II) which is only associ-ated with 06 and 08 serotypes and is involvedin MR-HA of only bovine RBC (7).We have shown that binding to human buccal

mucosal cells of ETEC strains isolated fromhumans with diarrheal disease correlates withtoxigenicity of human strains (36; Deneke,Thorne, and Gorbach, manuscript in prepara-tion). We report here the isolation of pili fromtoxigenic E. coli isolated from human beingswith diarrhea. These pili bind both to RBC and

362

on May 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

VOL. 26, 1979

to human buccal mucosal cells. Such surfacestructures may play a role in adherence ofETECto the small bowel of humans.

MATERIALS AND METHODSBacterial strains. E. coli strains used in this study

were isolated from a variety of human sources includ-ing infants, travelers, and indigenous populations. Thesources of these strains are detailed in a previouspaper (36). The prototype strain for this work was E.coli 334 (015:H1l), originally isolated by one of us(S.L.G.) from a case of acute diarrhea in Calcutta,India. Nontoxigenic control strains included thosefrom a healthy person (CD-1), a well-described strain(H10405), and a plasmid-free derivative of 334, termed334LL. The individual strains are detailed in Table 1.

Isolation of pili. Bacteria were grown overnight at370C on peptone salt agar, composed of 2% peptone(Difco Laboratories, Detroit, Mich.), 0.5% NaCl, and1.5% agar. The cells were harvested in phosphate-buffered saline, pH 7.2 (PBS). The bacterial cells werewashed once and resuspended in PBS containing 0.5%mannose (mPBS). Pili were removed from the washedbacteria by blending for 3 min with short (about 30-s)bursts; the blender was cooled in ice to mnimizethermal denaturation. Intact bacteria and large debriswere removed by centrifugation at 27,000 x g for 10min, leaving the pili in the supernatant. This mPBSsupernatant, containing pili, was mixed with freshlydrawn, washed guinea pig RBC and cooled on ice toallow attachment of the pili to the RBC. In the pres-ence of mannose, type 1 pili could be removed withmPBS in the cold by low-speed centrifugation. Onlythose bacterial components which bound to RBC inthe presence of mannose and sedimented at low speedwere retained.Attachment pili were eluted from RBC under con-

ditions which reversed the HA reaction, i.e., resuspen-sion in PBS and incubation for 15 min at 37°C. TheRBC were removed by centrifugation at 500 x g for 10mini with a Dynascan clinical centrifuge at room tem-perature. This washing-elution procedure was re-peated with temperatures of 37, 42, and 50°C. Finally,RBC membrane fragments and debris were removedby ultracentrifugation at 45,000 rpm for 2 h, leavingpartially purified pili in the supernatant.

Electron microscopy. Bacteria were prepared fornegative staining by growing them for either 6 h orovernight. Bacteria were resuspended in saline andapplied for 2 min to carbon-coated grids (E. F. Fullam,Schenectady, N.Y.) which had been pretreated for 1min with a 0.1% solution of bovine serum albumin.The grids were washed with deionized filtered waterbefore staining with 2% (wt/vol) phosphotungstic acid.Pili preparations were similarly treated with a 3-minabsorption time and either 2 or 4% uranyl acetatestain. The preparations were examined in a JelcoJEM-100B electron microscope.SDS-PAGE. Molecular weight determinations of

the pili subunits were determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by the method of Laemmli (23) with thefollowing modifications. For the separation gel, theacrylamaide concentration was increased to 16.8% with

ATTACHMENT PILI FROM E. COLI 363

0.45% bisacrylamide; for the stacking gel, the acryl-amide concentration was 5.8% with 0.15% bisacrylam-ide. The separating gel was allowed to polymerizeovernight. The molecular weight standards were my-oglobin (molecular weight 17,200), lactalbumin (mo-lecular weight 18,400), and lysozyme (molecularweight 14,300), all obtained from Sigma Chemical Co.(St. Louis, Mo.).Samples were dissolved in 2% SDS, 5% mercapto-

ethanol, and 10% glycerol (pH 8.8) and were heated ina steam bath for 20 min to ensure complete denatur-ation. The samples were electrophoresed for 4 to 6 hwith a current of 50 mA. After electrophoresis, gelswere stained overnight in a solution of 225 ml ofethanol, 45 ml of glacial acetic acid, and 225 ml ofwater containing 0.5 g of Coomassie brilliant blue R(Sigma). The destaining solution was ethanol-aceticacid-water (2:3:35). Staining and destaining were donein a shaking water bath at 50°C.Buccal cell binding by pili. Buccal cells were

collected from 10 to 15 volunteers by scraping thebuccal epithelium with wooden tongue depressors.The buccal cells were then washed three times withmPBS. Isolated pili preparations were mixed withwashed buccal cells, incubated for 5 min, and washedfour times with mPBS with a Beckman microfuge.Each wash supernatant and final precipitate weredissolved in an equal volume of the SDS solubilizationreagent, heated for 20 min in a steam bath, and ana-lyzed by SDS-PAGE.

Antisera. Antisera were prepared in rabbits withpurified pili preparations. By the method of Stirm etal. (35), pili were injected into the marginal ear vein ofadult New Zealand white rabbits twice weekly for 3weeks. After a 1-week resting period, the animals werebled. Immunoglobulin G was isolated from the wholeserum by sequential sodium sulfate precipitation, firstwith 18% and then 14% (both wt/vol). The precipitatewas dissolved in 15 mM phosphate buffer (pH 8.0) andwas passed through diethylaminoethyl-Sephadex.Monovalent Fab fragment was prepared by papaincleavage. The remaining immunoglobulin G was re-moved from the Fab by gel chromatography with Seph-adex G-100.

RESULTS

Electron microscopy. Short, needle-like pili(5 to 10 by 300 nm) were present on the surfaceof a number of different ETEC strains (Fig. 1Ato F). These include 334, 193-4, TX-1, D481, andD563. An electron micrograph of the attachmentpili ofETEC strain 193-4 isolated by the methoddescribed above is shown in Fig. 2. Pili fromstrain 334, obtained in the same manner, areshown in Fig. 3. The pili preparations appearedto be homogeneous, without the presence of thelarger structures which were visible on the intactcells. The isolated pili had the same diameter asthe small pili on the intact bacterial cells, sup-porting the impression that the smaller needle-like structures are the attachment pili. It is these

on May 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

364 DENEKE, THORNE, AND GORBACH

B

C

E

D

F

FIG. 1. Five strains ofETEC and one non-enterotoxigenic strain negatively stained with 2%phosphotung-stic acid. (A) 334; (B) 193-4; (C) D563; (D) D481; (E) TX-I; (F) 334LL (negative control). Attachment pili aresmall, needle-like structures (4 to 6 nm). Bar = 1.0 ,nm.

INFECT. IMMUN.

on May 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

ATTACHMENT PILI FROM E. COLI 365

.el .- q

a'twso'~'*b..y

FIG. 2. Pili preparation ofETEC strain 193-4 stained with 4% uranyl acetate. Bar = 1.0 ,um.

purified attachment pili which we have furthercharacterized.The purified pili of E. coli 334 were found to

possess two polypeptide chains by SDS-PAGE(Fig. 4A). These doublet bands were present ineach of the supernatant fractions of the purifi-cation procedures and in each of the cold mPBSwashes. After the first supernatant, which con-tained material not bound to RBC, there wereno protein bands other than the pili doublet. Pili-with identical molecular weights were also foundon the other E. coli strains (Table 1).The presence of pili polypeptides throughout

the cold mPBS washes suggests that the piliexisted in an equilibrium between unattachedand free states, even in the cold. This equilib-rium was probably not seen in the macroscopicscale ofHA because we were observing the over-all reaction of a large number of pili on eachbacterium and multiple attachment sites on eachRBC. When the incubation temperature wasraised, this equilibrium was apparently shiftedtoward the unbound state. Thus, both reversalof the HA reaction and desorption of isolatedpili were observed when the temperature wasraised to 370C.

Pili prepared with the above isolation andpurification procedure bind to isolated humanbuccal cells (Fig. 4). Buccal cells without bacte-rial products did not demonstrate a band at thecharacteristic position of the pili doublet in theSDS-PAGE system (Fig. 4B). Moreover, no pilibands appeared in subsequent washes; this sug-gests that those pili which were bound existedin a relatively tight association and were notremoved by washing, in contrast to the RBCbinding reaction.

Finally, when the buccal cells were dissolvedin SDS, pili were still present (Fig. 4H). Thus,although not all of the pili isolated by our RBCadsorption method were reactive, at least someof them attached rather tightly to human buccalcells. This implies that some of the pili involvedin HA and buccal cell binding are identical andthat the two subunit polypeptide chains bind toboth types of eucaryotic cells.The effect of the Fab fragment of anti-pili

immunoglobulin G antibody on buccal bindingby whole bacterial cells is shown in Table 2. Theaddition of the Fab fragment inhibits the bindingof intact organisms to human buccal cells. Themonovalent Fab fragment was used because in-

VOL. 26, 1979

on May 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

366 DENEKE, THORNE, AND GORBACH

t'34

FIG. 3. Pili preparation ofETEC strain 334 negatively stained with 2%o uranyl acetate. Bar = 1.0 ,um.

A B C D E F G H

FIG. 4. Isolated pili attach to buccal cells. Pili,isolated by attachment to RBC, also attach to buccalcells as the characteristic pili bands are present inthe buccal cell preparation after washing. SDS-PAGE. (A) Purifiedpili alone; (B) buccal cells alone;(C) initial mixture of buccal cells and pili; (D) super-natant from first wash; (E) supernatant from secondwash; (F) supernatant from third wash; (G) super-natant from fourth wash; (H) final buccal cellprecip-itate, arrow points to pili bands.

tact anti-pili immunoglobulin G agglutinates thewhole bacteria, interfering with the filter assay.We have further shown that the isolated pili areprecipitated by this antibody.

DISCUSSIONThe pili of ETEC which appear to be respon-

sible for the attachment to human mucosal cellsare biochemically different from those associ-ated with attachment in animals, i.e., K88 andK99 pili. The pili reported here had a diameterof 5 to 10 nm compared with a diameter of 8 to13 nm for K88 and 7 to 8 nm for K99. Thepolypeptide chain subunits had a lower molec-ular weight, being 12,500 and 13,100 for thehuman attachment pili and 22,500 for K99. Themolecular weights that we report are closer tothat observed for the F sex pilus, which was11,800. Brinton (3) has reported type 2 pili in thesame size range as our preparation, but theywere not chemically characterized.The attachment pili discussed here differ in

HA pattern from CFA/I and CFA/TI. For ex-ample, we were unable to show a direct correla-tion between mannose-resistant HA of humantypes A or B, guinea pig, and bovine RBC witheither the presence of the attachment pili de-scribed here or buccal binding as discussed pre-viously (36). This is unlike the case of CFA/Iand CFA/II which mediate MR-HA human Aand bovine RBC or only bovine RBC, respec-tively.

INFECT. IMMUN.

on May 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

ATTACHMENT PILI FROM E. COLI 367

TABLE 1. Detection of K-like pili on strains ofETEC isolated from humans

MRHAa Pili detected by

Entero Buccal Electron micros-Strains Serotype toxin cell ad- SDS- copy

herence Human puea Bovine PAGEbpig ~~~~5tolO0 13 to 20

nm nmeETEC

334 015:H11 LT/ST + + + + + + +193-4 NTd LT/ST + + + + + + +TX-1 078:K80:H12 ST + + - + + + +D542 NT LT/ST + + - + + + +D481 NT LT/ST + + - + + + +D563 NT LT/ST + + - + + + +

Controlnontoxigenic

334LL 015:H11 - - - - - 9 - -CD-1 NT - - - - - 9 - -

K12,K88 NT - - - + - + + -a Performed at 00C.b Proteins of 12,500 and 13,100 molecular weight detected by SDS-PAGE after RBC attachment and elution

procedure.eThese larger structures (13 to 20 nm) morphologically resemble flagella.d NT, Not typable.

TABLE 2. Inhibition of buccal binding by anti-334pili Faba

Amt of Mean no. ofBacterial Type of an- anti- bacteria perstrain tiserum sera buccalccell

334 100334 Fah 200 60334 Fah 400 10334 Normal rab- 200 110

bit serum(heat inac-tivated)

a Bacteria alone were pretreated for 15 min with either F.bor heat-inactivated normal rabbit serum before the additionof buccal cells.

Although we found that the same pili ap-peared to be binding to RBC and buccal cells,the receptors on these cell types are unlikely tobe the same since pili remain bound to buccalcells at 370C, whereas they are eluted from RBCunder the same conditions. It seems likely thata cross-reacting receptor structure is involved.Our studies show that these pili are found on

ETEC strains isolated from humans, they arephysically and chemically distinct from previ-ously reported pili types, and the same isolatedpili attach to human mucosal cells. Further evi-dence which suggests that these pili are involvedin mucosal cell attachment is that the Fab frag-ment from anti-pili immunoglobulin G blocksthe attachment of whole bacteria to buccal cells.Other workers have also shown inhibition ofbacterial binding by antibodies (13, 15, 22). Be-cause of potential changes in surface conforma-tion, charge, and other steric factors, the inhi-

bition of buccal cell binding by anti-pili Fab doesnot preclude additional attachment mecha-nisms.

Previously (36) we have reported that theability to adhere to human buccal mucosal cellsoccurs to a greater extent and more frequentlyin human ETEC strains than in E. coli strainsisolated from healthy people or from animals.Many, but not all, of the ETEC strains exhibitMR-HA in the cold, but we have not found, ashave Evans et al. (9), a direct correlation be-tween HA of RBC from a particular animalspecies and the presence of these attachmentpili. Attachment of human ETEC strains tobuccal mucosal cells appears to be mediated viathese attachment pili, which are not found oncontrol strains, and it is possible that these pilimight also function in the attachment of ETECto human intestinal mucosa, the initial event inthe pathophysiology of acute diarrheal disease.

ACKNOWLEDGMENTSThis investigation was supported in part by Public Health

Service research grant R22AI13164-02 from the National In-stitute of Allergy and Infectious Diseases, by contract 223-75-2116 from the Food and Drug Administration, and by contractDAMA 17-76-C6007 from the United States Department ofthe Army.We thank Clifford M. Chapman for electron microscopy.

We acknowledge the excellent technical assistance of LindaCabibbo and Elaine Unemori and the typing assistance ofLouise Kelly, Constance Adler, and Ardis Ono.

LITERATURE CITED

1. Arbuckle, J. B. R. 1971. Enteropathogenic Escherichiacoli on the intestinal mucopolysaccharide layer of pigs.J. Pathol. 104:93-98.

2. Brinton, C. C., Jr. 1959. Non-flagellar appendages ofbacteria. Nature (London) 183:782-786.

VOL. 26, 1979

on May 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

368 DENEKE, THORNE, AND GORBACH

3. Brinton, C. C., Jr. 1965. The structure, function, synthe-sis and genetic control of bacterial pili and a molecularmodel for DNA and RNA transport in gram negativebacteria. Trans. N. Y. Acad. Sci. 27:1003-1054.

4. Brinton, C. C., Jr. 1971. The properties of sex pili, theviral nature of "conjugal" genetic transfer systems, andsome possible approaches to the control of bacterialdrug resistance. Crit. Rev. Microbiol. 1:105-160.

5. Burrows, M. R., R. Sellwood, and R. A. Gibbons.1976. Haemagglutinating and adhesive properties asso-ciated with the K99 antigen of bovine strains of Esch-erichia coli. J. Gen. Microbiol. 96:269-275.

6. Duguid, J. P., I. W. Smith, G. Dempster, and P. N.Edmunds. 1955. Nonflagellar filamentous appendages("fimbriae") and haemagglutinating activity in Bacte-rium coli. J. Pathol. Bacteriol. 70:335-348.

7. Evans, D. G., and D. J. Evans, Jr. 1978. New surface-associated heat-labile colonization factor antigen (CFA/II) produced by enterotoxigenic Escherichia coli ofserotypes 06 and 08. Infect. Immun. 21:638-647.

8. Evans, D. G., D. J. Evans, Jr., and H. L. DuPont. 1977.Virulence factors of enterotoxigenic Escherichia coli. J.Infect. Dis. 136:S118-S123.

9. Evans, D. G., D. J. Evans, Jr., and W. Tjoa. 1977.Hemagglutination of human group A erythrocytes byenterotoxigenic Escherichia coli isolated from adultswith diarrhea: correlation with colonization factor. In-fect. Immun. 18:330-337.

10. Evans, D. G., D. J. Evans, Jr., W. S. Tjoa, and H. L.DuPont. 1978. Detection and characterization of colo-nization factor of enterotoxigenic Escherichia coli iso-lated from adults with diarrhea. Infect. Immun. 19:727-736.

11. Evans, D. G., T. K. Satterwhite, D. J. Evans, Jr., andH. L. DuPont. 1978. Differences in serological re-sponses and excretion patterns of volunteers challengedwith enterotoxigenic Escherichia coli with and withoutthe colonization factor antigen. Infect. Immun. 19:883-888.

12. Evans, D. G., R. P. Silver, D. J. Evans, Jr., D. G.Chase, and S. L. Gorbach. 1975. Plasmid-controlledcolonization factor associated with virulence in Esche-richia coli enterotoxigenic for humans. Infect. Immun.12:656-667.

13. Freter, R. 1970. Mechanism of action of intestinal anti-body in experimental cholera. II. Antibody-mediatedantibacterial reaction at the mucosal surface. Infect.Immun. 2:556-562.

14. Freter, R., and G. W. Jones. 1976. Adhesive propertiesof Vibrio cholerae: nature of the interaction with intactmucosal surfaces. Infect. Immun. 14:246-256.

15. Gibbons, R. J. 1974. Bacterial adherence to mucosalsurfaces and its inhibition by secretary antibodies. Adv.Exp. Med. Biol. 45:315-325.

16. Hohmann, A., and M. R. Wilson. 1975. Adherence ofenteropathogenic Escherichia coli to intestinal epithe-lium in vivo. Infect. Immun. 12:866-880.

17. Isaacson, R. E. 1977. K99 surface antigen of Escherichiacoli: purification and partial characterization. Infect.Immun. 15:272-279.

18. Isaacson, R. E., P. C. Fusco, C. C. Brinton, and H. W.Moon. 1978. In vitro adhesion of Escherichia coli toporcine small intestinal epithelial cells: pili as adhesivefactors. Infect. Immun. 21:392-397.

19. Isaacson, R. E., B. Nagy, and H. W. Moon. 1977.Colonization of porcine small intestine by Escherichiacoli: colonization and adhesion factors of pig entero-pathogens that lack K88. J. Infect. Dis. 135:531-539.

20. Jones, G. W., and J. M. Rutter. 1972. Role of the K88antigen in the pathogenesis of neonatal diarrhea caused

by Escherichia coli in piglets. Infect. Immun. 6:918-927.

21. Jones, G. W., and J. M. Rutter. 1974. The associationof K88 antigen with haemagglutinating activity in por-cine strains of Escherichia coli. J. Gen. Microbiol. 84:135-144.

22. Jones, G. W., and J. M. Rutter. 1974. Contribution ofthe K88 antigen of Escherichia coli to enteropatho-genicity; protection against disease by neutralizing theadhesive properties of the K88 antigen. Am. J. Clin.Nutr. 27:1441-1449.

23. Laemmli, U. K. 1970. Cleavage of structural proteinsduring the assembly of the head of bacteriophage T4.Nature (London) 227:680-685.

24. Lawn, A. M. 1966. Morphological features of the piliassociated with Escherichia coli K12 carryingR factorsor the F factor. J. Gen. Microbiol. 45:377-383.

25. Moon, H. W., B. Nagy, and R. E. Isaacson. 1977.Intestinal colonization and adhesion by enterotoxigenicEscherichia coli: ultrastructural observations on adher-ence to ileal epithelium of the pig. J. Infect. Dis. 136:S124-S129.

26. Moon, H. W., B. Nagy, R. E. Isaacson, and L. 0rskov.1977. Occurrence of K99 antigen on Escherichia coliisolated from pigs and colonization of pig ileum by K99'enterotoxigenic E. coli from calves and pigs. Infect.Immun. 15:614-620.

27. Morris, J. A., A. E. Stevens, and W. J. Sojka. 1978.Isoelectric point of cell-free K99 antigen exhibiting he-magglutinating properties. Infect. Immun. 19:1097-1098.

28. Nagy, B., H. W. Moon, and R. E. Isaacson. 1976.Colonization of porcine small intestine by Escherichiacoli: ileal colonization and adhesion by pig enteropath-ogens that lack K88 antigen and by some acapsularmutants. Infect. Immun. 13:1214-1220.

29. 0rskov, I., and F. 0rskov. 1977. Special O:K:H sero-types among enterotoxigenic E. coli strains from diar-rhea in adults and children. Occurrence of the CF(Colonization factor) antigen and of hemagglutinatingabilities. Med. Microbiol. Immunol. 163:99-110.

30. Rivier, D. A., and M. R. Darekar. 1975. Inhibitors ofthe adhesiveness of enteropathogenic E. coli. Experi-mentia 31:662-664.

31. Salit, I. E., and E. C. Gotschlich. 1977. Hemagglutina-tion by purified type 1 Escherichia coli pili. J. Exp.Med. 146:1169-1181.

32. Salit. L. E., and E. C. Gotschlich. 1977. Type 1 Esche-richia coli pili: characterization of binding to monkeykidney cells. J. Exp. Med. 146:1182-1194.

33. Smith, H. W., and M. A. Linggood. 1971. Observationson the pathogenic properties of the K88, mly, and Entplasmids of Escherichia coli with particular referenceto porcine diarrhea. J. Med. Microbiol. 4:467485.

34. Smith, H. W., and M. A. Linggood. 1972. Further ob-servations on Escherichia coli enterotoxins with partic-ular regard to those produced by atypical piglet strainsand by calf and lamb strains: the transmissible natureof these enterotoxins and of a K antigen possessed bycalf and lamb strains. J. Med. Microbiol. 5:243-250.

35. Stirm, S., F. 0rskov, L. 0rskov, and A. Birch-Ander-sen. 1967. Episome-carried surface antigen K88 ofEscherichia coli. III. Morphology. J. Bacteriol. 93:740-748.

36. Thorne, G. M., C. F. Deneke, and S. L. Gorbach. 1979.Hemagglutination and adhesiveness of toxigenic Esch-erichia coli isolated from humans. Infect. Immun. 23:690-699.

37. Tomoeda, M., M. Inuzuka, and T. Date. 1975. Bacterialsex pili. Prog. Biophys. Mol. Biol. 30:23-56.

INFECT. IMMUN.

on May 10, 2020 by guest

http://iai.asm.org/

Dow

nloaded from