INFECTION AND IMMUNITY, June 1984, p. 592-598 Vol. 44, No. 30019-9567/84/060592-07$02.00/0Copyright C) 1984, American Society for Microbiology

Characterization of Nonfimbrial Mannose-Resistant ProteinHemagglutinins of Two Escherichia coli Strains Isolated from Infants

with EnteritisPETER H. WILLIAMS,'* STUART KNUTTON,2 MOIRA G. M. BROWN,'t DAVID C. A. CANDY,2 AND

ALEXANDER S. McNEISH2

Department of Genetics, University of Leicester, Leicester LE] 7RH,1 and Institute of Child Health, University ofBirmingham, Birmingham B16 8ET,2 England

Received 27 October 1983/Accepted 22 February 1984

Escherichia coli strains 444-3 and 469-3, isolated from patients with severe infantile enteritis, are able toadhere to and penetrate human epithelial cells in culture. In addition to type 1 fimbriae and glycocalyces,both strains elaborate mannose-resistant nonfimbrial protein hemagglutinins specific for human erythro-cytes. Purified agglutinins are aggregates (greater than 4 x 106 daltons) of a single protein subunit ofapparent M, 14,000 (469-3) to 14,500 (444-3). The optimal temperature for expression of the agglutinins is37°C. Bacteria grown at 22°C, which show 1% or less of maximal activity, and mutants deficient in theability to agglutinate human erythrocytes do not synthesize detectable levels of these surface proteins and,moreover, do not adhere to cultured epithelial cells. Coupled with the observation that purified agglutininscompetitively inhibit bacterial adherence to cultured cells, these data indicate that the nonfimbrial surfaceproteins expressed by strains 444-3 and 469-3 are essential for adherence both to erythrocytes and tocultured epithelial cells.

We previously described two independent strains of Esch-erichia coli, designated 444-3 and 469-3, isolated from casesof severe dysentery-like diarrheal disease of infants (17).These strains adhere to cultured human epithelial cells in thepresence of D-mannose, showing remarkably close apposi-tion of the bacterial membrane with cell surfaces and micro-villi (16). Subsequently, penetration of the epithelial mono-layer occurs, and the bacteria exist intracellularly withinmembrane-bound vesicles (16). In this paper we report thecharacterization of chromosome-specified protein structurespresent on the bacterial surface (in addition to type 1fimbriae and glycocalyces) that are essential both for aggluti-nation specifically of human erythrocytes and for adherenceto cultured human epithelial cell surfaces as a prerequisitefor penetration.

MATERIALS AND METHODSBacterial strains and culture medium. Bacterial strains

used in this study are described in Table 1. Pathogenic E.coli strains 444-3 (O?:H4) and 469-3 (021:H-), which canadhere to and penetrate cultured human epithelial cells (16),have been described previously (17). Plasmid-cured deriva-tives were obtained by treatment with ethidium bromide aspreviously described (1, 25). Strain 01 was isolated from thefeces of a healthy person and is used here as a "control"strain (17). Culture medium for all strains was Luria broth(10 g of Bacto-Tryptone [Difco Laboratories] per liter, 5 g ofyeast extract [Difco] per liter, 0.5 g of NaCl per liter,adjusted to pH 7.0 with 1 M NaOH) solidified with 1.5% agarif required.

Hemagglutination. Fresh erythrocytes were washed twiceand suspended to 10% of their original concentration in 0.9%(wt/vol) NaCl (saline). To 25 plI of erythrocyte suspension

* Corresponding author.t Present address: Dental Research Unit, Royal College of Sur-

geons of England, Downe, Orpington, Kent, England.

592

were added 25 ,ul of 3% (wt/vol) D-mannose and 100 ,ul of thesuspension or solution to be tested. Mixtures were incubatedon ice for 30 min before inspection for hemagglutination.Quantitation of activity was achieved by assaying serialdilutions of hemagglutinating samples.

Mutagenesis. Bacterial cells growing logarithmically inLuria broth were harvested by centrifugation, washed, andsuspended in 0.1 M citrate buffer (pH 5.5). N-Methyl-N'-nitro-N-nitrosoguanidine was added to a final concentrationof 2 ,ug/ml, and the suspension was incubated for a timeestimated from preliminary experiments to kill approximate-ly 50% of the cell population.Enrichment for hemagglutination-defective mutants. To

facilitate screening for mutants defective in hemagglutina-tion in mutagenized bacterial populations, the followingenrichment protocol was adopted. Fresh human group Aerythrocytes were washed twice and suspended to theiroriginal concentration in sterile saline. Enrichment mixtures(5 ml) were composed of up to 107 mutagenized bacteria,20% (vol/vol) erythrocyte suspension, and 0.5% (wt/vol) D-mannose in saline. Mixtures were incubated on ice for 15 to20 min and then centrifuged at low speed to pellet theerythrocytes and adherent bacteria. The supernatant frac-tion was gently removed to another tube containing a freshsample of erythrocytes, and the process was repeated.Reconstruction experiments with mixtures of strainsLG1354 (a genetically marked derivative of control strain01) and 444-3 indicated that an increase in the nonadheringcomponent of the population from 10-6 initially to about10-2 was achieved with eight cycles of enrichment. Isolatesderived by this enrichment procedure were checked fornonselected resistance markers characteristic of the parentstrain.

Purification of hemagglutinins. Bacteria were grown over-night on Luria agar at 37°C. Cells were scraped off the platesand suspended in saline to a density of approximately 1011cells per ml. Suspensions were blended for 2 min in an MSE

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NONFIMBRIAL MRE HEMAGGLUTININS OF E. COLI 593

TABLE 1. Bacterial strainsStrain Characteristics' and references

444-3 Apr pcr Sur (17)LG1391 Cured (ethidium bromide) derivative of 444-3LG1407 As LG1391, Nalr Rif' (spontaneous)LG1412 Hemagglutination-deficient mutant of LG1407 (NTG)469-3 Apr pcr Smr Spr Sur, cryptic plasmid (17)LG1505 Hemagglutination-deficient mutant of 469-3 (NTG)LG1507 Mutant of 469-3 with reduced hemagglutination

(NTG)01 Non-hemagglutinating, nonadhering (17)LG1354 As 01, Nalr Rif (spontaneous)

a Plasmid-determined resistances: Ap, ampicillin; Pc, penicillin;Sm, streptomycin; Sp, spectinomycin; Su, sulphonamides. Chromo-somal resistances: Nal, nalidixic acid; Rif, rifampicin. NTG, N-Methyl-N'-nitro-N-nitrosoguanidine.

Ato-Mixer at the highest speed setting or for 10 min in aSorvall Omnimixer at setting 7.5 (this procedure had noeffect on bacterial viability). After blending, whole cellswere pelleted by centrifugation, and the supernatant fluidwas sterilized by membrane filtration. Preparations werecentrifuged at 40,000 rpm for 2 h at 15°C in a Beckman 50 Tirotor, and the supernatant fluid, which contained most of thehemagglutinating activity, was retained. After concentrationby dialysis against 40% polyethylene glycol (Mr, 20,000),agglutinin preparations were effectively purified of contami-nating protein by passage through a Sepharose 4B-CL col-umn (34 by 1.6 cm). Samples (5 ml) were applied and elutedwith saline at a flow rate of 20 ml/h; hemagglutinationactivity of either strain eluted as a single peak. Typical yields

of purified hemagglutinin were approximately 1 mg of pro-tein per g (wet weight) of bacteria. The protein concentrationof preparations was determined by the method of Bradford(2) with bovine serum albumin as a standard, and carbohy-drate was estimated by the method of Dubois et al. (5) withD-glucose as a standard.

Bacterial adherence to tissue culture cells. The preparationof HeLa and HEp-2 monolayers and light microscopicanalysis of bacterial adherence have been described previ-ously (17). The adherence index of a bacterial culture isdefined as the average number of adherent bacteria percultured cell scored.

SDS-polyacrylamide gel electrophoresis. Agglutinin prepa-rations, concentrated by dialysis against polyethylene gly-col, were mixed with sodium dodecyl sulfate (SDS; 0.2%,wt/vol), boiled for 5 min, and applied to a polyacrylamide gel(16%, with an 8% stacking gel; monomer/dimer ratio, 44:0.8)containing 0.2% SDS. Electrophoresis buffer was 25 mMTris (pH 8.8)-200 mM glycine, also containing 0.2% SDS.Proteins used as molecular weight markers were bovineserum albumin (Me, 68,000), chymotrypsinogen (25,700),lysozyme (14,300), and RNase A (13,700). Coomassie bril-liant blue was used to stain for proteins. The presence ofcarbohydrate in gels was determined by the periodic acid-Schiff reaction (3).Amino acid analysis. Duplicate samples of purified aggluti-

nin preparations were hydrolyzed for 24 or 48 h in 6 Nhydrochloric acid at 110°C in evacuated tubes. Analysis wasperformed with a Locarte amino acid analyzer.

Estimation of pL. Isoelectric focusing of SDS-denaturedagglutinin preparations was performed with an LKB Multi-phore horizontal gel system (LKB Instruments, Inc.) andLKB ampholine carrier ampholytes giving pH ranges 3.5 to

TABLE 2. Hemagglutination by strains 444-3 and 469-3

Hemagglutinating activityaBlood Assay conditions, treatment

444-3 469-3

Human group A 128 128Human group A D(+)-Mannose (0.5%, wt/vol) 128 128Human group B D(+)-Mannose (0.5%, wt/vol) 64 128Human group AB D(+)-Mannose (0.5%, wt/vol) 128 128Human group 0 D(+)-Mannose (0.5%, wt/vol) 64 64Human group A N-Acetyl-neuraminic acid (1%, wt/vol) 128 128Human group A N-Acetyl-D-glucosamine (1%, wt/vol) 128 128Human group A N-Acetyl-D-galactosamine (1%, wt/vol) 128 128Human group A a-L(-)-Fucose (1%, wt/vol) 128 128Human group A D(+)-Glucose (1%, wt/vol) 128 128Human group A Lactose (1%, wt/vol) 128 128Human group A a-D(+)-Melibiose (1%, wt/vol) 128 128Chicken D(+)-Mannose (0.5%, wt/vol) 0 0Guinea pig D(+)-Mannose (0.5%, wt/vol) 0 0Horse D(+)-Mannose (0.5%, wt/vol) 0 0Monkey D(+)-Mannose (0.5%, wt/vol) 0 0Mouse D(+)-Mannose (0.5%, wt/vol) 0 0Ox D(+)-Mannose (0.5%, wt/vol) 0 0Pig D(+)-Mannose (0.5%, wt/vol) 0 0Rabbit D(+)-Mannose (0.5%, wt/vol) 0 0Rat D(+)-Mannose (0.5%, wt/vol) 0 0Sheep D(+)-Mannose (0.5%, wt/vol) 0 0Human group A D(+)-Mannose (0.5%, wt/vol), heatb 128 128Human group A D(+)-Mannose (0.5%, wt/vol), formaldehydec 64 32Human group A D(+)-Mannose (0.5%, wt/vol), proteased 32 32

a Values are the reciprocals of the highest dilutions of bacterial suspension in which hemagglutination was detectable.b Heat at 65°C, 4 h.C Formaldehyde concentration of 0.5% (vol/vol), 37°C, 4 h.d Autodigested Pronase (1 mg/ml), 37°C, 2 h.

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10 and 4 to 6. Gels were stained with Coomassie brilliantblue to locate polypeptide bands.

RESULTSHemagglutination by E. coli 444-3 and 469-3. E. coli 444-3

and 469-3 both exhibited mannose-resistant hemagglutina-tion of human erythrocytes of several blood groups (Table2). No reaction was observed in the presence of mannosewith the blood of a number of other animal species, includingmost of those used by Duguid et al. (6) in their hemagglutina-tion typing scheme. The mannose-resistant reaction withhuman group A blood was progressively lost (eluted) atassay incubation temperatures up to about 50°C, but activitywas recovered by cooling the mixtures of bacteria anderythrocytes on ice (Fig. 1). None of a range of sugarsknown to inhibit lectin-like interactions had any observableeffect on hemagglutination by either strain (Table 2). Incuba-tion of bacterial cells with formaldehyde did not result in thecomplete loss of activity characteristic of the mannose-resistant eluting agglutinins of E. coli (6, 7). High concentra-tions of Pronase significantly reduced hemagglutinating ac-tivity by both strains, but exposure of the bacteria to heathad no effect (Table 2).The optimum growth temperature for expression of agglu-

tinins of both 444-3 and 469-3 was 37°C (Fig. 2); themaximum activity per viable cell was similar whether thebacteria were grown in aerated liquid culture or on agarsurfaces. However, agar-grown cells showed much greatervariation in activity, so that at a growth temperature of 220Cstrain 444-3 showed 1% and strain 469-3 less than 0.1% of theoptimal level of hemagglutination.

Isolation and characterization of hemagglutination-deficientmutants. Strain LG1407 (a plasmid-free derivative of 444-3)and strain 469-3 were treated with N-methyl-N'-nitro-N-nitrosoguanidine, washed thoroughly with phosphate buffer(pH 7.0), and suspended in Luria broth. This suspension wasdivided into workipg samples, which were incubated sepa-rately at 37°C overnight and then subjected to eight cycles of

1.0-

c

0,

m 0.5 \E

0

10 30 50

Temperature (0c)

FIG. 1. Effect of temperature on hemagglutination of humangroup A erythrocytes by strain 469-3. Assay mixtures were incubat-ed at the temperatures indicated for up to 2 h (0) and subsequentlyincubated on ice for a further 1 h (O). Hemagglutination by strain444-3 showed the same response to variations in assay temperature.

I

m0 00

CL 0.1-

0~~~~~~~~~~~

0.001-(a) (b)

.I30 40 30 40

Temperature (cc)

FIG. 2. Effect of growth temperature on expression of aggluti-nins of (a) strain 444-3 and (b) strain 469-3. Bacteria were grown tothe stationary phase on Luria agar surfaces (0) or in aerated Luriabroth cultures (O) at the temperatures indicated. Agar-grown cellswere suspended in saline. Hemagglutination of human group Aerythrocytes and bacterial viable counts were determined by serialdilution of bacterial suspensions.

incubation with human erythrocytes as described above toenrich for nonadherent bacteria. Samples of supernatantfluid after the seventh and eighth enrichment cycles werediluted and plated onto Luria agar. Individual colonies werepicked into 2-ml portions of Luria broth, grown at 37°Covernight, and tested for the ability to agglutinate humangroup A erythrocytes. Of the colonies tested after enrich-ment, up to 4% were deficient in mannose-resistant hemag-glutination. All of these were also found to have lost theability to adhere to HeLa cells in culture (Table 3). Mostmutants showed undetectable levels of agglutination andadherence; however, mutant strain LG1507 displayed mea-surable, but significantly reduced, levels of both processes.

Unlike the parental strains 444-3 and 469-3, populations ofhemagglutination-deficient mutants did not contain a signifi-cant proportion of cells elaborating glycocalyces (16). How-

TABLE 3. Properties of hemagglutination-deficient mutants

Hemagglutinating AdherenceStrain activity' ~ indeXb

LG1354 0 0LG1407 128 6.8LG1412' 0 0.4469-3 128 35.9LG1505c 0 0.2LG1507 4 1.2

a See footnote a of Table 2.b Average number of bacteria adhering to each HeLa cell.c Eight other independently isolated mutants of strain LG1407

showed the same properties as LG1412; two other independentmutants of 469-3 showed the same properties as LG1505.

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TABLE 4. Properties of purified hemagglutininsHemagglutinating activitya

Treatment444-3 469-3

Control 1,024 512pH 3b 1,024 512pH 10b 1,024 5122°C, 6 mo 512 512650C, 4 h 1,024 512100°C, 2 min 0 0

a Values are the reciprocals of the highest dilutions of prepara-tions in which hemagglutination was detectable.

b Incubated at 37°C for 30 min and neutralized before the assay.

ever, since the glycocalyx promotes mannose-resistant ad-herence of bacteria to erythrocytes, the method used herefor enrichment of mutants would tend to select for individ-uals in which glycocalyx synthesis is usually repressed.Apart from this, electron microscopy did not reveal anyobvious differences between the surfaces of hemagglutina-tion-defective mutants and their parental strains (16).

Purification and properties of hemagglutinating activity.Agglutinins were purified from agar-grown cultures (37°C) ofstrains 444-3 and 469-3 as described above. Such prepara-tions were found to be strikingly stable (Table 4); hemagglu-tinating activity was unaffected by incubation in conditionsof either high or low pH or at 65°C for several hours orindeed at 2°C for several months. However, activity wasirreversibly destroyed by boiling for 2 min.

Agglutinins of both strains were purified by using theability to bind to human erythrocytes to cause hemagglutina-tion as an indicator of activity. Purified agglutinins also bindto tissue culture cells, as demonstrated by the fact that theycompetitively inhibit bacterial adherence (Table 5). Notethat concentrations of 444-3 agglutinin that inhibited bindingof the producing strain had no effect on strain 469-3, and viceversa, suggesting that the individual surface protein aggre-gates of strains 444-3 and 469-3, although functionally simi-lar, are structurally distinct and recognize different receptorson the mammalian cell surface.

Characterization of purified hemagglutinins. Concentratedpreparations of agglutinins of strains 444-3 and 469-3 wereanalyzed by SDS-polyacrylamide gel electrophoresis as de-scribed above. In gels stained to show proteins, purifiedpreparations contained a single major band, which was

TABLE 5. Effect of purified agglutinins on bacterial adherence tocultured HEp-2 cells

Agglutinina Adherence indexb( g) 444-3 469-3

None 22 67444-3 (0.1) 25 63444-3 (1.0) 10 66444-3 (10.0)c <1 59469-3 (0.1) 21 51469-3 (1.0) 25 8469-3 (10.0) 19 2

a Purified agglutinins were added to washed cell monolayers andincubated at 37°C for 30 min. Monolayers were washed once morebefore the addition of bacteria.

b See footnote b of Table 3.c High levels of 444-3 agglutinin were found to be slightly cytotox-

ic.

68Mol. Wt.x 10--3

143~~~~~

13.7

a b c d e 9 h

FIG. 3. SDS-polyacrylamide gel electrophoresis of hemaggluti-nin preparations of strains 444-3 and 469-3. Samples of hemaggluti-nating activity in blended culture supernatants (lanes b, c, and d)and eluted from a Sepharose 4B column (lanes f, g, and h) wereapplied to gels as described in the text. Preparations from strain 444-3 grown at 37°C (lanes b and f), and strain 469-3 grown at 37°C (lanesc and g) or 22°C (lanes d and h) were analyzed by using themolecular weight standards listed in the text (lanes a and e).

clearly visible also in the supernatant fraction of blendedbacterial suspensions (Fig. 3). The apparent Mr of the 444-3polypeptide is 14,500, and that of the 469-3 product is 14,000.These bands were absent in similar preparations from mu-tant derivatives of either strain that were defective in hemag-glutination. Furthermore, the 14,000-dalton polypeptide wasundetectable in preparations from strain 469-3 grown at 22°C(Fig. 3). When gels were stained to show the presence ofcarbohydrates, no difference was observed between sampleswith or without hemagglutinating activity, and there was noevidence of staining material in any lane of the gel in theregion of the 14,000- and 14,500-dalton polypeptides associ-ated with purified agglutinins. Similar estimates for subunitsize were obtained by gel filtration (Fig. 4) in the presence ofguanidine hydrochloride to maintain dissociation, althoughthis technique was not sufficiently sensitive to determine adifference between preparations from the two strains. Puri-fied agglutinins of both strains eluted as single protein peaks;

3

to

x 2

0

75 80 85Elution Volume (ml)

FIG. 4. Gel filtration of purified hemagglutinin preparations ofstrains 444-3 and 469-3. Samples (2 ml) of protein in 5 mM Tris buffer(pH 8.5) containing guanidine hydrochloride (8 M) were incubated at37°C for 2 h to allow dissociation of subunits before application to a

Sephadex G-100 superfine column (1.6 by 30-cm bed volume) in thesame buffer (flow rate, 8 ml/h). In separate runs, the elution volumeof molecular weight markers (2 mg of protein per sample) chymo-trypsinogen (0), lysozyme (A), and RNase A (V) was determined as

absorption at 280 nm, and that of agglutinin subunits (approximately150 p.g of protein per sample, OI) by biochemical estimation ofprotein concentration in collected fractions (1 ml).

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no associated carbohydrate was detectable chemically inpeak fractions after extensive dialysis against saline toremove guanidine hydrochloride.

Isoelectric focusing of purified agglutinins each showed asingle polypeptide band of pI 4.7 in the case of the 444-3protein and pl 5.0 for the 469-3 product. Amino acid analysis(Table 6) indicated that the purified proteins are similar inthat, like the fimbrial agglutinins of E. coli and of otherbacterial species (18), they are markedly hydrophobic; non-polar amino acids comprise 43 and 45% of the residues of the444-3 agglutinin and the 469-3 agglutinin, respectively.

Denaturation and renaturation of agglutinin aggregates.Hemagglutinating activity from strains 444-3 and 469-3 wasexcluded by Sepharose 6B-CL (data not shown), which hasan exclusion limit for globular proteins of about 4 x 106daltons. Both agglutinins, however, were retarded by Se-pharose 4B-CL (Fig. 5), which excludes proteins larger than20 x 106 daltons. Denaturation of purified agglutinins byheating to 65°C in SDS (0.2%, wt/vol) was followed byrestoration of up to 75% of the original hemagglutinatingactivity when SDS was removed by passage of the mixturethrough a Sephadex G50 column. Reaggregated activity wasexcluded by Sepharose 4B-CL (Fig. 5), indicating an appar-ent molecular mass in excess of 20 x 106 daltons.

DISCUSSIONBacterial adherence is an important early stage in pathoge-

nicity. Pathogenic strains of bacteria usually elaborate sur-face structures whose primary function is interaction withreceptors in the membranes of target cells, but which mayalso cause agglutination of erythrocytes of particular speciesof animal. Although this interaction with erythrocytes is,perhaps, fortuitous, it is clear that it reflects the presence ofsurface structures whose role in adherence to epithelial cellsmay be crucial to the progress of an infection. Thus,hemagglutination typing schemes have been developed withstandard panels of blood from a variety of animals to classifythese surface structures (6, 8, 9). Most strains of E. colipossess an agglutinin (associated with the presence of type 1fimbriae) active against erythrocytes from many animals (7).

TABLE 6. Amino acid composition of 444-3 and 469-3 agglutininprotein subunitsa

No. of residuesAmino acid

444-3 469-3

Aspartic acid 17 15Threonine 10 8Serine 25 21Glutamic acid 20 17Proline 2 4Glycine 24 20Alanine 10 12Valine 6 7Methionine 2 2Isoleucine 4 5Leucine 7 8Tyrosine 4 2Phenylalanine 5 5Histidine 4 3Lysine 3 4Tryptophan ND NDArginine 0 2

a The total number of amino acid residues were as follows: forstrain 444-3, 143 residues (14,428 daltons); for strain 469-3, 135residues (13,671 daltons). ND, Not determined.

l

. 3 m

c

2-c

c

C

0~~~~~

0~0 ,l- Y- -.-

5 10 15 20 25

Fraction NumberFIG. 5. Sepharose 4B-CL column chromatography of purified

444-3 hemagglutinin. Samples (1 ml) of native agglutinin (0) and ofSDS-denatured and renatured activity (El, see the text) were appliedto a Sepharose 4B-CL column (1.2- by 20-cm bed volume) andeluted with saline at a flow rate of approximately 0.4 ml/min.Fractions (2 ml) were collected and assayed for protein concentra-tion and hemagglutination activity.

Since this hemagglutination can be inhibited by the additionof D-mannose or its analogs, the reaction is described asmannose sensitive. Some strains of E. coli promote man-nose-resistant hemagglutination, frequently in addition to amannose-sensitive reaction. Although they may vary widelyin the specificity of blood types affected, mannose-resistantagglutinins have a number of general characteristics, includ-ing the reversible loss (elution) of agglutination at tempera-tures up to about 50°C (hence the designation mannoseresistant and eluting [MRE]; 6, 7). In most examples exam-ined, MRE activity is associated with the presence offimbriae (collectively designated type MRE fimbriae; 6, 7).However, it is characteristic of strains that show unusualsubstrate specificity (for example, promoting mannose-resis-tant hemagglutination of blood of only a single animalspecies) that they do not have fimbriae (6, 12); the surfacesof some such strains have been described as having irregularexcrescences (12).

In this paper we describe nonfimbrial hemagglutininsexpressed by two independent strains of E. coli, 444-3 and469-3. The fact that these strains were originally isolatedfrom patients with dysentery-like disease (17, 19) and theobservation that they can penetrate cultured human epitheli-al cells and exist intracellularly within membrane-boundvesicles (16) suggest that invasion in the manner describedfor Salmonella spp. (15) may be an important contribution totheir virulence. However, as with some urinary tract isolatesof E. coli (24), penetration of cultured cells does not corre-late reliably (17) with the ability to cause keratoconjunctivi-tis in guinea-pig (Sereny test; 23). Strains 444-3 and 469-3 donot, as far as we can judge (17), possess a high-molecular-weight plasmid such as those reported to be essential forinvasion by Shigella sp. (10, 20, 21), by strains of E. colidesignated invasive on the basis of the Sereny test (11), or bySalmonella typhimurium (14). Furthermore, the difference inreceptor specificity between the hemagglutinins of 444-3 and469-3 infers significant structural and, therefore, geneticdiversity and argues against there being a common plasmid

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basis for expression of surface structures. Indeed, strains444-3 and 469-3 are probably best described as facultativelyenteropathogenic (4), rather than truly enteroinvasive or

Shigella sp.-like.Each strain promotes mannose-resistant agglutination

only of human erythrocytes (out of a wide range tested), a

reaction which progressively elutes at temperatures abovethe optimum (0°C). As is typical for MRE hemagglutinins ofE. coli (6, 7), the optimal growth temperature for expressionof both activities is 37°C, but expression is temperaturedependent so that cells grown at 22°C show 1% or less ofmaximum hemagglutination. However, the level of activityis the same for cells grown at 37°C on agar or in liquidculture. Furthermore, in contrast to the classical MREhemagglutinins, those we describe here are particularlyresistant to incubation at elevated temperatures or in thepresence of formaldehyde. Since the term type MRE fimbri-ae undoubtedly embraces a widely diverse and heteroge-neous group of structures (witness the obvious dichotomy offimbrial and nonfimbrial forms), such departures from theusual reaction to diagnostic tests (7) are not at all surprising.Indeed, similar characteristics have been described for someof the fimbrial MRE hemagglutinins of Hemophilus sp. (22).

Purified agglutinins of strains 444-3 and 469-3 are globularprotein aggregates with an apparent Mr, estimated by gelfiltration, of between 4 x 106 and 20 x 106. Gel filtration andgel electrophoresis of denatured agglutinins indicated thepresence of single polypeptide subunits, without associatedcarbohydrate, having apparent molecular weights of 14,500for the 444-3 agglutinin and 14,000 for that of 469-3. Thesevalues are near the lower end of the range of subunit sizesobserved among the fimbriae of pathogenic E. coli strains(13), but in terms of the low isoelectric point and markedhydrophobicity of their subunits, the nonfimbrial agglutininsof strain 444-3 and 469-3 are by no means unusual incomparison with fimbrial agglutinins reported in the litera-ture (see reference 18 for review).There is unequivocal evidence that the hemagglutinins

whose purification and characterization are reported hereare required for adherence to cultured cells as a preliminaryto penetration. Mutants defective in hemagglutination thatfail to express surface agglutinins are unable to adhere tocultured cell monolayers. Indeed this intimate correlationbetween agglutination and adherence is confirmed by our

finding of a mutant with reduced activity in, rather than atotal lack of, both processes. An extension of these observa-tions is that expression of hemagglutination by wild-typestrains is temperature dependent, so that strain 469-3 grown

at 22°C, for example, is phenotypically both hemagglutina-tion deficient and nonadhering. Furthermore, purified agglu-tinins competitively inhibit adherence of the producing bac-teria to epithelial cells in culture. The unusual stability of thehemagglutinating activities of strains 444-3 and 469-3 may bedue in part to the ability of agglutinin subunits to renatureinto complexes that, although not necessarily identical withnative aggregates, still exhibit significant hemagglutinatingactivity. It is possible that self-assembly of secreted subunitpolypeptides on the bacterial cell surface is important in vivoin the formation of active agglutinins.

ACKNOWLEDGMENTS

We are very grateful to Maureen Burns for her excellent technicalassistance and to Goff Sargent for his help and advice. We thankJohn Keyte for performing the amino acid analyses and Patrick Hawfor isolating some of the mutants as part of an undergraduateresearch project.

This work was supported by MRC Project grant no. G977/255/S,awarded to P.H.W. and A.S.M.

LITERATURE CITED

1. Bouanchaud, D. H., M. R. Scavizzi, and Y. A. Chabbert. 1969.Elimination by ethidium bromide of antibiotic resistance inenterobacteria and staphylococci, J. Gen. Microbiol. 54:417-425.

2. Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72:248-254.

3. Clark, J. T. 1964. Simplified "disc" (polyacrylamide) gel elec-trophoresis. Ann. N.Y. Acad. Sci. 121:428-436.

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