INFECTION AND IMMUNIT-Y, Apr. 1992, p. 1577-15880019-9567/92/041577-12$02.00/0Copyright © 1992, American Society for Microbiology

Adherence to Respiratory Epithelia by Recombinant Escherichia coliExpressing Klebsiella pneumoniae Type 3 Fimbrial Gene Products

DOUGLAS B. HORNICK,* BRADLEY L. ALLEN, MARK A. HORN, AND STEVEN CLEGG

Departments of Internal Medicine and Microbiology, University ofIowa College of Medicine, Iowa City, Iowa 52242

Received 25 October 1991/Accepted 30 January 1992

We examined the role of Klebsiella fimbrial types 1 and 3 in mediating adherence to human buccal andtracheal cells and to lung tissue sections. We found that clinical isolates ofKlebsiella pneumoniae producing type3 fimbriae and Escherichia coli HB101 containing a recombinant plasmid encoding expression ofKkebsiella type3 fimbriae (pFKIO) demonstrated increased adherence to tracheal cells, trypsinized buccal cells, and lung tissuesections, in contrast to nonfimbriate and to type 1 fimbriate bacteria. Adherence by type 3 fimbriate bacteriawas inhibited by purified type 3 fimbriae and Fab fragments derived from type 3 fimbrial-specific polyclonalimmunoglobulin G. Type 3 fimbriae mediated attachment to the basolateral surface of tracheal cells and to thebasal epithelial cells and the basement membrane regions of bronchial epithelia. Using an E. coli transformant(pDC17/pFK52), which expresses nonadherent P fimbrial filaments, along with the type 3 fimbrial adhesin(MrkD), we demonstrated that type 3 fimbrial attachment to respiratory cells was attributable to the MrkDadhesin subunit. Subsequent experiments demonstrated that the epithelial target of the type 3 fimbrial adhesinwas most likely a peptide molecule rather than a carbohydrate. The results of this study demonstrate that, invitro, the KlebsieUla type 3 fimbrial adhesin mediates adherence to human respiratory tissue.

Members of the family Enterobacteriaceae account for atleast one-third of reported nosocomial pneumonias, accord-ing to data accumulated as part of the National NosocomialInfection Survey (14). Gram-negative bacterial colonizationoften precedes nosocomial pneumonia (3, 13, 16). To colo-nize the respiratory tract, these bacteria must overcomemucociliary clearance and adhere to the epithelium. Mem-bers of Kiebsiella, the genus of Enterobacteriaceae fre-quently causing nosocomial pneumonia, commonly producetype 1 and/or type 3 fimbriae. Fimbriae are filamentousorganelles which may abrogate mechanical host clearancemechanisms and facilitate attachment to epithelial receptorsby fimbria-associated adhesins. This study was designed toexamine the ability of the Kiebsiella type 1 and type 3fimbriae to mediate adherence to human respiratory epithe-lia.Most species of the Enterobacteriaceae can produce type

1 fimbriae. Organisms expressing this fimbrial type exhibitmannose-sensitive hemagglutination (MS HA) in vitro, andthe receptors for the type 1 fimbrial adhesin are believed tobe mannose-containing glycoproteins. Type 3 fimbriae be-long within the heterogeneous group of fimbrial typesbroadly classified as mannose-resistant (MR) hemaggluti-nins. Organisms expressing type 3 fimbriae demonstrate MRagglutination of erythrocyte suspensions only after theerythrocytes have been pretreated with 0.01% tannic acid.Klebsiella organisms commonly exhibit this phenomenon(27) and, therefore, the type 3 fimbria-mediated HA isreferred to as MR Klebsiella-like HA (MR/K HA). In con-

trast to the type 1 fimbriae, type 3 fimbriae are not expressedby Escherichia coli but are frequently produced by speciesof Enterobacter, Proteus, Providencia, Morganella, Yer-sinia, and Serratia (2). Recent investigations have shownthat type 3 fimbrial adherence is mediated by the MrkDpolypeptide (12), that type 3 fimbrial adherence is inhibitedby cationic compounds such as spermidine (9), and that renal

* Corresponding author.

tubular basement membrane and, specifically, type V colla-gen may be targets for attachment by the type 3 fimbrialadhesin (32).

In this study, we examined the ability of Klebsiella fim-brial types to attach in vitro to buccal and tracheal cells andto lung tissue derived from normal human volunteers. Theresults demonstrated that (i) increased adherence was asso-ciated with type 3 fimbriae, in contrast to type 1 fimbrialexpression by clinical strains of Klebsiella pneumoniae andE. coli HB101 containing recombinant plasmids encoding theexpression of Klebsiella fimbriae, (ii) attachment was local-ized to cryptic sites on epithelial cells or sites normally notexposed on intact buccal or airway epithelium, (iii) adher-ence by type 3 fimbriae was attributable to the MrkD adhesinsubunit, and (iv) the epithelial target for the type 3 adhesinwas most likely a peptide rather than a carbohydrate.

MATERIALS AND METHODS

Bacterial strains and plasmids. Clinical isolates, K pneu-moniae UIR040 and IA565, were obtained from the Univer-sity of Iowa Hospitals and Clinics Clinical MicrobiologyLaboratory. K pneumoniae UIRO40 was isolated from thetracheal aspirate of a patient with nosocomial pneumonia,and K pneumoniae LA565 had been previously used as a

source of DNA to clone bothK pneumoniae type 1 and type3 fimbrial gene clusters (8, 10). The construction of thefollowing recombinant plasmids used to transform E. coliHB101 has been described elsewhere: pFK10 encodes Kpneumoniae type 3 fimbriae (8), pGG101 encodes K pneu-moniae type 1 fimbriae (10), and pDC1 encodes an E. coliP fimbria (1) (Fig. 1). The type 3 and type 1 fimbrial geneclusters were cloned into medium-copy-number vectorspACYC184 and pBR322, respectively. In addition, plasmidpFK25 is a deletion derivative of the cloned type 3 fimbrial(mrk) gene cluster (Fig. 1), and E. coli(pFK25) transformantsare nonfimbriate. Plasmid pFK52 contains only the mrkD(adhesin) gene and E. coli(pFK52) is also nonfimbriate, andbecause of the lack of accessory fimbrial genes, this trans-

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formant does not hemagglutinate. Plasmid pDC17 is apapGdeletion derivative of pDC1 (Fig. 1), and E. coli(pDC17)therefore expresses nonadhesive P fimbriae (Fll serotype).The double transformant E. co1i(pDC17/pFK52) expresses Pfimbrial filaments associated with the type 3-specific ad-hesin, which is detectable by MR/K HA (12). Organismscontaining recombinant plasmids were maintained on Luria(L) agar supplemented with the appropriate antibiotics (20).Prior to use in the binding assays, bacteria were grown for 20h at 37°C on minimal medium agar plates supplemented with3% glycerol and 0.1% Casamino Acids (G-CAA), which havebeen previously shown to optimize expression of type 3fimbriae (8). The phenotypic expression of appropriate fim-brial types was confirmed by HA and reactivity with fimbria-specific sera.

Bacterial labeling. Bacteria were harvested, after over-night culture, into 0.2% glucose-M-9 minimal salts solutionand were labeled metabolically with [35S]methionine (Amer-sham, Chicago, Ill.) by standard techniques (23). For theassays with frozen tissue sections, bacterial suspensionswere labeled with fluorescein isothiocyanate according tothe method of Nowicki et al. (26).

Purification of type 3 fimbriae and fimbrial antiserum.Cell-free fimbrial appendages were purified from E.coli(pFK10) as previously described by Gerlach and Clegg(11). Fimbrial antiserum against purified type 3 fimbriae wasraised in rabbits. Immunoglobulin G (IgG) was isolated fromhyperimmune serum by protein G column chromatographyand subsequently digested with solid-phase papain accordingto the manufacturer's protocol (Pierce, Chicago, Ill.). TheFab fragments were purified by multiple elutions over aprotein A column (ChromatoChem, Missoula, Mont.). Thepurity of the resulting material was confirmed by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis. TheA280 for this material was measured, and the Fab concentra-tion was derived from the appropriate extinction coefficient.The purified Fab fragments inhibited MR/K HA when type 3fimbriate bacteria were preincubated with the Fab fractionfor 15 min at 25°C.

Epithelial cell procurement. Buccal and tracheal cells wereobtained from nonsmoking normal adult human volunteers.

(i) Tracheal epithelial cell isolation. Tracheal cells wereobtained from normal volunteers by bronchoscopicallyguided tracheal brushing (3-mm bronchial brush; Bard, Bil-lerica, Mass.) according to the methods of Niederman et al.(25). Cell counts were performed with a hemocytometer, andmicroscopic examination for viability was checked by trypanblue exclusion. The cells were used immediately for bacte-rial attachment assays.

(ii) Buccal epithelial cell isolation. Buccal cells were ob-tained from laboratory personnel with low levels of indige-nous flora, via gentle curettage (sterile cotton-tipped appli-cator) of buccal mucous membranes. Cells were washedthree times in phosphate-buffered saline (PBS) at pH 7.4.Cell counts and assessment of viability were performed asdescribed above.Human lung tissue. Normal human lung tissue was ob-

tained from uninvolved areas of surgical biopsy materialprovided by the University of Iowa Hospitals and ClinicsSurgical Pathology Laboratory. Tissue was snap-frozen inliquid nitrogen and stored at -70°C. Thin frozen sections (5to 6 ,um) were cut at -20°C with a Reichert-Jung 2800Frigocut Cryostat and placed on glass slides which had beentreated with siliconizing solution (1% dimethyldichlorosilanein carbon tetrachloride) and stored at -70°C until used.Simple hematoxylin-eosin staining confirmed the presence ofbronchial epithelium and underlying histologic structureswithin the sections.Whole bacterium-epithelial cell adherence assay. The ad-

herence assays were adapted from previous studies by otherinvestigators (7, 25, 33) and differed minimally when buccalinstead of tracheal cells were used. The major exception wasthat the buccal cells were, in some experiments, pretreatedwith trypsin. Preliminary results showed that maximal ad-herence was obtained when buccal cells were pretreatedwith trypsin by established techniques (36), in which 2.5 ,ugof trypsin (bovine pancrease; Sigma) per 105 buccal cells wasused and cells were incubated at 37°C for 30 min. After

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ADHERENCE BY KLEBSIELLA TYPE 3 FIMBRIAE 1579

pretreatment in all cases, buccal cells were washed in PBS,recounted, and checked for viability by trypan blue exclu-sion prior to use in the bacterial adherence assays.

(i) Radiolabeled-bacterium adherence assay. Washed epi-thelial cells (0.5 ml) and 35S-labeled bacteria (2 x 108/ml)were combined in a bacteria-to-cell ratio of 1,000:1. Thisratio was found to be optimum in preliminary studies usingboth tracheal and buccal cells. All adherence assays weredone in triplicate. Cells and bacteria were incubated for 90min at 37°C with gentle continuous end-over-end agitation.Cells with adherent bacteria were collected on 22-mm-diameter (8-,um pore size for buccal cells or 3-,um pore sizefor tracheal cells) polycarbonate filters (Nucleopore), whichhad been pretreated with 3% bovine serum albumin (BSA) inPBS. Nonadherent bacteria were washed through the filterwith 50 volumes of PBS. The filtered cells with adherentbacteria were then solubilized (1 N NaOH, 16 h, 37°C), andthe radiolabel was counted in a liquid scintillation counter.The number of adherent bacteria per cell was calculated asdescribed previously by Old et al. (27). Initially, bacterialcounts were confirmed by quantitative cultures of one filterfrom each triplicate.

(ii) Unlabeled-bacterium adherence assay. The unlabeled-bacterium adherence assay was performed simultaneouslywith, and identically to, the radiolabeling assay, with theexception that cells with adherent bacteria were Gramstained and permanently mounted on a glass microscopicslide. The slides were examined by using bright-field micros-copy by personnel who were unaware of the fimbrial typebeing tested. Adherence was recorded as the mean numberof bacteria per epithelial cell (in the tracheal cell assay, onlythe ciliated cells were counted) after the examination of 20consecutive cells in the central section and four peripheralsections of the filter (total cells counted per filter = 100).Results were expressed as the number of adherent bacteriaper cell. Binding distribution per cell was also noted. Whenthe binding distribution was significant, we confirmed theresults by examining cells, prior to filtering, with phase-contrast microscopy to rule out artifacts that may have beenintroduced by staining and mounting filters.

Tissue section adherence assay. The adherence assay withhuman lung tissue sections was carried out according to themethod of Virkola (34). Briefly, snap-frozen tissue sectionsmounted on glass slides were allowed to come to roomtemperature, immediately prior to the assay, in a humiditychamber. The sections were then fixed with fresh 3.5%(wt/vol) paraformaldehyde for 10 min at ambient tempera-ture and washed three times in PBS. The sections were thenoverlaid with a PBS-fluorescein isothiocyanate-labeled bac-terial suspension (50 ,ul of 108 to 109 organisms per ml) andincubated at 4°C for 45 min in a humidity chamber. Nonad-herent and loosely adherent bacteria were then removed bythree 5-min washes with PBS-0.05% Tween 20 (vol/vol),with shaking. Slides were allowed to dry before the additionof glycerol mounting solution and the glass coverslip. Slideswere observed by light microscopy, using a reflected-fluo-rescence attachment.

(i) Inhibition of adherence by Fab fragments of anti-type 3IgG and purified fimbriae. Inhibition of adherence wasexamined by addition of a 1:10 or 1:40 dilution of the Fabsolution to the bacterial suspensions. These suspensionswere incubated for 15 min at 25°C, with shaking, and washedonce with an equal volume of PBS prior to incubation withthe tissue sections. Equivalent concentrations of IgG puri-fied from normal rabbit serum were used as controls.

In some experiments, immediately prior to incubation

with the bacterial suspension, tissue sections were preincu-bated with purified type 3 fimbriae (100 or 300 ,ug/ml) or acontrol protein (BSA) at a similar concentration. Slides weresubsequently washed three times for 5 min each with PBS-0.05% Tween 20.

(ii) Collagenase, acetic acid, and sodium metaperiodatepretreatment of tissue sections. Tissue sections were overlaidwith 50 ,lI of chromatographically purified collagenase de-rived from Clostridium histolyticum (4 U/ml; WorthingtonBiochemicals, Freehold, N.J.) and incubated at 37°C for 5 hin either the presence or absence of inhibitor (10 mMEDTA). Sections were washed three times for 1 min each inPBS and fixed with paraformaldehyde before incubationwith bacteria.

In separate experiments, by using the methods describedpreviously (21), the slides were overlaid with a 0.1 Msolution of acetic acid for 30 min at 25°C. Control slides wereincubated with the acetic acid along with 10 mM EDTA and2 ,ug of pepstatin A per ml (Sigma, St. Louis, Mo.). Slideswere subsequently washed three times in PBS and fixed withparaformaldehyde prior to the bacterial overlay.To attenuate potential carbohydrate receptors, tissue sec-

tions were exposed to a suspension of 100 mM sodiummeta-periodate (Sigma) for 30 min at 37°C and washed threetimes for 5 min each in PBS before the adherence assay wasconducted. The control for these experiments used P fimbri-ate E. coli(pDC1), which adhere well to respiratory cells andthe adherence of which is dependent on a galabiose gly-colipid receptor (14a).

Statistical analysis. Arithmetic means and standard devia-tions (except where indicated as the standard error of themean) were calculated for the quantitative adherence assayswith buccal and tracheal cells. Results were analyzed byusing the Student's t test for independent variables, and theP values reported are for two-tailed tests.

RESULTS

Fimbria-mediated adherence to respiratory epithelia wasdetermined by utilizing buccal cells, tracheal cells, andfrozen lung tissue sections obtained from normal humansubjects.

Buccal and tracheal cell adherence assay. The mean ±standard error of the mean number of buccal cells obtainedfrom laboratory personnel was 1.0 x 106 + 0.1 x 106 cells.Only 10% or fewer of the cells were viable, as determined bytrypan blue exclusion. The mean ± standard error of themean number of tracheal cells obtained at bronchoscopy was3.1 x 106 ± 1.2 x 106. The majority (64.7% ± 2.7%) oftracheal cells were ciliate, and 47.6% ± 9.4% were observedto be viable by trypan blue exclusion. In initial studies, wefound that by the end of the adherence assay, viability of thetracheal cells had decreased further and that bacteria wereadhering in equal numbers to both the nonviable and viablecells.

(i) Organisms expressing Kiebsiella fimbriae. Adherence byfimbriate strains of K pneumoniae to epithelial cells isshown in Table 1. K pneumoniae IA565, when cultured onL agar, expresses both MS and MR/K HA. K pneumoniaeUIR040, however, produces a very weak MR/K HA activityand does not cause MS HA when subcultured on L agar.Consistent with previous observations, both isolates demon-strate increased MR/K HA when subcultured on G-CAAagar (8). When the organisms were tested for adherence toboth human tracheal cells and trypsinized buccal cells, theorganisms demonstrating the greatest MR/K HA, associated

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TABLE 1. Adherence to buccal and tracheal cells by clinical strains ofK pneumoniae expressing type 3 fimbriae

Mean no. of bacteria/cell ± SDK MR/K

pneumoniae Passage HA Buccal cells' Trachea cellsstrain medium titee

Radiolabeling assay nc Microscopic assay n Radiolabeling assay n Microscopic assay n

IA565 L 6 4.37 ± 0.29 4 4.86 ± 1.24 5 2.01 ± 0.81 5 0.52 ± 0.37 6G-CAA 19 10.89 ± 2.39 (P = 0.003)" 4 14.42 ± 0.74 (P < 0.001) 5 7.96 ± 1.97 (P < 0.001) 5 2.46 - 0.96 (P =0.001) 6

UIRO40 L 3 1.94 ± 0.42 6 1.34 ± 1.11 6 1.17 ± 0.72 5 0.47 ± 0.39 6G-CAA 22 10.51 ± 2.82 (P < 0.001) 6 11.96 ± 5.13 (P < 0.001) 6 10.02 + 2.81 (P = 0.001) 5 3.96 ± 1.59 (P < 0.001) 6

a Expressed as the mean of the reciprocal of the highest dilution mediating MR/K HA.b Adherence values for trypsin-pretreated buccal cells.c n is the number of experiments with different samples of epithelial cells.d P values for comparison to the same strain cultured on L agar.

with type 3 fimbriae, attached to the epithelial cells insignificantly greater numbers than did nonfimbriate or type 1fimbriate bacteria (Table 1).

Table 2 shows the adherence to epithelial cells by E. coliHB101 transformed with recombinant plasmids encodingKlebsiella fimbriae. In these experiments, the type 3 fimbri-ate transformant, E. coli(pFK10), demonstrated significantlygreater adherence than the other organisms tested. Pheno-typically nonfimbriate E. coli HB101 and E. coli(pFK25)demonstrated no significant adherence.The transformant producing type 1 fimbriae, E. coli

(pGG101), adhered in low numbers to human tracheal cellsbut did not adhere significantly to trypsinized buccal cells.Also, the microscopic assay confirmed all observationsmade for the radiolabeled-bacterium adherence assay, ex-

cept that low-level adherence to tracheal cells by E. coli(pGG101) was not observed.

(ii) Effect of trypsin on adherence to buccal cells. The datashown in Table 3, derived from the radiolabeled-bacteriumadherence assay (confirmed by the unlabeled-bacterium ad-herence assay [data not shown]), demonstrate that pretreat-ment of the buccal cells with trypsin resulted in significantlyenhanced binding by the type 3 fimbriate organisms. Thegreatest enhancement was demonstrated by organisms pro-ducing relatively large amounts of the type 3 fimbriae (i.e.,those grown on the G-CAA medium or the E. coli recombi-nant). Also, no significant enhancement of adherence wasnoted by the type 1 fimbriate E. coli(pGG101) and thenonfimbriate E. coli HB101 or E. coli(pFK25).

(iii) Inhibition assays. Preincubation of the type 3 fimbriaterecombinant strain, E. coli(pFK10), with a 1:25 dilution ofFab fragments of anti-type 3 IgG resulted in markedlyreduced adherence to both the normal tracheal cells and totrypsinized buccal cells (Fig. 2). The Fab fragments isolated

from the anti-type 3 fimbrial serum also inhibited the MR/Khemagglutinating activity of bacteria.

Consistent with previous observations demonstrating thatspermine and spermidine inhibit MR/K HA (9), E. coli(pFK10) attachment to both normal human tracheal cells andtrypsinized buccal cells was inhibited by addition of sper-mine or spermidine to the assay mixture (Table 4). Com-pounds that showed no inhibition, within a similar molarconcentration range, included a-methylmannoside (50 and150 mM), glucose (50 and 150 mM), and D-lactose (73 and146 mM).

Interestingly, P,L-lysine (145 mM) and, particularly, thepolycations poly-L-lysine (0.25% [wt/vol]) and protamine(0.25% [wt/vol]) resulted in enhancement of adherence bythe type 3 fimbriate E. coli(pFK10) (Table 4). However,nonfimbriate and type 1 fimbriate organisms also adhered inlarge numbers (means of 24 and 19 bacteria per cell, respec-tively) when the epithelial cells were pretreated with prota-mine. Similar data were obtained by using poly-L-lysine. Wealso observed epithelial cell clumping, which was not com-pletely reversible when cells were washed, suggesting thatthese adhesive compounds likely coat or, possibly, evenalter the epithelial cell surfaces. Thus, the increase inadherence observed when the epithelial cells were pre-treated with poly-L-lysine and protamine may be explained,in part, by the adhesive characteristics of these compounds.

(iv) Role of the MrkD polypeptide in type 3 fimbrial-mediated adherence. The results presented in Table 5 indi-cate that the double transformant, E. coli(pDC17/pFK52),which produces the type 3 fimbrial adhesin (mrkD geneproduct) with the P fimbrial filament, adheres to humantracheal cells and trypsinized buccal cells in significantlygreater numbers than E. coli(pDC17), which produces onlythe nonhemagglutinating P fimbrial phenotype (Fll sero-

TABLE 2. Adherence to human buccal and tracheal cells by recombinant E. coli HB101 expressing K pneumoniae fimbriaea

Mean no. of bacteria/cell + SD

Plasmid Type of Buccal cellsb Trachea cellsfimbriae

Radiolabeling Microscopic Radiolabeling Microscopicassay assay assay assay

(pFK25)c None 2.22 ± 0.99 1.11 ± 1.16 1.72 + 1.49 0.30 ± 0.25pGG101 Type 1 1.75 + 0.72 1.80 ± 0.76 3.44 1.15d 0.32 ± 0.30pFK10 Type 3 25.93 ± 10.06e 22.58 ± 12.04e 16.84 ± 7.87e 8.23 ± 3.14e

a All adherence assays were performed at least ten times with different samples of epithelial cells.b Adherence values are for trypsin-pretreated buccal cells.c Both E. coli HB101 and E. coli HB101(pFK25) were nonfimbriate, and adherence values for both strains were similar.d Value differed significantly from the corresponding value for E. coli HB101 (P = 0.016).Value differed significantly from the corresponding values for E. coli(pGG101) and the nonfimbriate control (P < 0.001).

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TABLE 3. Effect of trypsin pretreatment of buccal cells on adherence by bacteria expressing Klebsiella fimbriaea

Mean no. of bacteria/cell ± SDBacterial strain or plasmid Passage Type of

medium fimbriae Nontrypsinized Trypsinized increase

E. coli HB101b None 1.22 + 0.33 2.22 ± 0.99 0.82 NSCE. coli(pGG101) Type 1 1.44 ± 0.17 1.75 ± 0.72 0.22 NSK pneumoniae IA565 L Types 1 and 3 1.76 + 0.41 4.37 + 0.29 1.48 <0.001K pneumoniaeUIR040 L Type 3d 0.90 ± 0.22 1.94 ± 0.42 1.16 0.003K pneumoniae IA565 G-CAA Types 1 and 3 3.28 ± 0.19 10.89 + 2.39 2.32 0.008K pneumoniae UIRO40 G-CAA Type 3 3.82 ± 0.87 10.51 + 2.82 1.75 0.004E. coli(pFK10) Type 3 4.20 + 0.37 25.93 ± 10.06 5.17 <0.001

a All adherence assays were performed at least four times with different samples of epithelial cells.b E. coli HB101 and E. coli(pFK25) were used interchangeably for these experiments.c NS, not significant.d Organisms grown on L agar produced low levels of type 3 fimbriae.

type). Interestingly, by using the radiolabeling assay, low-level attachment by E. coli(pDC17) to tracheal cells wasobserved.

Site-specific adherence mediated by K. pneumoniae type 3fimbriae. Adherence to both the washed, isolated epithelialcells and the human lung tissue sections demonstrated alocalized pattern of attachment by organisms expressingtype 3 fimbriae.

(i) Adherence to isolated tracheal cells. The followingorganisms expressing the type 3 fimbrial adhesin (MrkD), Kpneumoniae IA565 and UIRO40, E. coli(pFK10), and E.coli(pDC17/pFK52), all demonstrated a localized attachmentto the basolateral surfaces of columnar ciliated tracheal cells(Fig. 3A to C). Both nonfimbriate E. coli(pFK25) and type 1fimbriate E. coli(pGG101) organisms demonstrated low-leveland nonspecific adherence (Fig. 3D).

(ii) Adherence to human lung tissue sections. Bacteriaexpressing type 3 fimbriae bound in high numbers to thetissue sections. The bacterial cells bound to basal cells of the

1200

1 I 1:100

[Feb)FIG. 2. Adherence by E. coli(pFK10) in the presence of rabbit

anti-type 3 fimbria Fab. Maximal inhibition is demonstrated at a 1:25dilution of Fab, and the inhibitory effect is negligible when the Fabdilution is increased to 1:100. Closed rectangle, tracheal cells;closed triangles, trypsinized buccal cells; open rectangle with a

horizontal line, attachment to tracheal cells following pretreatmentof bacteria with nonimmune rabbit IgG instead of Fab; asterisk,attachment to trypsinized buccal cells following pretreatment ofbacteria with nonimmune rabbit IgG. Results shown are the means

standard errors of the means.

pseudostratified columnar epithelium or to the basementmembrane region of the bronchial epithelium (Fig. 4B andC). In addition, E. coli(pDC17/pFK52) also adhered to thebasement membrane region of intact epithelium (Fig. 4D).Type 1 fimbriate E. coli(pGG101) (Fig. 4E), nonfimbriate E.coli strains (Fig. 4F), and E. coli(pDC17) exhibited noappreciable binding to the tissue sections when used atconcentrations identical to those used for the adherentfimbriate organisms.

(iii) Inhibition assays. When E. coli(pFK10) was preincu-bated with a 1:10 dilution of Fab fragments of anti-type 3fimbrial IgG, adherence to the tissue surface was greatlydecreased. This inhibition was abrogated by using a fourfoldlower concentration of the anti-type 3 Fab solution (Fig. SAand B). Preincubation of type 3 fimbriate bacteria with asimilar concentration of nonimmune rabbit IgG did not alterbinding.

E. coli(pFK10) bound less avidly to tissue sections prein-cubated with purified type 3 fimbriae, whereas preincubationof the tissue sections with a similar concentration of BSAdemonstrated no effect on the level or distribution of binding(Fig. SC and D). Both spermidine and spermine dislodgedtissue sections after fixation to the slide, and therefore, nodata was obtainable in this assay for these known inhibitorsof type 3 fimbria-mediated HA.

Chemical nature of the type 3 adhesin target in lung tissue.Chemical treatment of lung tissue sections with sodiummeta-periodate had no effect on the adherence of E.coli(pFK10) (Fig. 6A) orK pneumoniae IA565. In contrast,treatment of lung tissue sections with collagenase resulted ina significant drop in the number and pattern of adherent E.coli(pFK10) (Fig. 6B) and K pneumoniae IA565 cells.Adherence by type 3 fimbriate bacteria was unaffected,however, when lung tissue sections were incubated withcollagenase and its inhibitor (10 mM EDTA) (Fig. 6C).Furthermore, preincubation of the tissue with a dilute solu-tion of acetic acid, which exposes collagen epitopes (19),resulted in greatly enhanced binding of type 3 fimbriateorganisms to the basement membrane area of the epitheliumand to the basolateral surfaces of the epithelial cells (data notshown). When the inhibitors EDTA and pepstatin A wereadded to the acetic acid solution, the enhanced level ofbinding caused by acetic acid was no longer observed.

DISCUSSION

Bacterial fimbriae have been shown to mediate attachmentto specific targets on epithelial surfaces in the lung and the

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TABLE 4. Effect of various compounds on adherence byE. coli(pFK10) to human buccal and tracheal cells

Concn Adherencea + SD (%) to:Compound (mM or %) Buccal cells' Tracheal cells

a-Methylmannoside 50 109 + 14 119 ± 29150 105 6 111 9

Glucosec 50 96 112150 113 108

D-LactoseC 73 89 102146 106 96

Spermidine 80 NDd 30 ± 8e40 22 ± 3e 47 ± 9e4 72 ± 4e 78 ± 9e2 ND 82 ± le

Spermine 60 ND 41 ± lle15 29 4e 58 12e2 41 le 71 12e

D,L-LysineC 145 155 160Poly-L-lysine 0.25% 204 ± 36e 193 ± 22eProtamine 0.25% 325 55e 388 ± 67e

a Adherence was expressed as a percentage of the adherence obtained incontrol assays performed with PBS alone.

* All experiments were performed with trypsinized buccal cells.c Results are expressed as the means of experiments performed only twice.d ND, not determined.e Value differed significantly compared with the value for the a-methylman-

noside control (P < 0.01).

gastrointestinal and urinary tracts (4, 5, 31, 36). Bacterialattachment is believed to be a prerequisite for colonizationor infection of the lung. Recent studies have supported a rolefor fimbriae in this initial interaction between the bacteriaand the host. For example, it has been demonstrated thattype 1 fimbriae of E. coli mediate attachment to humanbuccal cells (30), type 1 fimbriate K pneumoniae attach toguinea pig tracheal cells maintained in a primary culturedmonolayer (6), and a recombinant type 1 fimbriate E. colistrain adheres to rat tracheal ring explants (4). The in vitroadherence data presented herein suggest that the type 3fimbrial adhesin may facilitate attachment to specific targetswithin human respiratory epithelia.

Prior studies, which examined the role that type 3 fimbriaeplay in attachment, have shown that this fimbrial type mayfacilitate adherence to plant root hairs (18), to urinarycatheters (24), and to specific sites in human renal tissue(32). In addition, a study that examined adherence to intes-tinal epithelial monolayers by clinical isolates of Klebsiellaspp. which expressed primarily MR/K HA showed low-leveladherence and no correlation to the HA phenotype (28).Several studies that examined the frequency of fimbrialtypes among Klebsiella species have found that the majorityof both environmental and clinical isolates produce type 3

fimbriae, while a smaller proportion of strains also producetype 1 fimbriae (9, 15, 27, 29). To date, however, there hasbeen no study examining the ability of Klebsiella type 3fimbriae to mediate adherence to human respiratory epithe-lia, yet Klebsiella species are frequently implicated in noso-

comial pneumonias.

Our initial experiments utilizing Klebsiella isolates, cul-tured under conditions that optimized expression of theMR/K HA (type 3 fimbrial production), suggested that theseorganisms attached to human buccal cells and tracheal cellsin larger numbers than did Klebsiella isolates producingdemonstrably less MR/K HA (Table 1). Since these experi-ments used clinical isolates, it is possible that the adherenceobserved might be attributable to other surface factors whichmay be expressed under the conditions which favor type 3fimbrial expression. Consequently, we also used E. coliHB101 transformants which produce fimbrial proteins en-coded by recombinant plasmids carrying either the Klebsi-ella type 1 (fim) or type 3 (mrk) gene clusters (8, 10). Type 3fimbriate E. coli(pFK10) adhered to human tracheal cells,trypsinized buccal cells, and human lung tissue sections ingreater numbers than did E. coli(pGG101) bearing Klebsiellatype 1 fimbriae and nonfimbriate E. coli cells. Although type1 fimbrial expression did facilitate adherence to trachealcells, the number of bacteria per cell was significantly lessthan that observed for the type 3 fimbriate recombinantorganism (Table 2). Attachment by type 3 fimbriate Klebsi-ella clinical isolates and E. coli(pFK10) was also site spe-cific. These organisms adhered to the basolateral surfaces ofbronchoscopically obtained tracheal cells and to basal cellsand the basement membrane region of bronchial epitheliumwithin the lung tissue sections (Fig. 3 and 4). Confirmation ofthe fimbria-mediated binding to airway epithelia was deter-mined by inhibition with Fab fragments of anti-type 3 IgG. Inaddition, spermine and spermidine, previously shown to beinhibitory for fimbria-mediated MR/K HA (9), also inhibitedepithelial binding, and tissue sections pretreated with puri-fied type 3 fimbriae did not bind whole bacteria.Type 3 fimbrial adherence to respiratory tissue appeared

to be mediated by the MrkD subunit of the type 3 fimbrialsystem. The double transformant, E. coli(pDC17/pFK52),which had been constructed in previous studies (12), wasused in these experiments. The double transformant allowsthe mrkD gene product (encoded by pFK52) to conferadherence upon E. coli(pDC17), an otherwise poorly adher-ent P fimbria-producing strain. The data from the adherenceassays with the double transformant (Table 5) showed thatthe MrkD subunit mediated attachment to respiratory epi-thelial cells. Our histologic experiments (Fig. 3 and 4) alsoshowed that the MrkD subunit was responsible for attach-ment by type 3 fimbriate organisms to the basolateral surface

TABLE 5. Role of the MrkD polypeptide in adherence to human buccal and tracheal cells

Mean no. of bacteria/cell ± SDE. coli HB101 HA

plasmid phenotype Buccal cellsa Tracheal cells"Radiolabeling assay Microscopic assay Radiolabeling assay Microscopic assay

(pDC17) None 3.42 1.20 2.66 ± 1.47 9.35 ± 1.91 0.95 ± 0.22(pDC17/pFK52) MR/K 22.55 ± 8.11 (P = 0.004)c 16.09 ± 1.78 (P < 0.001) 22.69 + 6.60 (P = 0.015) 4.38 ± 0.61 (P < 0.001)

a All experiments (n = 4) were done with trypsinized human buccal cells.* For tracheal cells, n = 4 for the radiolabeling assay and n = 7 for the microscopic assay.c Values compared to corresponding value for E. coli(pDC17).

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FIG. 3. Photomicrographs showing tracheal cells with adherent bacteria, which were stained in situ on a polycarbonate filter. (A) Kpneumoniae UIR040 (passaged on G-CAA); (B) E. coli(pFK10); (C) the double transformant, E. coli(pDC17/pFK52); (D) E. coli(pGG101).All organisms shown in panels A to C express the type 3 fimbrial adhesin and attach to the basolateral surfaces of ciliated human tracheal cells.No significant attachment to tracheal cells by type 1 fimbriate E. coli(pGG101) was observed (panel D). Arrowheads, selected adherentbacteria; arrows, ciliate and apical portions of the tracheal cells.

of tracheal cells and to the basal cells and the basementmembrane region of bronchial epithelium.

Despite the fact that the double transformant showedsignificantly increased adhesive capabilities compared withthe nonhemagglutinating P fimbriate E. coli(pDC17), it wasalso noted that E. coli(pDC17) demonstrated low-level ad-hesion to tracheal cells, particularly in the radiolabeled-bacterium adherence assay. A recent report has shown thatP fimbriae exhibit low-affinity attachment to fibronectin,which is notpapG mediated (35). Since fibronectin is foundin the extracellular matrix of basal epithelial cells and as acomponent of epithelial basement membrane (21, 22), thepapG deletion mutant, E. coli(pDC17), may adhere to thebasal nonciliated tracheal epithelial cells, which may include

remnants of basement membrane. Despite this low-affinityattachment to tracheal cells by E. coli(pDC17), the data fromthe adherence assays with the double transformant supportthe hypothesis that the MrkD subunit of the type 3 fimbrialsystem mediates attachment to respiratory epithelial cells.The frozen-lung-tissue adherence assay was utilized to

examine the nature of the target molecule(s) recognized bythe type 3 fimbrial adhesin. Pretreatment of tissue sectionswith sodium periodate, which cleaves most surface-associ-ated carbohydrates, had no effect on the binding character-istics of type 3 fimbriate organisms. Conversely, tissuepretreated with a collagen-specific protease proved to be lesssuitable for binding by the type 3 fimbriate organisms. Whencollagenase activity was inhibited by EDTA, however, ad-

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FIG. 4. Binding of fluorescein isothiocyanate-labeled bacteria to human lung tissue sections. (A) Hematoxylin-eosin-stained section fromthe tissue sample; (B) type 3 fimbriate K. pneumoniae IA565 (passaged on G-CAA); (C) E. coli(pFK10); (D) E. coli(pDC17/pFK52); (E) E.coli(pGG101) expressingK pneumoniae type 1 fimbriae; (F) nonfimbriate E. coli(pFK25). Note the localization of bacterial binding over thebasement membrane region for bacteria expressing the type 3 fimbrial adhesin (B, C, and D) in contrast to those of E. coli(pGG101) (E) andE. coli(pFK25) (F), which demonstrate low-level, nonspecific adherence. L, lumen of small airway; E, airway epithelium; BM, basementmembrane region; S, submucosa. Selected bacteria are indicated by arrowheads. The bright fibrillar material seen in the submucosa isautofluorescent connective tissue fibers.

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FIG. 6. Chemical nature of the type 3 adhesin target in lung tissue. (A) The tissue section was incubated with 100 mM sodium periodatefor 30 min prior to the testing for adherence by the type 3 fimbriate E. coli(pFK10). Adherence to the basement membrane region (arrowheads)appeared to be unaffected. Tissue sections were also incubated with 4 U of collagenase per ml either alone (B) or in the presence of its inhibitor(10 mM EDTA) (C) prior to the addition of E. coli(pFK10). Note the decreased binding to the basement membrane region in panel B versusthat in panel C. See Fig. 4 for the identification of figure labels.

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ADHERENCE BY KLEBSIELLA TYPE 3 FIMBRIAE 1587

herence by type 3 fimbriate organisms remained unaffected.Dilute-acetic-acid treatment is postulated to stimulate activ-ity of native acid proteases in the tissue and has been used inimmunohistochemical studies to enhance antibody recogni-tion of a collagen epitope (19). Interestingly, we observed anincrease in the level of binding to the basal cells and thebasement membrane region of the bronchial epithelium afterpretreating the tissue sections with acetic acid. Thus, theevidence supports a protein-protein interaction for the type 3fimbrial adhesin rather than a protein-carbohydrate interac-tion more typically described for fimbrial ligand-receptorinteractions (i.e., the type 1 fimbrial interaction with man-nose and the P fimbrial interaction with galabiose).

Purified type V collagen provides a target for the type 3fimbrial adhesin. Although fibronectin, laminin, and type IVcollagen are proteins more commonly associated with epi-thelial intercellular matrix and bronchial basement mem-branes, assays with these purified proteins as solid-phasetargets have failed to show attachment by the type 3 fimbrialadhesin in prior studies (32). Type V collagen has beenidentified in fibrils anchoring basement membrane to thesubmucosa (21), and in intercellular locations in humanepithelia (17, 21). Type V collagen, however, is not likely tobe found on erythrocytes and has not been directly demon-strated as intimately associated with human buccal or tra-cheal cells. Thus, the target for the type 3 adhesin mayinvolve several proteins that share a common domain. Also,since the polyamines spermine and spermidine inhibit type 3fimbrial attachment, it is possible that such a shared epitopemay be rich in positively charged amino acids.A characteristic of type 3 fimbrial adherence is that the

target compound is frequently not exposed on the intact cellsurface. For example, tannic acid pretreatment of erythro-cytes is necessary for detecting MR/K HA and is believed tomodify or remove normal surface proteins and uncover acryptic target molecule. The experiments reported hereusing human buccal cells demonstrated that the most effec-tive adherence by type 3 fimbriate organisms occurred afterpretreatment of buccal cells with trypsin (Table 3), which isanalogous to that observed in experiments with erythro-cytes. Similarly, our adherence assays showed that the type3 adhesin-mediated attachment to sites on human trachealcells and bronchial epithelium not exposed on intact mucosa.It is possible that damage to the epithelial lining must occurbefore type 3 fimbriate Klebsiella spp. may adhere to therespiratory mucosal surface.Tarkkanen et al. have also shown that type 3 fimbriae

mediate adherence to renal tubular basement membranes,Bowman's capsule, and renal vessel walls (32). In addition,uropathogenic E. coli producing the 075X (Dr) adhesinadheres to the amino-terminal portion of type IV collagen,and binding is localized to the basement membranes oftubules in renal tissue sections (26). These observations,along with our results obtained with respiratory tissue,broaden the possibilities for fimbria-mediated attachment tohuman tissues and suggest that adherence factors producedby bacterial pathogens may also mediate attachment toextracellular matrix components, which may be exposedonly after epithelial injury.

In summary, we have presented data suggesting thatKiebsiella spp. adherence to human respiratory epithelialtissue in vitro is mediated by the type 3 fimbrial adhesin. Theinteraction between this adhesin and the respiratory epithe-lium was specific for cryptic sites on buccal cells, within thebasal layers of airway epithelium, and the basement mem-brane region. The adhesin-ligand interaction appeared to be

a protein-protein interaction, and the data support the find-ing of previous experiments showing that type V collagen isa potential target for the type 3 adhesin (32). Further studiesare needed, however, to clarify the role of the Klebsiellatype 3 fimbrial adhesin in the pathogenesis of lung infectionsand the virulence of this organism. To exclude any effect ontype 3 fimbrial adherence by expression of the Klebsiella-derived organelle in a heterologous host, such studies shouldevaluate adherence utilizing isogenic Klebsiella strains spe-cifically mutated within the chromosomal mrk gene cluster.Furthermore, the virulence of such strains would also needto be evaluated in an animal model of Klebsiella lunginfection.

ACKNOWLEDGMENTSWe thank Jeneva Ford for expert secretarial assistance and A.-M.

Tarkkanen and T. K. Korhonen for technical assistance in estab-lishing the tissue adherence assay.D.B.H. is supported, in part, by the Edward Livingston Trudeau

Award from the American Lung Association and the AmericanLung Association of Iowa. B.L.A. is supported, in part, by the NIHMedical Scientist Training Program Grant (GM07337) and by thepulmonary training grant from the NHLBI (NRSA HL07638).

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