Vol. 34, No. 3INFECTION AND IMMUNITY, Dec. 1981, p. 980-9860019-9567/81/120980-07$02.00/0

Participation of Pili and Cell Wall Adhesin in the YeastAgglutination Activity of Escherichia coli

YUVAL ESHDAT,1 VOLKER SPETH,2 AND KLAUS JANN2*Department ofBiophysics, The Weizmann Institute of Science, Rehovot, Israel' and Max-Planck-Institut

fir Immunbiologie, 7800 Freibrug-Zahringen, Federal Republic of Germany2

Received 26 February 1981/Accepted 6 August 1981

Escherichia coli strain 2699 (06:K13) which had been isolated from a case ofurinary tract infection exhibited pili during the stationary phase (24 to 40 h), butnot during the exponential phase (4 h), when grown in static broth culture. Thebacteria were also piliated when grown for 24 h on agar. They agglutinatedSaccharomyces cerevisiae (bakers' yeast) in the piliated as well as in the nonpi-liated state. The agglutinations were mannose sensitive, i.e., they could beinhibited with 50 mM methyl-a-mannoside. The bacteria were first depiliated byshearing and then used for the isolation of outer membrane vesicles with anOmnimnixer. Purified pili and outer membranes were characterized by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy. Thepili could be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis only after treatment at low pH or in saturated guanidine hydrochloridewhich is typical of the common type 1 pili. Depiliated bacteria, purified pili, andpurified outer membranes gave mannose-sensitive agglutination of S. cerevisiae.The findings are discussed with respect to possible mechanisms of cell aggluti-nation.

The adherence of bacteria to host tissue is anearly event in host-parasite interactions leadingto bacterial colonization and infection ofthe host(8, 12, 19, 23). It is often paralleled by the abilityof the bacteria to agglutinate erythrocytes oryeast cells (6, 13, 17, 24). Adherence and agglu-tination may or may not be inhibited by a-mannose or a-mannosides and are then termedmannose sensitive or mannose resistant (1, 6,20). It has been shown that adherence and ag-glutination are frequently caused by bacterialpili (1, 6, 11, 22, 26). Mannose-sensitive adher-ence of Escherichia coli is mostly due to com-mon type 1 pili (1, 2, 18, 22), although bacterialflagella have also been implicated in the adhe-sion of E. coli to human buccal cells, mousespleen lymphocytes, and yeast cells (4). We havefound that E. coli 2699, isolated from a case ofurinary tract infection (L. A, Hanson, Goteborg,personal communication), exhibited mannose-sensitive adherence to a number of tissue culturecells, to erythrocytes, and to yeast cells, and thatthe binding occurs not only with piliated butalso with nonpiliated bacteria (11). In this com-munication we show that E. coli 2699 (06:K13)exhibits agglutinins not only in the pili but alsoin the outer membrane. A putative role of theseadhesins and their role in adhesion of the bac-teria are discussed.

MATERLALS AND METHODS

Materials. Tris(hydroxymethyl)aminomethaneand sodium dodecyl sulfate (SDS) were obtained fromRoth, Karlsruhe. Ultrapure guanidine hydrochloride,methyl-a-mannoside, methyl-a-glucoside, and trypsinwere purchased from Serva, Heidelberg, and phenyl-methylsulfonyl fluoride was from Sigma, Munchen.Other chemicals, purchased from commercial sources,were of the highest purity available and were usedwithout further purification.

Bacteria and media. E. coli 2699 (06:K13:H-)was isolated from a case of urinary tract infection andwas obtained from L. A. Hanson, University of Gote-borg. It agglutinated cells ofSaccharomyces cerevisiae(bakers' yeast), guinea pig erythrocytes, and humanerythrocytes independent of ABO blood group. Theagglutinations could be inhibited with methyl-a-man-noside (11). The bacteria were grown either in Luriabroth (containing 1% tryptone, 0.1% yeast extract, and0.5% sodium chloride) or on Loeb agar (containing 1%tryptone, 0.1% yeast extract, 0.1% glucose, 0.8% sodiumchloride, 0.028% calcium chloride, and 1.5% agar).Yeast agglutination assay. Quantitative assays

were done in duplicate in microtiter plates, each wellcontaining 109 cells of S. cerevisiae per ml and dilu-tions of the samples to be tested in a final volume of50 pl of phosphate-buffered saline. Methyl-a-manno-side (50 mM) and methyl-a-glucoside (50 mM) wereused as putative inhibitors (3, 11, 17).

Analytical methods. Polyacrylamide gel electro-phoresis in the presence of SDS was performed in

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slabs by the method of Laemmli and Favre (14) with15% running gels as described previously (10). Samplesof pill were treated with saturated guanidine hydro-chloride before their application to the gel (Y. Eshdat,F. J. Silverblatt, and N. Sharon, submitted for publi-cation). Protein determinations were done by themethod of Markwell et al. (15) with bovine serumalbumin as a standard. The concentrations of bacterialand yeast cell suspensions were determined by count-ing in a Petroff-Hausser chamber.

Electron microscopy. Preparations were stainedwith 0.1% phosphotungstic acid on copper grids coatedwith Formvar and carbon. The samples were examinedin a Phillips EM 400 microscope.

Isolation of pili and membranes. E. coli 2699was harvested from 25 agar plates (14-cm diameter) in30 ml of ice-cold phosphate-buffered saline and cen-trifuged at 7,500 x g for 10 min. Unless otherwisestated, all manipulations were performed at 4°C. Thewashed bacteria (7 g, wet weight) were suspended in35 ml of phosphate-buffered saline containing 850 mMsodium chloride, 100mM methyl-a-mannoside, and 30nM phenylmethylsulfonyl fluoride, and the suspensionwas agitated first with a Vortex mixer (setting 2, 3 x5 min) and then in a sonication bath (room tempera-ture, 5 min). Centrifugation at 8,000 x g for 10 minresulted in a pellet of depiliated bacteria which wasused for the preparation of outer membranes and asupernatant from which the pili were isolated by dif-ferential centrifugation and precipitation with mag-nesium chloride.From the above supernatant residual bacteria were

first removed by two centrifugatons at 6,000 x g (10min each), and the supernatant was then centrifugedat 40,000 x g to remove membrane vesicles. From thesupernatant the pili were obtained either by ultracen-trifugation at 150,000 x g for 2 h or by precipitationwith magnesium chloride (0.1 M final concentration).The pili were suspended in phosphate-buffered salineand purified by a repeated centrifugation at 150,000x g or by repeated precipitations with magnesiumchloride. Both preparations were equally pure, as de-termined by electron microscopy and SDS-polyacryl-amide gel electrophoresis.

For the preparation of outer membranes, the bac-terial pellet obtained after depiliation was subjectedto a second treatment with the Vortex mixer, centri-fuged at 7,500 x g for 10 min, and then washed bycentrifugation of the pellet in 0.5% NaCl (7,500 x g, 10min). The pellet was suspended in 35 ml of 20 mMsodium acetate buffer (pH 4.4) containing 3 nM phen-ylmethylsulfonyl fluoride, and the suspension was ag-itated for 20 min in a Sorvall omnimnixer at position 5.The bacteria were removed by repeated centrifugationat 10,000 x g, and the membranes were then obtainedby centrifugation at 40,000 x g for 2 h.

Proteolytic digestion with trypsin. Bacteria (5x 108/ml), bacterial membranes, or purified pili (1 mgof protein per ml) were suspended in 0.1 M tris-(hydroxymethyl)aminomethane-hydrochloride buffer(pH 8), and trypsin (1 mg/ml in 1 mM hydrochloricacid) was added to a final concentration of 100 ,ug/ml.After 2 h at room temperature, samples were takenfor the yeast agglutination assay and electron micro-

scopic examination. The trypsin incubation of mem-branes in samples taken for SDS-polyacrylamide gelelectrophoresis was terminated by the addition of anequal volume of the SDS disintegration mixture (10)followed by boiling for 5 min. To the trypsin-treatedpili, guanidine hydrochloride (saturation) and mercap-toethanol (final concentration, 4%) were added. Themixtures were then incubated at 37°C for 2 h, dialyzedagainst 5 mM tris(hydroxymethyl)aminomethane-hy-drochoride buffer (pH 8), and added to an equal vol-ume of the SDS disintegration mixture (10).

RESULTSInfluence of growth conditions on the de-

velopment of bacterial pili. When E. coli2699 was grown on agar at 370C, the cells ex-hibited pili, but not flagella, as shown by electronmicroscopy (Fig. 1). The piliation was slightlydenser after 40 h than after 24 h. Inspection ofthe bacteria grown in Luria broth showed thatthey were neither piliated nor flagellated after 4h of growth and that they had acquired pili butnot flagella after 24 h. The appearance of thecells had not changed after 40 h of growth. Whengrown on agar or in liquid culture at 200C, onlyabout 5% of the bacteria were slightly piliated.Influence of growth conditions on yeast-

agglutinating activity. The capacity of E. coli2699 to agglutinate S. cerevisiae was tested aftergrowth of the bacteria under the conditions de-scribed above. E. coli 2699 agglutinated theyeast cells after growth under all conditions. Theagglutinations could be inhibited with 50 mMmethyl-a-mannoside, but not with 50 mMmethyl-a-glucoside. It is noteworthy that unpi-liated bacteria also agglutinated the yeast cells.Thus, bacteria from 4-h cultures in Luria brothat 370C as well as stationary cultures at 200Cwere active. Since after growth at 200C stillabout 5% of the bacteria were piliated, wewanted to know whether activity was due to thefew piliated bacteria in the culture. The adher-ence of E. coli 2699 grown at 20 and 370C to S.cerevisiae was therefore determined. To 109yeast cells per ml 2.4 x 10' bacteria per ml wereadded. The mixture was incubated at room tem-perature for 10 min and then centrifuged at 750x g to remove the yeast cells and their aggre-gates. When the bacteria in the supernatantwere counted, the concentration of the bacteriagrown at 200C was 2.7 x 107/ml. When theexperiment was performed in the presence of 10mM methyl-a-mannoside, all bacteria were re-covered from the supernatant. Methyl-a-gluco-side did not interfere with the binding of thebacteria to S. cerevisiae. The same results wereobtained with the bacteria grown at 370C. Thisshowed that 90% of the bacteria were attachedto the yeast cells, also when only 5% of them

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FIG. 1. Electron micrograph of E. coli 2699 grown on Loeb agar at 37°C for 40 h. Bar, I ,im.

were piliated. Table 1 summarizes the influenceof growth conditions on the agglutinating activ-ity of E. coli. The results show that E. coli 2699,which is normally piliated, can be obtained in anunpiliated state in which it is still active.Isolation of pili and outer membrane ves-

icles. To elucidate the role of pili and cell wallsin the adherence of E. coli 2699 to yeast cells,these bacterial components were isolated as in-dicated in Fig. 2. Agitation of the bacteria in aVortex mixer followed by sonic irradiation in asonication bath removed the pili from the bac-terial cells, as evidenced by electron microscopy.The pili were purified from the supernatanteither by differential centrifugation, where theysedimented at 105,000 x g, or by precipitationwith magnesium chloride (0.1 M final concentra-tion). Both methods yielded preparations of piliwhich appeared to be pure in electron micro-scopic observation (Fig. 3A).

Suspensions of the depiliated bacteria wereagitated at pH 4.4 with an Omnimixer. Thisresulted in partial release of outer membraneswhich were purified by differential centrifuga-tion. After the final centrifugation at 40,000 x gthe membranes appeared as pure vesicles when

TABLE 1. Influence ofgrowth conditions on thefornation of bacterial appendages and on yeast

agglutination activity of E. coli 2699aYeast ag-

Growth me- glutinatingdium Growth conditions Piliation activity

(bacteria/ml)

Loeb agar 37°C, 24 h pil+ 1.2 x 108Loeb agar 37°C, 40 h pil+ 5 x 107Loeb agar 20°C, 24 h (pil) 5 x 107Luria broth 37°C, 4 h pil- 2.1 x 107Luria broth 37°C, 5 x 24 h pil+ 1 x 107

a Activity is expressed as the lowest bacterial con-centration agglutinating 109 yeast cells at room tem-perature. Bacterial appendages were observed by elec-tron microscopy: pil+, all bacteria were densely pi-hated; (pil), only about 5% of the bacteria were pi-liated; pil-, all bacteria were unpiliated.

inspected with the electron microscope (Fig. 3B).Figure 4 shows the SDS-polyacrylamide gel

electrophoresis of pili (Fig. 4B) and outer mem-branes (Fig. 4E). The bands in Fig. 4B couldonly be seen after pretreatment of the pili withsaturated guanidine hydrochloride (Eshdat etal., submitted for publication) which seems to be

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Bacteria

IBacterial pellet

I Agitation with OmnimixerDifferential centrifugation

Outer membrane vesicles

Agitation with Vortex and sonicationbath

Centrifugation

.1ISupernatant

Differential centrifugation orprecipitation with magne-sium chloride

Bacterial pili

FIG. 2. Isolation of bacterial pili and outer membrane vesicles from E. coli 2699.

characteristic for type 1 pili (unpublished obser-vations). It has been reported that untreatedcommon type 1 pili cannot be demonstrated bySDS-polyacrylamide gel electrophoresis (16).The outer membrane preparation obtained bymixing the depiliated bacteria in an Omnimixerexhibited the same pattern as outer membranesisolated by the method of Uemura and Mizush-ima (25) from urinary tract-infective E. coli 06:K13 (10). When the membrane preparationswere saturated with guanidine hydrochloride, noadditional bands due to pili could be seen onsubsequent SDS-polyacrylamide gel electropho-resis.Yeast agglutinating activity ofdepilated

bacteria, pili, and outer membranes. Thedepiliated bacteria, obtained by agitation with aVortex mixer and low-energy sonication, wereused in the yeast agglutination test. As shown inTable 2, they had about the same activity as thepiliated bacteria. Outer membranes and pili,which were both pure preparations as shown byelectron microscopy (Fig. 3) and SDS-polyacryl-amide gel electrophoresis (Fig. 4), also agglutin-ated the yeast cells. The respective minimalagglutinating concentrations are included in Ta-ble 2. The agglutinating activity of all prepara-tions was completely inhibited by 50 mMmethyl-a-mannoside. Treatment of bacteria,pili, and outer membranes with trypsin abol-ished the agglutinating ability of the prepara-tions. The SDS-polyacrylamide gel electropho-resis pattern of pili was unchanged when theaction of trypsin was stopped before deaggrega-tion of the pili into their subunits (Fig. 4C, ascompared with Fig. 4B). When the action oftrypsin was allowed to proceed with the pilussubunits, complete degradation was observed(Fig. 4D). Trypsinization of outer membranesresulted in cleavage of the 33K protein and inthe appearance of a smaller split product (Fig.4F). Trypsinization of the yeast cells before theagglutination test as well as the presence oftrypsin during the agglutination test did notinterfere with the agglutinating capacity of thepreparations.

DISCUSSION

E. coli 2699, which had been isolated from acase of urinary tract infection, agglutinated cellsof S. cerevisiae. The bacteria also agglutinatedguinea pig and human erythrocytes and adheredto a number of tissue culture cells (11). All ofthese cell interactions could be inhibited bymethyl-a-mannoside.

Since mannose-sensitive agglutination ofyeast cells (1, 13, 21) and guinea pig erythrocytesis typically exhibited by bacteria with commontype 1 pili, E. coli 2699 was subjected to electronmicroscopic examination. It was found that thebacteria which did not exhibit pili after 4 h ofgrowth in Luria broth at 370C were denselypiliated after prolonged growth in liquid cultureor on agar at 37°C. Growth at 20°C largelyprevented the formation of pili. The bacteriahad a smooth surface; structures indicating in-complete pili could not be seen. However, E. coli2699 agglutinated yeast cells whether or not thebacteria were piliated. This raised the questionwhether or not the pili played a role in aggluti-nation by or adherence of E. coli 2699. Pureisolated pili were also capable of agglutinatingyeast cells; this agglutination could be inhibitedby methyl-a-mannoside. The pili could be dem-onstrated by SDS-polyacrylamide gel electro-phoresis only after subjection to treatments orig-inally developed for common type 1 pili, viz.,treatment at low pH (16) or with saturated guan-idine hydrochloride (Eshdat et al., submitted forpublication). SDS-polyacrylamide gel electro-phoresis of the pili revealed a major and a minorpeptide band of about 17K. The same patternwas obtained with common type 1 pili (data notshown). Therefore, 2699 pili resemble commontype 1 pili with respect to the tightness of sub-unit assembly and the SDS-gel pattern of theirsubunits.

It was found that outer membranes isolatedfrom depiliated E. coli 2699 agglutinated cells ofS. cerevisiae, and that this agglutination couldbe inhibited with methyl-a-mannoside. This isin accord with the fact that unpiliated bacteria

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0

xI-

(DLu

.3r

-J

LL

0

604336

29

20 -

7.8-

14.3_ _

TD

A B C D E F

FIG. 3. Electron micrographs of purified pili (A)and purified outer membranes (B) of E. coli 2699.Bar, 0.2 ,im.

also agglutinated the yeast cells in a mannose-

sensitive way-presumably via components ofthe outer membrane. Electron microscopic andgel electrophoretic examinations did not revealpili in the outer membrane preparation. Short,rigid structures which could be interpreted as

residues of pili broken during depiliation byshearing were also absent from the membranes.This and the lack of incomplete stubs of pili inunpiliated E. coli 2699 raised the possibility thatthe cell wall agglutinins are pilus monomers

inserted into the outer membrane without po-

lymerization. Alternatively, the cell wall agglu-tinin could be a protein different from pilinwhich contains the same mannose recognitionsite as pilin. SDS-polyacrylamide gel electropho-

FIG. 4. SDS-gel electrophoresis in 15% polyacryl-amide separation gel of molecular weight markers(A), purified pili (B), purified pili incubated withtrypsin followed by the addition ofguanidine hydro-chloride and mercaptoethanol (C), purified pili afterthe incubation with trypsin without subsequent ad-dition of mercaptoethanol (D), purified outer mem-branes (E), and purified outer membranes after theincubation with trypsin (F). The molecular weightmarkers were lysozyme (14,300), sperm whale myo-globulin (17,800), soybean trypsin inhibitor (20,000),carbonic anhydrase (29,000), glyceraldehyde-3-phos-phate dehydrogenase (36,000), ovalbumin (43,000),and beef liver catalase (60,000). Tracking dye (TD)was bromophenol blue. The gel was stained withCoomassie brilliant blue R250.

TABLE 2. Agglutinating activities ofpiliatedbacteria, depiliated bacteria, pili, and outermembranes of E. coli 2699 in the absence and

presence of methyl-a-mannoside (aMM) and aftertrypsinizationa

Minimal agglutinating concn(per ml)

PurifiedPrepn Piliated Depi- Purified outer

E. coli liated E. pili (tug mem-2699 coli 2699 of pro- branes(cells) (cells) tein) (jAg of

protein)

With aMM >1 x 1010 >1 x 1010 >500 >500Without aMM 2 x 107 5 X 107 100 25After >1 x 1010 >1 x 1010 >500 >500

trypsinization

aSerial dilutions of the preparations were incubated with109 yeast cells per ml in microtiter plates.

resis under the conditions which we have usedcannot differentiate between these possibilities.The band seen in the outer membrane pattern

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at about 17K may be due to piln monomers;

however, it could be another, unrelated proteinsuch as protein III (7, 9).The agglutinating activity of E. coli 2699 was

lost after incubation of the bacteria with trypsin.Trypsinization also abolished the agglutinatingactivity of purified pili and outer membranes.The SDS-polyacrylamide gel electrophoreticpattern of trypsin-treated pili was the same as

that of untrypsinized pili. When the pili were

exposed to the action of trypsin under conditionswhich disaggregated the pili into subunits, com-

plete enzymatic digestion occurred. One possibleexplanation for these findings is that enzymaticdigestion occurred only at the terminal pilussubunit (tip), and the internal subunits were

completely protected from the action of trypsinby their association. Only their disaggregationrendered them vulnerable to proteolysis. Sincetrypsin treatment of the complete pili abolishedtheir agglutination activity, such an interpreta-tion leads to the assumption that the strain 2699pili have exposed mannose-binding sites only atthe tip and that they are monovalent. Alterna-tively, although pilus subunits are protectedfrom complete enzymatic digestion, each subunitmay have an exposed mannose-binding sitewhich is trypsin sensitive. In this case the piliwould be polyvalent, and the destruction of theirbinding sites must have occurred by a shorteningof the primary peptide chain too insignificant tobe observed in SDS-polyacrylamide gel electro-phoresis. When outer membranes were analyzedby SDS-polyacrylamide gel electrophoresis afterincubation with trypsin, a major outer mem-

brane protein had disappeared and a new

smaller protein band was observed. It had beenshown by Haller and Henning (9) that proteinII*, one of the major outer membrane proteinsof E. coli K-12, is split by trypsin with theformation of protein II. Both proteins II* and IIhave the same electrophoretic mobility in SDS-gel as the proteins of E. coli 2699 before andafter trypsin treatment of the outer membranes.Since protein II* is also a major outer membraneprotein in E. coli strains which do not aggluti-nate yeast cells, the effect of trypsin observed bySDS-polyacrylamide gel electrophoresis is prob-ably unrelated to its effect on the cell wall agglu-tinin, especially since many outer membraneproteins seem to be trypsin sensitive (9).Our results show that E. coli 2699 possesses

agglutinins (adhesins) both in the pili and in theouter surface of the cell wall, and that bothexhibit the same specificity for attachment. Thismay indicate the operation of a two-stage adhe-sion ofE. coli 2699 to target cells, a long-distanceinteraction via pili and a short-distance interac-tion via the cell wall causing final adherence.

Such an interpretation would favor the assump-tion of monovalent pili with the mannose-bind-ing site only in the tip. There is of course thepossibility that the mannose-binding protein inthe cell wall has no function in binding, which isstabilized by multiple attachment of polyvalentpili to the target cell. In the latter case the cellwall adhesin would represent a portion of pilussubunits which had not or not yet assembledinto pili. To study this phenomenon further weare attempting to isolate mutants which lackone or the other of the agglutinins. This will notonly enable us to analyze the phenomenon ofbacterial adherence further, but it may also behelpful in understanding the assembly of bacte-rial pili.

LITERATURE CIMD1. Duguid, J. P., and R. R. Gillies. 1957. Fimbriae and

adhesive properties in bacilli. J. Pathol. Microbiol. 74:397-411.

2. Duguid, J. P., L. W. Smith, G. Dempster, and P. N.Edmunds. 1955. Non flagellar filamentous appendages("fimbriae") and hemagglutinating activity in Bacte-rium coli. J. Pathol. Bacteriol. 70:3335-3348.

3. Eshdat, Y., L. 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 epithellal cells. Biochem. Bio-phys. Res. Commun. 85:1551-1559.

4. Eshdat, Y., N. Sharon, I. Ofek, and D. Mirelman. 1980.Structural association of the outer surface mannose-specific lectin ofEscherichia coli with bacterial flagella.Israel J. Med. Sci. 16:479.

5. Evans, D. G., D. J. Evans, S. Clegg, and J. Panley.1979. Purification and characterization of the CFA/Iantigen of enterotoxigenic Escherichia coli. Infect. Im-mun. 25:738-748.

6. Evans, D. J., D. G. Evans, and K. Z. Dupont. 1979.Hemagglutination patterns of enterotoxigenic and en-teropathogenic Escherichia coli determined with hu-man bovine chicken and guinea pig erythrocytes in thepresence and absence of mannose. Infect. Immun. 23:336-346.

7. Garten, W., I. Hindenach, and U. Henning. 1975. Themajor proteins of Escherichia coli outer cell envelopemembranes. Characterization of proteins II* and III,comparison of all proteins. Eur. J. Biochem. 59:215-221.

8. Gibbons, R. J. 1973. Bacterial adherence in infection andimmunity. Rev. Microbiol. 4:48-60.

9. Haller, I., and U. Henning. 1974. Cell envelope andshape of Escherichia coli K-12. Cross linking withdimethyl imidoesters of the whole cell wall. Proc. Natl.Acad. Sci. U.S.A. 71:2018-2021.

10. Jann, B., and K. Jann. 1980. SDS polyacrylamide gelelectrophoresis pattern of the outer membrane proteinsfrom E. coli strains of different pathogenic origin.FEMS Microbiol. Lett. 7:19-22.

11. Jann, K., G. Schmidt, E. Blumenstock, and K. Vos-beck. 1981. Adhesion ofEscherichia coli to mammalianand yeast cells. The role of piliation and surface hydro-phobicity. Infect. Immun. 32:484-489.

12. Jones, G. W. 1977. The attachment of bacteria to thesurface of animal cells, p. 139-176. In J. Reissig (ed.),Microbial interactions. Chapman and Hall, London.

13. Korhonen, T. K. 1979. Yeast agglutination of purifiedenterobacterial pili. FEMS Microbiol. Lett. 6:421-425.

14. Laemmli, U. K., and M. Favre. 1973. Maturation of the

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head of bacteriophage T4. J. Mol. Biol. 80:575-599.15. Markwell, S. M. H., L. L. Bieber, and N. Z. Tolbert.

1978. A modification of the Lowry procedure to simplifyprotein determination in membrane and lipoproteinsamples. Anal. Biochem. 87:206-210.

16. McMichael, J. C., and T. J. Ou. 1979. Structure ofcommon pili from Escherichia coli. J. Bacteriol. 138:969-975.

17. Mirelman, D., G. Altmann, and Y. Eshdat. 1980.Screening of bacterial isolates for mannose-specific lec-tin activity by agglutination of yeasts. J. Clin. Microbiol.11:328-331.

18. Nagy, B., H. W. Moon, R. E. Isaacson, C. C. To, andC. C. Brinton. 1978. Immunization of suckling pigsagainst enteric enterotoxigenic Escherichia coli infec-tion by vaccinating dams with purified pili. Infect. Im-mun. 21:269-274.

19. Ofek, I., and E. H. Beachey. 1980. General concepts andprinciples of the adherence of bacteria to animal cells,p. 1-27. In E. H. Beachey (ed.), Bacterial adherence:receptors and recognition, vol. B6. Chapman and Hall,London.

20. Ofek, I., D. Mirelman, and N. Sharon. 1977. Adherence

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21. Old, D. C. 1972. Inhibition of the interaction betweenfimbrial hemagglutinins and erythrocytes by D-man-nose and other carbohydrates. J. Gen. Microbiol. 71:149-157.

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24. 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.

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