INFECTION AND IMMUNITY, Nov. 1980, p. 531-5370019-9567/80/11-0531/07$02.00/0

Vol. 30, No. 2

Effect of Carbohydrates on Adherence of Escherichia coli toHuman Urinary Tract Epithelial Cells

ANTHONY J. SCHAEFFER,* SUSAN K. AMUNDSEN, AND JOANNE M. JONESDepartment of Urology, Northwestern University Medical School, Chicago, Illinois 60611

Adherence of Escherichia coli cells to voided uroepithelial cells from healthywomen was measured by use of [3H]uridine-labeled bacteria filtered through apolycarbonate membrane filter (5-rm pore size). At a concentration of 2.5% (wt/vol), D-mannose, D-manmtol, a-methyl-D-mannoside, and yeast mannan com-pletely inhibited adherence of the bacteria to the epithelial cells. At this sameconcentration, D-fructose, D-lyxose, D-arabinose, and D-glyceraldehyde partiallyinhibited adherence. Reducing the concentration of D-mannose, or its derivatives,to between 1.0 and 0.1% resulted in partial inhibition in the adherence of thebacteria; a further reduction in the concentration to between 0.01 and 0.001%caused an enhancement of adherence up to 160% of the control level. Bacterialpreincubation in 2.5% D-mannose for 1 min before epithelial cells were addedcompletely inhibited adherence; similar treatment of the epithelial cells had nosignificant effect on subsequent adherence of the bacteria. Bacteria that werepreincubated for 1 h with D-mannose at concentrations between 0.1 and 0.75%showed enhanced adherence. The inhibitory effect of D-mannose was decreasedif bacterial adhesive ability, or cell receptivity, increased. A variety of othercarbohydrates tested had no effect on the adherence of E. coli to the uroepithelialcells. These results suggest that adherence can be altered by interactions)between specific carbohydrate molecules and receptors on the bacterial surface.

Attachment of bacteria to mucosal surfaces ofa susceptible host is now recognized as an im-portant step in colonization and infection (11,21, 25). The ability of bacteria to adhere toepithelial cells in vitro appears to be mediatedby the surface components of both cell types. Toprobe the biochemical nature of the structuresinvolved, investigators have focused on the sen-sitivity of bacterial agglutinins to carbohydrateswhich compete with identical or related struc-tures on the cell surface (5, 6, 17). Studies show-ing that D-mannose or its derivatives specificallyinhibit adherence of Escherichia coli to epithe-lial cells support the hypothesis that E. coliligands can recognize and bind D-mannose or D-mannose-like receptors on the epithelial cellsurface (8, 13, 16, 20). Recent identification of D-mannose-insensitive strains of E. coli suggeststhat other bacterial ligands may also mediateattachment to epithelial cells (4, 9).

Investigations of E. coli strains isolated fromurine suggest that several adherence factors areinstrumental in the attachment of bacteria tohuman uroepithelial cells. Svanborg Eden andHansson (26) demonstrated D-mannose- and a-methyl-D-mannose-sensitive E. coli agglutina-tion to guinea pig erythrocytes, but D-mannose-insensitive adherence to uroepithelial cells. In

subsequent studies which assessed adherencewith radioisotopic rather than visual techniques,the addition of 2.5% D-mannose to E. coli-uro-epithelial cell suspensions completely inhibitedadherence, even when the epithelial cells werehighly receptive (22). The purpose of this studywas to further define D-mannose-sensitive ad-herence by investigating the effect of variouscarbohydrates on the adherence of E. coli tohuman uroepithelial cells. The results supportthe concept that adherence can be altered byspecific carbohydrate molecules which competewith epithelial cell receptors for bacterial bind-ing sites. This complex interaction is influencedby the ability ofbacteria to adhere, the epithelialcell receptivity, and the concentration and timeof exposure to the carbohydrates.

MATERIALS AND METHODSBacteria. The E. coli strain used in all experiments

was isolated from infected urine. It was serotyped(018) by the method of Brent and Vosti (3). Thebacteria were grown in brain heart infusion broth(Difco Laboratories) for 72 h, harvested by centrifu-gation, and washed once in phosphate-buffered saline(Difco; pH 7.2). Variation between assays was mini-mized by suspending the bacteria in brain heart infu-sion broth plus dimethyl sulfoxide (Fisher ScientificCo.) at a final concentration of 5% (vol/vol) and freez-

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532 SCHAEFFER, AMUNDSEN, AND JONES

ing them immediately in an acetone-dry ice bath.Frozen bacteria were stored in small portions at -20'Cin screw-capped tubes. For use in the assay, a portionof bacteria was thawed at 37"C, washed once, sus-

pended in yeast nitrogen base (Difco) buffered to pH7 with phosphate buffer (Na2HPO4, 8 mM; KH2PO4, 2mM), and labeled with [3H]uridine as previously de-scribed (22).

Epithelial cells. Uroepithelial cells were obtainedfrom freshly voided midstream specimens provided bypremenopausal women who had no history of urinaryor vaginal infections and were not taking contraceptiveor antimicrobial agents. The epithelial cells were har-vested by centrifugation, washed once in phosphate-buffered saline, and suspended in minimal essentialmedium containing Earle salts (MEM; InternationalScientific Industries) at pH 5 to a concentration ofapproximately 2 x 105 cells per ml by use of a hema-cytometer. Cells with similar receptivity for use indifferent experiments were obtained by pooling epi-thelial cell suspensions and dispensing 1-ml portionsinto screw-cap tubes. Dimethyl sulfoxide (final con-centration, 5%, vol/vol) was gradually added prior toquick freezing in an acetone-dry ice bath and storageat -20'C. For each experiment, cells were thawed at370C and diluted in MEM to a concentration of 105cells per ml. Cell viability was determined by thetrypan blue exclusion technique, and total and viablecell counts were compared with the counts prior tofreezing.

Bacterial adherence assay. The adherence assaywas done as previously described (22). Samples (0.2mi) containing 107 bacteria and 104 epithelial cellswere combined, and control tubes containing bacteriaplus MEM or epithelial cells and MEM were alsoprepared. After incubation at 370C for 30 min, 0.2 mlof each sample was filtered under vacuum through a

polycarbonate membrane filter (5-owm pore size, Nucle-pore Corp.). The number of adhering bacteria wascalculated by substracting the number of bacteriaattaching non-specifically to the filter in the absenceof epithelial cells from the number of bacteria presenton the filter in the assay. Adherence was calculated bydividing the number of bacteria adhering by the num-ber of epithelial cells in the assay.

Inhibition experiments. A variety of carbohy-drates (Sigma Chemical Co.) and concanavalin A(ConA, Sigma) were tested as adherence blockingagents. D-mannose, a-methyl-D-mannoside, D-fruc-tose, D-mannitol, D-glyceraldehyde, L-glyceraldehyde,D-arabinose, D-lyxose, D-ribose, L-fucose, L-mannose,L-rhamnose, dihydroxyacetone, D-glucose, a-methyl-D-glucoside, D-galactose, D-xylose, yeast mannan, andConA were added to E. coli suspensions in MEM toachieve a final concentration of 2.5% (wt/vol) whencombined with epithelial cells. Carbohydrate-treatedbacterial suspensions were compared with nontreatedcontrols. Those carbohydrates demonstrating inhibi-tion were diluted in MEM (final concentrations usedwere between 2.5 and 0.0001%) to determine howvarious concentrations affected adherence.

Statistical analysis. The x2 test was used for allstatistical comparisons, and P values were determinedby reference to a standard table of critical values ofthe x2 distribution.

RESULTSCeli storage technique. The effect of the

storage technique on adherence and viabilitywas investigated by performing the assay withfresh cells and subsequently with samples fromthe same cell pools after the addition ofdimethylsulfoxide freezing. Adherence and viability werenot affected when cells were stored in dimethylsulfoxide at -20'C for up to 3 months. Cellsstored at 40C or in glycerol showed decreasedadherence and viability.Inhibition of adherence by carbohy-

drates and lectins. To determine the mosteffective carbohydrate and lectin inhibitor ofbacterial binding to uroepithelial cells, we madesuspensions of E. coli in MEM with twice thefinal carbohydrate concentration. After 1 min,the suspensions were diluted in half by the ad-dition of the epithelial cells. Bacterial adherencewas compared with that in nontreated controls.None of the compounds used were toxic to theepithelial cells, as determined by trypan blueexclusion, or to the bacteria, as determined bygrowth curves. Maximal inhibition of bacterialadherence occurred with yeast mannan and thesix-carbon carbohydrates D-mannose and D-mannitol (Table 1). A threefold increase in themolar concentration of a-methyl-D-mannosideor D-fructose was required to bring about thesame inhibitory effect. Five-carbon (D-arabi-nose, D-lyxose) and three-carbon (L-glyceralde-hyde) carbohydrates were less effective inhibi-tors of bacterial adherence and required a 6-foldand 13-fold increase, respectively, in their con-centrations to produce the 50% inhibitionachieved with D-mannose or D-mannitol.Adherence was inhibited completely by all

carbohydrates with the hydroxyl group at car-bon 2 in the same configuration as D-mannose

TABLE 1. Inhibition of E. coli adherence touroepithelial cells by carbohydrates or ConA

Concn required for 50% in-Compound hibition

(mM)

Six-carbonD-Mannose .. 27.8D-Mannitol ..27.4a-Methyl-D-mannoside .. 77.0D-Fructose ..83.0

Five-carbonD-Arabinose . ............ 166.0D-Lyxose ........ ............. 166.0

Three-carbonL-Glyceraldehyde .. 350.0

Yeast mannan ..0.05%aConA ......................... 0.0093

a Molecular weight not determined.

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E. COLI ADHERENCE TO UROEPITHELIAL CELLS 533

with the exception of L-fucose, which had noinhibitory effect. All carbohydrates with the hy-droxyl group at carbon 2 in the opposite config-uration (D-glucose, L-mannose, a-methyl-D-glu-coside, L-rhamnose, D-ribose, D-galactose, D-Xy-lose, D-glyceraldehyde, dihydroxyacetone) wereineffective inhibitors of adherence even at con-centrations of 2.5%. The plant lectin ConA,which binds to D-mannose (23), was also a veryeffective inhibitor of adherence.Effect of preincubation of E. coli or uro-

epithelial cells with MEM, D-mannose, orConA. The site ofD-mannose and ConA bindingwas investigated by adding these compounds tothe bacterial or epithelial cell suspensions priorto performing the adherence assay. After a 30-min preincubation, the E. coli or uroepithelialcell suspensions were washed once with phos-phate-buffered saline and then incubated withuntreated epithelial or bacterial cells, respec-tively. The adherence was compared with thatof control cells preincubated in MEM (Table 2).Preincubation of the bacteria with D-mannosecompletely prevented their adherence to epithe-lial cells, whereas preincubation of bacteria withConA was much less inhibitory. When epithelialcells were preincubated with ConA, subsequentE. coli adherence was inhibited by 92.6%,whereas preincubation of epithelial cells with D-mannose inhibited adherence by 29.9%.

Influence of variation in bacterial or ep-ithelial cell adhesive characteristics on D-mannose inhibition. The possibility that celladhesive characteristics alter the inhibiting ef-fect of D-mannose was investigated by incuba-ting E. coli cells exhibiting different degrees ofreceptivity with a common epithelial cell pooland a fixed concentration of D-mannose. One

TABLE 2. Effect on adherence ofpreincubation ofE. coli or uroepithelial cells with MEM, D -mannose,

or ConAAdherence Inhibi-

Cells and preincubation medium (bacteria/ tionepithelialcell) a

E. coliMEM .. 17.8 0D-Mannose (1%) 0........... 100Concanavalin A (0.05%) 8.5 52.3

Uroepithelial cellsMEM ..... 17.4 0D-Mannose (1%) 12.2 29.9ConA (0.05%) 1.3 92.6a After a 30-min preincubation, the E. coli or uro-

epithelial cells were washed once with phosphate-buffered saline and then incubated with untreateduroepithelial or bacterial cells, respectively. The re-sults are representative of three experiments.

strain of E. coli was repeatedly transferred inbrain heart infusion broth to select for presum-ably heavily piliated organisms (7). Adherenceto epithelial cells from a common pool increasedfrom 8 to 80 bacteria per cell and was completelyinhibited by 2.5% D-mannose. As shown in Fig.1, inhibition by 0.1% D-mannose ranged from 1%for bacteria that were good adherers (presum-ably heavily piliated) to 100% for bacteria thatadhered poorly. The possibility that the adhe-sive characteristics of epithelial cells alter theinhibiting effect of D-mannose was investigatedby incubating epithelial cells with different de-grees of receptivity with the same E. coli strainand a fixed concentration of D-mannose. Epithe-lial cells from different individuals have variousdegrees of receptivity (22). Epithelial cells werecollected from several individuals, pooled, andtested with the same strain of E. coli to deter-mine their receptivity. Adherence for the cellpools collected at different times ranged from 13to 93 bacteria per cell and was completely in-hibited by 2.5% D-mannose. As shown in Fig. 2,inhibition by 0.1% D-mannose ranged from 100%for cells with poor receptivity to 45% for cells towhich large numbers of bacteria adhered in theabsence of mannose.Effect of various concentrations of car-

bohydrates on adherence. E. coli and epithe-lial cells were incubated with D-mannose, D-mannitol, a-methyl-D-mannoside, D-fructose, orD-glucose at concentrations of 2.5 to 0.001%.Depending on the carbohydrate, adherencecould be completely inhibited, partially in-hibited, or enhanced. For example, when theconcentrations of D-mannose added to the sus-pensions of bacteria and epithelial cells was var-ied, adherence could be completely inhibited(2.5%), partially inhibited (between 1.0 and0.1%), or significantly enhanced (between 0.01and 0.001%) to up to 160% of control levels (P

100-

o 80I

a:1 60-

S 40-

z

w 20-

-1ID 20 40 60

MANNOSE INHIBITION (%)

80 100

FIG. 1. Influence of variation in E. coli adhesivecharacteristics on D -mannose (0.1%) inhibition ofad-herence. Repeated subculturing of E. coli increasedadherence to uroepithelial cells from a common poolfrom 8 to 80 bacteria per cell. Each point representsthe mean of two experiments.

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6:

I-

-

w

<a

100

90

80-70-60-50'

40

30

2010

0 40 60 80

MANNOSE INHIBITION (%)

FIG. 2. Influence of variation in uroepitreceptivity on D -mannose (0.1%) inhibitionence. Epithelial cells from different individvarious degrees of receptivity. Adherence o4strain of E. coli to epithelial cell pools fronindividuals varied from 13 to 93 bacteriaEach point represents the mean of two expo

< 0.05, Fig. 3). It is important to nglucose had no inhibitory or enhancirThe curves for a-methyl-D-mannosidimannitol were similar to the D-manncshown in Fig. 3, except that no enhawas observed for D-mannitol.The possibility that interactions of

drates might modify their effects onadherence was investigated by addingtions of inhibitory and non-inhibitorydrates to the adherence assay. The ceffect of inhibitory carbohydrates aptbe additive. For example, 1% D-manncD-fructose inhibited adherence by 80%respectively. When combined, 1% D-and 1% D-fructose showed 100% inhibit:inhibitory carbohydrates had no effe(degree of inhibition of an inhibitorydrate. When 1% glucose (which had rtory effect on adherence) was addedmannose or 1% D-fructose, the inhibit(was the same as that of 1% D-mannosefructose alone.Effect of time of bacterial prein(

with D-mannose on adherence. Theity of increasing the inhibitory effect cnose was investigated by increasing thbacterial preincubation with various cotions of the carbohydrate prior to theof uroepithelial cells. Adherence waswith that observed for bacteria preincuthe same length of time in MEM. AsFig. 4, complete inhibition was obser2.5% D-mannose preincubated with ba30 or 60 min. Initially, concentrations (nose (between 1.5 and 0.75%) showeinhibition of adherence (15 to 50% ofAs the time of preincubation increaconcentrations of D-mannose between

0.75%, adherence increased significantly to levels120 to 260% of the control (P < 0.05). With0.001% D-mannose, the initial level of enhancedadherence (190% of control) was maintained.The possibility that higher levels of adherencewere due to an increased bacterial concentrationwas excluded by the observation that the colony-forming units remained virtually constant duringthe time ofpreincubation. Furthermore, bacteriapreincubated in 2.5% glucose or in MEM for 60min showed no increase in adherence comparedwith control bacteria that had not been prein-

afeliadhcerl cubated. Lastly, the possibility of enhanced bac-tuals have terial adherence through mannose metabolismf the same was investigated. Preincubation of E. coli for 5a different min with a-methyl-D-mannoside produced levelsX per cell. of enhancement similar to those observed for D-eriments. mannose, even though growth curves demon-

strated that the test strain could not use a-iote that methyl-D-mannoside as a sole carbon source.

ng effect. DISCUSSIONe and D-)se curve Further insight into the stereochemical spec-Lncement ificity of the bacterial ligand can be obtained by

examining the extent to which a large numbercarbohy- of monosaccharides, yeast mannan, and ConAbacterial inhibit the adherence of bacteria to uroepithelialcombina- cells. The carbohydrate combining site of the E.carbohy- coli ligand investigated in these experimentscombined appears to be directed primarily toward the un-)eared to modified hydroxyl group at carbon 2 of D-man-se or 1% nose and its derivatives. Thus, D-mannose, D-and 20%,-mannoseion. Non- 220-At on the 22carbohy- Z\

0io inhibi- r 180-to 1% D- I 'zory effect 0or 1% D- u 140-

0

cubation-possibil- uo- 100- ... .

f D-man- zwietime ofoncentra- 60additionala-omparedbated forshown inved withLcteria forAf D-man-d partialcontrol).

sed, withn 1.5 and

10 1.0 0.1 0.01 0.00 1 0.0001CONCENTRATION (%)

FIG. 3. Effect of various concentrations ofD -man-nose (-), D -fructose (E), and D -glucose (0) on adher-ence of E. coli to uroepithelial cells. Each pointrepresents the mean ± standard deviation of fourexperiments.

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E. COLI ADHERENCE TO UROEPITHELIAL CELLS 535

0

z 0.001%0

u- 160 /

w 120- /1.5%z

w 80/

O- ; v v 2.5%

0 30 60TIME OF BACTERIAL PREINCUBATION (MINUTES)

FIG. 4. Effect of time ofE. colipreincubation withD -mannose on subsequent adherence to uroepithelialcells. The results are representative of three experi-ments.

mannitol, and yeast mannan (a polysaccharidethat is polymer of mannose) were potent inhib-itors of E. coli adherence to uroepithelial cellsin vitro. D-Fructose was a twofold less effectiveinhibitor of adherence than D-mannose. Al-though the open-chain configuration of D-fruc-tose has an aldehyde group at the second carbon,in solution rearrangement occurs such that D-fructose assumes the pyranose form with thesame configuration as D-mannose at carbon 2(12, 18). Although L-fucose has the same config-uration at carbon 2 as D-mannose, it did notinhibit adherence. These findings are in agree-ment with those of other investigators who havereported inhibition with D-mannose and no in-hibition with L-fucose (2, 15, 17, 19, 20). Thiscould be due to the replacement of the hydroxylgroup on carbon 6 with a hydrogen atom. Anymodification of the carbon 2 hydroxyl groupcompletely abolished the inhibitory action ofthecarbohydrate. Thus D-glucose, which differsfrom D-mannose only in the hydroxyl group atcarbon 2, did not inhibit adherence even at aconcentration of 2.5%. The length of the carbonchain also appears to be an important determi-nant of the inhibitory effect of a carbohydrate.Five-carbon and three-carbon carbohydrates,with the same configuration as D-mannose atthe second carbon, were 6-fold and 13-fold lesseffective inhibitors of adherence, respectively.Presumably, the pyranose ring of D-mannose isnot active in this interaction because the open-chain hexitol D-mannitol was a very effectiveinhibitor of adherence. The a linkage also seems

to be unimportant since the a-methyl glycosideof D-mannose reduced the inhibitory effect ob-served for D-mannose and the a-methyl glyco-side of D-glucose had no effect on adherence.The concept that the hydroxyl group at carbon2 in the D-mannose configuration may interferewith binding of E. coli to uroepithelial cells issupported by the inhibitory effect of D-mannoseon E. coli adherence to buccal cells (16), renalcells (20), erythrocytes (27), and leukocytes (2,13). Other investigators, using mannose-sensi-tive E. coli strains (2, 15, 20, 27), have reportedresults on the inhibitory effect of other carbo-hydrates, and even the mannose derivatives,which differ from our findings. These inconsist-encies may be attributable to differences in thespecific surface characteristics of the bacteria, aswell as the eucaryotic cells, that were used.

Preincubation of bacteria with D-mannosecompletely inhibited their subsequent bindingto epithelial cells. Conversely, preincubation ofepithelial cells with the mannose-binding lectinConA significantly reduced their receptivity by92.6%. These observations support the conceptthat attachment of E. coli to uroepithelial cellsis mediated by a mannose-specific bacterial li-gand which binds to mannose or mannose-likereceptors on the epithelial cell surface. Studieswith ConA suggest that mannose is widely dis-tributed on the surface of other mammalian cells(23) and may be an integral part of the receptorsite required for E. coli attachment to epithelialcells (2, 16).When bacteria were incubated in broth to

increase their binding capacity (and presumablypiliation), the inhibitory effect of a fixed concen-tration of D-mannose was inversely proportionalto the degree of bacterial adherence. These re-sults suggest that the mannose-binding ligandcan be altered by growth conditions which areconducive to increased pilus formation and maybe associated with pili. Ofek and Beachey (15)showed that the bacterial population which ad-hered to epithelial cells consisted of heavily pi-liated cells that possessed increased mannosebinding activity, and Salit and Gotschlich (20)demonstrated mannose-sensitive binding of pu-rified E. coli type I pili to monkey kidney cells.More recent evidence suggests, however, thatother mannose-sensitive adhesions, biochemi-cally distinct from type I pili, may also be in-volved in the mannose-specific adherence of E.coli to eucaryotic cells (8, 13). Mannose-insen-sitive adhesions also appear to participate in E.coli-epithelial cell interactions. Svanborg Eden(26) described mannose-insensitive, pilus-asso-ciated adherence of E. coli to uroepithelial cells,and mannose-insensitive binding of enteropath-

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536 SCHAEFFER, AMUNDSEN, AND JONES

ogenic E. coli to intestinal cells has been iden-tified (4, 10).We also observed that the inhibitory effect of

D-mannose was influenced by the degree of re-ceptivity of the epithelial cells. When 0.1% D-mannose was added to incubation systems con-taining the same bacteria and epithelial cellswith different degrees of receptivity, the inhibi-tory effect of D-mannose decreased as the recep-tivity of the epithelial cells increased. This sug-gests that D-mannose inhibits bacterial adher-ence by competing with mannose-like receptorson the epithelial cell surface and that there maybe a difference among uroepithelial cells in thenumber ofreceptor sites or the degree of affinity,or both, that these receptors have for bacteria.The inhibitory effect of D-mannose, and its

derivatives, on the adherence of E. coli to uro-epithelial cells was dose related and was linearbetween concentrations of 2.5 and 1%. The ob-servation that adherence was enhanced by lowconcentrations (between 0.01 and 0.001%) of D-mannose, D-fructose, or a-methyl-D-mannosideis intriguing and may be representative of phys-iological conditions. That this phenomenon wasdue to the production ofadditional receptor siteson the epithelial cells is unlikely because onlyapproximately 20% of the epithelial cells used inthe assay were viable. Furthermore, enhance-ment was observed when the bacteria werepreincubated in D-mannose prior to incubationwith the epithelial cells. It also seems unlikelythat bacterial metabolism ofa carbohydrate dur-ing preincubation is necessary for enhancementsince enhancement was observed after preincu-bation with a-methyl-D-mannoside, a carbohy-drate that cannot be utilized as a sole carbonsource by the E. coli strain used in these exper-imnents. Enhancement of adherence in the pres-ence of 0.001% D-mannose or after prolongedbacterial preincubation with 0.75 to 1.5% D-man-nose may be due to cross-linking of bacteriaprior to their attachment to the epithelial cellreceptors. Thus, many bacteria could attach toa single binding site, increasing the total numberofbacteria per epithelial cell. Cross-linking couldbe due to nonspecific effects of mannose, suchas alterations in the electrostatic charge of pili,which make the bacteria more susceptible tobinding with epithelial cell receptors. Alterna-tively, bacteria could link themselves to oneanother through shared mannose molecules.McMichael and Ou (14) observed that piliatedE. coli cells formed aggregates in the presenceof lysozyme (one lysozyme molecule per twopilus subunits) or divalent cations such as Ca2"or Mg2e. Although high concentrations of man-nose might induce maximal cross-linking of bac-

INFECT. IMMUN.

teria, all available bacterial ligands would beblocked, thereby resulting in complete inhibitionof adherence to the epithelial cells.The relationship between mannose-mediated

adherence of E. coli to uroepithelial cells andurinary tract infections is not clear, but somestudies have suggested that it may have clinicalsignificance. Duguid (6) screened 108 strains ofE. coli isolates from humans by hemagglutina-tion and mannose-sensitive inhibition of hem-agglutination. He found that the majority (74%)of the strains possessed mannose-binding activ-ity and that all of the mannose-sensitive strainswere piliated. Aronson et al. (1) found that a-methyl-D-mannoside prevented urinary tract in-fections with E. coli in mice, presumably byblocking bacterial adherence to the urinarytract. Although it appears that receptors otherthan mannose could bind E. coli to uroepithelialcells, continued investigation of the mannose-binding activity of E. coli to uroepithelial cellsshould greatly increase our insight into the path-ogenesis and treatment of urinary tract infec-tions.

ACKNOWLEDGMENTSWe are grateful to Byron Anderson, Department of Bio-

chemistry and Otolaryngology and Maxillofacial Surgery,Northwestern University Medical School, for helpful advice.

This investigation was supported by Public Health Servicegrant RR-05370 from the National Institutes of Health.

LITMRATURE CITED1. Aronson, M., 0. Medalia, L. Schori, D. Mirelman, N.

Sharon, and I. Ofek. 1979. Prevention of colonizationof the urinary tract of mice with Escherichia coli byblocking of bacterial adherence with methyl-a-D-man-nopyranoside. J. Infect. Dis. 139:329-332.

2. Bar-Shavit, Z., I. Ofek, R. Goldman, D. Mirelman,and N. Sharon. 1977. Mannose residues on phagocytesas receptors for the attachment of Escherichia coli andSalmonella typhi. Biochem. Biophys. Res. Commun.78:455-460.

3. Brent, P., and K. L. Vosti. 1973. Micromethod for sero-grouping Escherichia coli. Appl. Microbiol. 25:208-211.

4. Cheney, C. P., E. C. Boedeker, and S. B. Formal. 1979.Quantitation of the adherence of an enteropathogenicEscherichia coli to isolated rabbit intestinal brush bor-ders. Infect. Immun. 26: 736-743.

5. Collier, W. A., and J. C. De Miranda. 1955. Bacterien-Haemagglutination. III. Die hummung der Coli-hae-magglutination durch mannose. Antonie van Leeuwen-hoek J. Microbiol. Serol. 21:133-140.

6. Duguid, J. P. 1964. Functional anatomy of Escherichiacoli with special reference to enteropathogenic E. coli.Rev. Latinoam. Microbiol. 7(Suppl. 13-4):1-16.

7. Duguid, J. P., and P. R. Gillies. 1957. Fimbriae andadhesive properties in dysentery bacilli. J. Pathol. Bac-teriol. 74:397-411.

8. Eshdat, Y., I. Ofek, Y. Yashouv-Gan, N. Sharon, andD. Mirelman. 1978. Isolation of a mannose-specificlectin from Escherichia coli and its role in the adher-ence of the bacteria to epithelial cells. Biochem. Bio-phys. Res. Commun. 85:1551-1559.

9. Evans, D. G., and D. J. Evans, Jr. 1978. New surface-

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associated heat-labile colonization factor antigen (CFA/II) produced by enterotoxigenic Escherichia coli ofserogroups 06 and 08. Infect. Immun. 21:638-647.

10. 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.

11. Jones, G. W. 1977. The attachment of bacteria to thesurfaces of animal cells, p. 141-176. In J. L. Reissing(ed.), Microbial interactions. Chapman and Hall Ltd.,London.

12. Karlson, P. 1970. Simple sugars (monosaccharides), p.278. In Charles H. Doering (ed.), Introduction to mod-em biochemistry, 3rd ed. Academic Press, Inc., NewYork.

13. Mangan, D. F., and I. S. Snyder. 1979. Mannose-sensi-tive interaction of Escherichia coli with human periph-eral leukocytes in vitro. Infect. Immun. 26:520-527.

14. McMichael, J. C., and J. T. Ou. 1979. Binding of lyso-zyme to common pili of Escherichia coli. J. Bacteriol.138:976-983.

15. Ofek, I., and E. H. Beachey. 1978. Mannose binding andepithelial cell adherence of Escherichia coli. Infect.Immun. 22:247-254.

16. Ofek, I., D. Mirelman, and N. Sharon. 1977. Adherenceof Escherichia coli to human mucosal cells mediatedby mannose receptors. Nature (London) 265:623-625.

17. Old, D. C. 1972. Inhibition of the interaction betweenfimbrial haemagglutinins and erythrocytes by D-man-nose and other carbohydrates. J. Gen. Microbiol. 71:

149-157.18. Oser, B. L. (ed.). 1965. Hawks physiological chemistry,

14th ed., p. 67-73. McGraw-Hill Book Co., New York.19. Salit, I. E., and E. C. Gotschlich. 1977. Hemagglutina-

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