Vol. 60, No. 4

INFECTION AND IMMUNIT-Y, Apr. 1992, p. 1577-1588 0019-9567/92/041577-12$02.00/0

Copyright © 1992, American Society for Microbiology

Adherence to Respiratory Epithelia by Recombinant Escherichia coli Expressing 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 of Iowa 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 and tracheal cells and to lung tissue sections. We found that clinical isolates of Klebsiella pneumoniae producing type 3 fimbriae and Escherichia coli HB101 containing a recombinant plasmid encoding expression of Kkebsiella type 3 fimbriae (pFKIO) demonstrated increased adherence to tracheal cells, trypsinized buccal cells, and lung tissue sections, in contrast to nonfimbriate and to type 1 fimbriate bacteria. Adherence by type 3 fimbriate bacteria was inhibited by purified type 3 fimbriae and Fab fragments derived from type 3 fimbrial-specific polyclonal immunoglobulin G. Type 3 fimbriae mediated attachment to the basolateral surface of tracheal cells and to the basal 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 MrkD adhesin subunit. Subsequent experiments demonstrated that the epithelial target of the type 3 fimbrial adhesin was most likely a peptide molecule rather than a carbohydrate. The results of this study demonstrate that, in vitro, the KlebsieUla type 3 fimbrial adhesin mediates adherence to human respiratory tissue.

Members of the family Enterobacteriaceae account for at least one-third of reported nosocomial pneumonias, according to data accumulated as part of the National Nosocomial Infection Survey (14). Gram-negative bacterial colonization often precedes nosocomial pneumonia (3, 13, 16). To colonize the respiratory tract, these bacteria must overcome mucociliary clearance and adhere to the epithelium. Members of Kiebsiella, the genus of Enterobacteriaceae frequently causing nosocomial pneumonia, commonly produce type 1 and/or type 3 fimbriae. Fimbriae are filamentous organelles which may abrogate mechanical host clearance mechanisms and facilitate attachment to epithelial receptors by fimbria-associated adhesins. This study was designed to examine the ability of the Kiebsiella type 1 and type 3 fimbriae to mediate adherence to human respiratory epithe-

lia. Most species of the Enterobacteriaceae can produce type 1 fimbriae. Organisms expressing this fimbrial type exhibit mannose-sensitive hemagglutination (MS HA) in vitro, and the receptors for the type 1 fimbrial adhesin are believed to be mannose-containing glycoproteins. Type 3 fimbriae belong within the heterogeneous group of fimbrial types broadly classified as mannose-resistant (MR) hemagglutinins. Organisms expressing type 3 fimbriae demonstrate MR agglutination of erythrocyte suspensions only after the erythrocytes have been pretreated with 0.01% tannic acid. Klebsiella organisms commonly exhibit this phenomenon (27) and, therefore, the type 3 fimbria-mediated HA is referred to as MR Klebsiella-like HA (MR/K HA). In contrast to the type 1 fimbriae, type 3 fimbriae are not expressed by Escherichia coli but are frequently produced by species of Enterobacter, Proteus, Providencia, Morganella, Yersinia, and Serratia (2). Recent investigations have shown that type 3 fimbrial adherence is mediated by the MrkD polypeptide (12), that type 3 fimbrial adherence is inhibited by cationic compounds such as spermidine (9), and that renal *

Corresponding author.

tubular basement membrane and, specifically, type V collabe targets for attachment by the type 3 fimbrial adhesin (32). In this study, we examined the ability of Klebsiella fimbrial types to attach in vitro to buccal and tracheal cells and to lung tissue derived from normal human volunteers. The results demonstrated that (i) increased adherence was associated with type 3 fimbriae, in contrast to type 1 fimbrial expression by clinical strains of Klebsiella pneumoniae and E. coli HB101 containing recombinant plasmids encoding the expression of Klebsiella fimbriae, (ii) attachment was localized to cryptic sites on epithelial cells or sites normally not exposed on intact buccal or airway epithelium, (iii) adherence by type 3 fimbriae was attributable to the MrkD adhesin subunit, and (iv) the epithelial target for the type 3 adhesin was most likely a peptide rather than a carbohydrate. gen may

MATERIALS AND METHODS

Bacterial strains and plasmids. Clinical isolates, K pneumoniae UIR040 and IA565, were obtained from the University of Iowa Hospitals and Clinics Clinical Microbiology Laboratory. K pneumoniae UIRO40 was isolated from the tracheal aspirate of a patient with nosocomial pneumonia, and K pneumoniae LA565 had been previously used as a source of DNA to clone both K pneumoniae type 1 and type 3 fimbrial gene clusters (8, 10). The construction of the following recombinant plasmids used to transform E. coli HB101 has been described elsewhere: pFK10 encodes K pneumoniae type 3 fimbriae (8), pGG101 encodes K pneumoniae type 1 fimbriae (10), and pDC1 encodes an E. coli P fimbria (1) (Fig. 1). The type 3 and type 1 fimbrial gene clusters were cloned into medium-copy-number vectors pACYC184 and pBR322, respectively. In addition, plasmid pFK25 is a deletion derivative of the cloned type 3 fimbrial (mrk) gene cluster (Fig. 1), and E. coli(pFK25) transformants are nonfimbriate. Plasmid pFK52 contains only the mrkD (adhesin) gene and E. coli(pFK52) is also nonfimbriate, and because of the lack of accessory fimbrial genes, this trans1577

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INFEC-F. IMMUN.

HORNICK ET AL. co

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FIG. 1. Genetic maps of recombinant plasmids encoding expression of K pneumoniae type 3 (pFK10) and type 1 (pGG101) fimbriae, E. coli P (pDC1) fimbriae, and the respective deletion derivatives used in these studies. Boxes with letters indicate the locations and designations of genes located on each cluster.

formant does not hemagglutinate. Plasmid pDC17 is a papG deletion derivative of pDC1 (Fig. 1), and E. coli(pDC17) therefore expresses nonadhesive P fimbriae (Fll serotype). The double transformant E. co1i(pDC17/pFK52) expresses P fimbrial filaments associated with the type 3-specific adhesin, which is detectable by MR/K HA (12). Organisms containing 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 20 h at 37°C on minimal medium agar plates supplemented with 3% glycerol and 0.1% Casamino Acids (G-CAA), which have been previously shown to optimize expression of type 3 fimbriae (8). The phenotypic expression of appropriate fimbrial types was confirmed by HA and reactivity with fimbriaspecific sera.

Bacterial labeling. Bacteria were harvested, after overnight culture, into 0.2% glucose-M-9 minimal salts solution and were labeled metabolically with [35S]methionine (Amersham, Chicago, Ill.) by standard techniques (23). For the assays with frozen tissue sections, bacterial suspensions were labeled with fluorescein isothiocyanate according to the 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 was raised in rabbits. Immunoglobulin G (IgG) was isolated from hyperimmune serum by protein G column chromatography and subsequently digested with solid-phase papain according to the manufacturer's protocol (Pierce, Chicago, Ill.). The Fab fragments were purified by multiple elutions over a protein A column (ChromatoChem, Missoula, Mont.). The purity of the resulting material was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The A280 for this material was measured, and the Fab concentration was derived from the appropriate extinction coefficient. The purified Fab fragments inhibited MR/K HA when type 3 fimbriate bacteria were preincubated with the Fab fraction for 15 min at 25°C.

Epithelial cell procurement. Buccal and tracheal cells were obtained from nonsmoking normal adult human volunteers. (i) Tracheal epithelial cell isolation. Tracheal cells were obtained from normal volunteers by bronchoscopically guided tracheal brushing (3-mm bronchial brush; Bard, Billerica, Mass.) according to the methods of Niederman et al. (25). Cell counts were performed with a hemocytometer, and microscopic examination for viability was checked by trypan blue exclusion. The cells were used immediately for bacterial attachment assays. (ii) Buccal epithelial cell isolation. Buccal cells were obtained from laboratory personnel with low levels of indigenous flora, via gentle curettage (sterile cotton-tipped applicator) of buccal mucous membranes. Cells were washed three times in phosphate-buffered saline (PBS) at pH 7.4. Cell counts and assessment of viability were performed as described above. Human lung tissue. Normal human lung tissue was obtained from uninvolved areas of surgical biopsy material provided by the University of Iowa Hospitals and Clinics Surgical Pathology Laboratory. Tissue was snap-frozen in liquid nitrogen and stored at -70°C. Thin frozen sections (5 to 6 ,um) were cut at -20°C with a Reichert-Jung 2800 Frigocut Cryostat and placed on glass slides which had been treated with siliconizing solution (1% dimethyldichlorosilane in carbon tetrachloride) and stored at -70°C until used. Simple hematoxylin-eosin staining confirmed the presence of bronchial epithelium and underlying histologic structures within the sections. Whole bacterium-epithelial cell adherence assay. The adherence assays were adapted from previous studies by other investigators (7, 25, 33) and differed minimally when buccal instead of tracheal cells were used. The major exception was that the buccal cells were, in some experiments, pretreated with trypsin. Preliminary results showed that maximal adherence was obtained when buccal cells were pretreated with trypsin by established techniques (36), in which 2.5 ,ug of trypsin (bovine pancrease; Sigma) per 105 buccal cells was used and cells were incubated at 37°C for 30 min. After

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ADHERENCE BY KLEBSIELLA TYPE 3 FIMBRIAE

pretreatment in all cases, buccal cells were washed in PBS, recounted, and checked for viability by trypan blue exclusion prior to use in the bacterial adherence assays. (i) Radiolabeled-bacterium adherence assay. Washed epithelial cells (0.5 ml) and 35S-labeled bacteria (2 x 108/ml) were combined in a bacteria-to-cell ratio of 1,000:1. This ratio was found to be optimum in preliminary studies using both tracheal and buccal cells. All adherence assays were done in triplicate. Cells and bacteria were incubated for 90 min at 37°C with gentle continuous end-over-end agitation. Cells with adherent bacteria were collected on 22-mmdiameter (8-,um pore size for buccal cells or 3-,um pore size for tracheal cells) polycarbonate filters (Nucleopore), which had been pretreated with 3% bovine serum albumin (BSA) in PBS. Nonadherent bacteria were washed through the filter with 50 volumes of PBS. The filtered cells with adherent bacteria were then solubilized (1 N NaOH, 16 h, 37°C), and the radiolabel was counted in a liquid scintillation counter. The number of adherent bacteria per cell was calculated as described previously by Old et al. (27). Initially, bacterial counts were confirmed by quantitative cultures of one filter from each triplicate. (ii) Unlabeled-bacterium adherence assay. The unlabeledbacterium adherence assay was performed simultaneously with, and identically to, the radiolabeling assay, with the exception that cells with adherent bacteria were Gram stained and permanently mounted on a glass microscopic slide. The slides were examined by using bright-field microscopy by personnel who were unaware of the fimbrial type being tested. Adherence was recorded as the mean number of bacteria per epithelial cell (in the tracheal cell assay, only the ciliated cells were counted) after the examination of 20 consecutive cells in the central section and four peripheral sections of the filter (total cells counted per filter = 100). Results were expressed as the number of adherent bacteria per cell. Binding distribution per cell was also noted. When the binding distribution was significant, we confirmed the results by examining cells, prior to filtering, with phasecontrast microscopy to rule out artifacts that may have been introduced by staining and mounting filters. Tissue section adherence assay. The adherence assay with human lung tissue sections was carried out according to the method of Virkola (34). Briefly, snap-frozen tissue sections mounted on glass slides were allowed to come to room temperature, immediately prior to the assay, in a humidity chamber. The sections were then fixed with fresh 3.5% (wt/vol) paraformaldehyde for 10 min at ambient temperature and washed three times in PBS. The sections were then overlaid with a PBS-fluorescein isothiocyanate-labeled bacterial suspension (50 ,ul of 108 to 109 organisms per ml) and incubated at 4°C for 45 min in a humidity chamber. Nonadherent and loosely adherent bacteria were then removed by three 5-min washes with PBS-0.05% Tween 20 (vol/vol), with shaking. Slides were allowed to dry before the addition of glycerol mounting solution and the glass coverslip. Slides were observed by light microscopy, using a reflected-fluorescence attachment. (i) Inhibition of adherence by Fab fragments of anti-type 3 IgG and purified fimbriae. Inhibition of adherence was examined by addition of a 1:10 or 1:40 dilution of the Fab solution to the bacterial suspensions. These suspensions were incubated for 15 min at 25°C, with shaking, and washed once with an equal volume of PBS prior to incubation with the tissue sections. Equivalent concentrations of IgG purified from normal rabbit serum were used as controls. In some experiments, immediately prior to incubation

with the bacterial suspension, tissue sections were preincubated with purified type 3 fimbriae (100 or 300 ,ug/ml) or a control protein (BSA) at a similar concentration. Slides were subsequently washed three times for 5 min each with PBS0.05% Tween 20. (ii) Collagenase, acetic acid, and sodium metaperiodate pretreatment of tissue sections. Tissue sections were overlaid with 50 ,lI of chromatographically purified collagenase derived from Clostridium histolyticum (4 U/ml; Worthington Biochemicals, Freehold, N.J.) and incubated at 37°C for 5 h in either the presence or absence of inhibitor (10 mM EDTA). Sections were washed three times for 1 min each in PBS and fixed with paraformaldehyde before incubation with bacteria. In separate experiments, by using the methods described previously (21), the slides were overlaid with a 0.1 M solution of acetic acid for 30 min at 25°C. Control slides were incubated with the acetic acid along with 10 mM EDTA and 2 ,ug of pepstatin A per ml (Sigma, St. Louis, Mo.). Slides were subsequently washed three times in PBS and fixed with paraformaldehyde prior to the bacterial overlay. To attenuate potential carbohydrate receptors, tissue sections were exposed to a suspension of 100 mM sodium meta-periodate (Sigma) for 30 min at 37°C and washed three times for 5 min each in PBS before the adherence assay was conducted. The control for these experiments used P fimbriate E. coli(pDC1), which adhere well to respiratory cells and the adherence of which is dependent on a galabiose glycolipid receptor (14a). Statistical analysis. Arithmetic means and standard deviations (except where indicated as the standard error of the mean) were calculated for the quantitative adherence assays with buccal and tracheal cells. Results were analyzed by using the Student's t test for independent variables, and the P values reported are for two-tailed tests.

1579

RESULTS Fimbria-mediated adherence to respiratory epithelia was determined by utilizing buccal cells, tracheal cells, and frozen lung tissue sections obtained from normal human subjects. Buccal and tracheal cell adherence assay. The mean ± standard error of the mean number of buccal cells obtained from laboratory personnel was 1.0 x 106 + 0.1 x 106 cells. Only 10% or fewer of the cells were viable, as determined by trypan blue exclusion. The mean ± standard error of the mean number of tracheal cells obtained at bronchoscopy was 3.1 x 106 ± 1.2 x 106. The majority (64.7% ± 2.7%) of tracheal cells were ciliate, and 47.6% ± 9.4% were observed to be viable by trypan blue exclusion. In initial studies, we found that by the end of the adherence assay, viability of the tracheal cells had decreased further and that bacteria were adhering in equal numbers to both the nonviable and viable cells. (i) Organisms expressing Kiebsiella fimbriae. Adherence by fimbriate strains of K pneumoniae to epithelial cells is shown in Table 1. K pneumoniae IA565, when cultured on L agar, expresses both MS and MR/K HA. K pneumoniae UIR040, however, produces a very weak MR/K HA activity and does not cause MS HA when subcultured on L agar. Consistent with previous observations, both isolates demonstrate increased MR/K HA when subcultured on G-CAA agar (8). When the organisms were tested for adherence to both human tracheal cells and trypsinized buccal cells, the organisms demonstrating the greatest MR/K HA, associated

HORNICK ET AL.

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INFECT. IMMUN.

TABLE 1. Adherence to buccal and tracheal cells by clinical strains of K pneumoniae expressing type 3 fimbriae Mean no. of bacteria/cell ± SD

K MR/K pneumoniae Passage HA medium titee strain

IA565

L G-CAA UIRO40 L G-CAA

6 19 3 22

Buccal cells'

Radiolabeling assay

4.37 10.89 1.94 10.51

± ± ± ±

nc

0.29 2.39 (P = 0.003)" 0.42 2.82 (P < 0.001)

Trachea cells

Microscopic assay

n

Radiolabeling assay

4 4.86 ± 1.24 5 2.01 4 14.42 ± 0.74 (P < 0.001) 5 7.96 6 1.34 ± 1.11 6 1.17 6 11.96 ± 5.13 (P < 0.001) 6 10.02

± ± ± +

n

Microscopic assay

0.81 5 0.52 1.97 (P < 0.001) 5 2.46 0.72 5 0.47 2.81 (P = 0.001) 5 3.96

n

± 0.37

6 - 0.96 (P =0.001) 6 ± 0.39 6 ± 1.59 (P < 0.001) 6

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.

a

with type 3 fimbriae, attached to the epithelial cells in significantly greater numbers than did nonfimbriate or type 1 fimbriate bacteria (Table 1). Table 2 shows the adherence to epithelial cells by E. coli HB101 transformed with recombinant plasmids encoding Klebsiella fimbriae. In these experiments, the type 3 fimbriate transformant, E. coli(pFK10), demonstrated significantly greater adherence than the other organisms tested. Phenotypically 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 cells but did not adhere significantly to trypsinized buccal cells. Also, the microscopic assay confirmed all observations made for the radiolabeled-bacterium adherence assay, except that low-level adherence to tracheal cells by E. coli (pGG101) was not observed. (ii) Effect of trypsin on adherence to buccal cells. The data shown in Table 3, derived from the radiolabeled-bacterium adherence assay (confirmed by the unlabeled-bacterium adherence assay [data not shown]), demonstrate that pretreatment of the buccal cells with trypsin resulted in significantly enhanced binding by the type 3 fimbriate organisms. The greatest enhancement was demonstrated by organisms producing relatively large amounts of the type 3 fimbriae (i.e., those grown on the G-CAA medium or the E. coli recombinant). Also, no significant enhancement of adherence was noted by the type 1 fimbriate E. coli(pGG101) and the nonfimbriate E. coli HB101 or E. coli(pFK25). (iii) Inhibition assays. Preincubation of the type 3 fimbriate recombinant strain, E. coli(pFK10), with a 1:25 dilution of Fab fragments of anti-type 3 IgG resulted in markedly reduced adherence to both the normal tracheal cells and to trypsinized buccal cells (Fig. 2). The Fab fragments isolated

from the anti-type 3 fimbrial serum also inhibited the MR/K hemagglutinating activity of bacteria. Consistent with previous observations demonstrating that spermine and spermidine inhibit MR/K HA (9), E. coli (pFK10) attachment to both normal human tracheal cells and trypsinized buccal cells was inhibited by addition of spermine or spermidine to the assay mixture (Table 4). Compounds that showed no inhibition, within a similar molar concentration range, included a-methylmannoside (50 and 150 mM), glucose (50 and 150 mM), and D-lactose (73 and 146 mM). Interestingly, P,L-lysine (145 mM) and, particularly, the polycations poly-L-lysine (0.25% [wt/vol]) and protamine (0.25% [wt/vol]) resulted in enhancement of adherence by the type 3 fimbriate E. coli(pFK10) (Table 4). However, nonfimbriate and type 1 fimbriate organisms also adhered in large numbers (means of 24 and 19 bacteria per cell, respectively) when the epithelial cells were pretreated with protamine. Similar data were obtained by using poly-L-lysine. We also observed epithelial cell clumping, which was not completely reversible when cells were washed, suggesting that these adhesive compounds likely coat or, possibly, even alter the epithelial cell surfaces. Thus, the increase in adherence observed when the epithelial cells were pretreated 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 fimbrialmediated adherence. The results presented in Table 5 indicate that the double transformant, E. coli(pDC17/pFK52), which produces the type 3 fimbrial adhesin (mrkD gene product) with the P fimbrial filament, adheres to human tracheal cells and trypsinized buccal cells in significantly greater numbers than E. coli(pDC17), which produces only the 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 Plasmid

Type of fimbriae

Buccal

Radiolabeling assay

(pFK25)c pGG101 pFK10

None Type 1 Type 3

2.22 ± 0.99 1.75 + 0.72 25.93 ± 10.06e

+

SD

cellsb

Trachea cells

Microscopic assay 1.11 ± 1.16 1.80 ± 0.76 22.58 ± 12.04e

Radiolabeling assay

Microscopic assay

1.72 + 1.49 3.44 1.15d 16.84 ± 7.87e

0.30 ± 0.25 0.32 ± 0.30 8.23 ± 3.14e

All adherence assays were performed at least ten times with different samples of epithelial cells. 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).

a

b

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ADHERENCE BY KLEBSIELLA TYPE 3 FIMBRIAE

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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 ± SD

Passage

Bacterial strain or plasmid

medium

E. coli HB101b E. coli(pGG101) K pneumoniae IA565 K pneumoniaeUIR040 K pneumoniae IA565 K pneumoniae UIRO40 E. coli(pFK10)

L L G-CAA G-CAA

Type of

fimbriae

None Type 1 Types 1 and 3 Type 3d Types 1 and 3 Type 3 Type 3

Nontrypsinized 1.22 1.44 1.76 0.90 3.28 3.82 4.20

+ ± + ± ± ± +

0.33 0.17 0.41 0.22 0.19 0.87 0.37

Trypsinized

increase

± ± + ± + + ±

0.82 0.22 1.48 1.16 2.32 1.75 5.17

2.22 1.75 4.37 1.94 10.89 10.51 25.93

0.99 0.72 0.29 0.42 2.39 2.82 10.06

NSC NS