INFECTION AND IMMUNITY, Dec. 1990, p. 4036-4044 0019-9567/90/124036-09$02.00/0 Copyright © 1990, American Society for Microbiology
Vol. 58, No. 12
Haemophilus influenzae Adheres to and Enters Cultured Human Epithelial Cells JOSEPH W. ST. GEME III1* AND STANLEY FALKOW12 Department of Microbiology and Immunology, Stanford University, Stanford, California 94305-5402,1 and
National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, Montana 598402 Received 20 June 1990/Accepted 27 September 1990
Haemophilus influenzae is a common commensal organism of the human respiratory tract that initiates infection by colonizing the nasopharyngeal epithelium. In some individuals, colonization is followed by localized respiratory tract or systemic disease. To gain insight into the mechanisms by which H. influenzae attaches to and persists within the nasopharynx, we examined the interactions between a nonpiliated clinical isolate of H. influenzae and human epithelial cells. We noted substantial adherence that occurred independently of pili and required viable bacteria capable of de novo protein synthesis. Comparison of profiles of outer membrane proteins synthesized during incubation with epithelial cells for adherent and nonadherent bacteria identified several candidate adhesin molecules. In addition, a small number of adherent bacteria were capable of entering epithelial cells in a process that was inhibited by cytochalasin D and colchicine. The suggestion from our studies is that one or more of several newly synthesized nonpilus bacterial proteins are required for maximal in vitro adherence and invasion. We speculate that H. influenzae entry into epithelial cells may provide a mechanism for evasion of host defenses, thereby allowing persistence in the nasopharynx.
nasopharynx, we examined the interactions between a clinical isolate of H. influenzae and tissue culture cells derived from normal human conjunctival epithelium. We noted substantial adherence that occurred independently of pili and required viable bacteria capable of de novo protein synthesis. In addition, we observed that a small percentage of adherent organisms were capable of epithelial cell entry in a process that involved actin filaments and microtubules. (This work was presented in part at the Society for Pediatric Research Meeting, Anaheim, Calif., 1990.)
Haemophilus influenzae is a common commensal organism of the human respiratory tract. In addition, this bacterium is an important cause of both localized respiratory tract and systemic (invasive) disease (27). Nonencapsulated, nontypeable strains account for the majority of local disease, including otitis media, sinusitis, bronchitis, and conjunctivitis. In contrast, serotype b strains are responsible for over 95% of cases of H. influenzae invasive disease (26). It is widely believed that disease due to H. influenzae begins with colonization of the nasopharyngeal epithelium, followed by either contiguous spread within the respiratory tract or invasion of the bloodstream (19). Despite the importance of nasopharyngeal colonization, characteristics of H. influenzae that promote its survival in the nasopharynx remain poorly defined. In 1982, Guerina et al. (10) and Pichichero et al. (21) independently reported the presence of pili in selected isolates of H. influenzae type b. These investigators noted a correlation between piliation and increased adherence to human oropharyngeal cells and erythrocytes, suggesting a role for pili as adhesins. More recently, several investigators have presented evidence that H. influenzae may possess nonpilus adhesins as well. Farley et al. noted that nonpiliated strains of H. influenzae were capable of mucosal attachment to nasopharyngeal tissue in organ culture (6, 7). In a comparison of piliated and nonpiliated strains of H. influenzae b, Sable and co-workers found that adherence to HEp-2 cells was greater by nonpiliated strains (24). Bakaletz et al. recently studied a series of clinical isolates of nontypeable H. influenzae and observed no correlation between the degree of piliation and the ability to adhere to human oropharyngeal cells or chinchilla tracheal epithelium in organ culture; all isolates adhered regardless of their state of piliation (1). In an effort to gain additional insight into the mechanisms by which H. influenzae attaches to and persists within the *
MATERIALS AND METHODS Bacterial strains and culture conditions. H. influenzae SUl is a clinical isolate that was obtained from the Clinical Microbiology Laboratory at Stanford University Hospital. It is nonencapsulated and appears to be a nontypeable strain, on the basis of multilocus enzyme electrophoresis (kindly performed by J. Musser, University of Pennsylvania) and outer membrane protein analysis. Negative-staining electron microscopy demonstrated that this strain is nonpiliated (data not shown). Furthermore, erythrocyte selection failed to enrich for a piliated variant (4). SUl was stored frozen at -80°C in brain heart infusion broth with 20% glycerol. For all assays, bacteria were streaked from frozen aliquots onto fresh chocolate agar supplemented with 1% Supplement VX (Difco Laboratories, Detroit, Mich.) and were grown at 37°C in a 5% CO2 incubator overnight. For growth in broth, bacteria were suspended from an 18-h chocolate agar plate in brain heart infusion broth supplemented with hemin (10 ,xg/ml) and NAD (3.5 jig/ml). Escherichia coli K-12 was obtained from the Stanford University collection and is the Stanford wild-type prototrophic strain (ATCC 10798) (15). It was stored frozen at -80°C in LB broth with 50% glycerol and was grown either on LB agar plates or in LB broth (GIBCO, Grand Island, N.Y.). Tissue culture cells. Chang epithelial cells (Wong-Kilbourne derivative, clone 1-5c-4 [human conjunctival) were
Corresponding author. 4036
VOL. 58, 1990
obtained from the American Type Culture Collection (ATCC CCL 20.2). Cells were maintained in modified Eagle medium with Earle salts and nonessential amino acids (Irvine Scientific) supplemented with 10%c heat-inactivated fetal calf serum (GIBCO) and 2.0 mM L-glutamine. They were subcultivated every 2 to 5 days and were used between passages 3 and 50. For adherence and invasion assays, approximately 1.5 x 105 cells suspended in 0.5 ml of tissue culture medium were seeded into each well of 24-well tissue culture plates (Becton Dickinson Labware), which were then incubated at 37°C in 5% CO, for 18 to 20 h. In the case of radiolabeled adherence assays, cells were seeded onto 12-mm-diameter glass cover slips placed on the bottom of each well of tissue culture plates. Adherence assays. (i) Radiolabeled assay. Bacteria were suspended in supplemented brain heart infusion broth and grown at 37°C with aeration to late log phase (109 CFU/ml). They were pelleted, washed once with phosphate-buffered saline (PBS), pH 7.0, and resuspended in methionine assay medium (Difco). After incubation at 37°C with aeration for 15 min, 50 pCi of [3-5SJmethionine (Amersham, Arlington Heights, Ill.) was added to the suspension along with hemin and NAD, and incubation was continued for another 45 min. While there was minimal bacterial growth during the period of radiolabeling, on average the level of incorporation of radioactivity was 0.01 cpm per bacterium. Radiolabeled bacteria were washed three times with PBS, resuspended in PBS, and inoculated (10 ,ul, 1 x 107 to 2 x 107 CFU) onto monolayers. Tissue culture plates were incubated at 37°C (5% CO) for various times. After the desired incubation period, the infected monolayers were rinsed three times with PBS with gentle rocking to remove nonadherent bacteria. Nonspecific adherence was determined by inoculating radiolabeled bacteria onto cover slips without epithelial cells, followed by rinsing with PBS. Cover slips were transferred to vials containing 5 ml of aqueous counting scintillant (ACS II; Amersham), and counts were determined and compared with those of the inoculum. The expression of adherence as a percentage of the original radioactivity provided a measure of bacterial attachment which was independent of later bacterial events, such as replication. Calculations of the average number of adherent bacteria per eucaryotic cell accounted for bacterial replication and assumed no epithelial cell division during the course of an assay. (ii) Viable-count assay. Bacteria were grown to log phase as for the radiolabeled adherence assay. Approximately 1 x 107 to 2 x 107 CFU (10 RI) was inoculated directly from the broth culture onto monolayers, and tissue culture plates were incubated at 37°C in 5% CO,. Following the appropriate incubation period, monolayers were rinsed four times with PBS and then treated with trypsin-EDTA (0.05% trypsin, 0.5 mM EDTA) in PBS to release them from the plastic support. Well contents were agitated, and dilutions were plated on supplemented chocolate agar for H. inflienzae or LB agar for E. coli, yielding the number of adherent bacteria per monolayer. Invasion assay. To determine the number of bacteria entering the cells of an epithelial monolayer, we adapted the assay of Isberg and Falkow (12). Briefly, bacteria were grown and monolayers were infected as outlined above for the viable-count adherence assay. After the desired incubation, monolayers were rinsed three times with PBS, and fresh tissue culture medium containing gentamicin (100 ,ug/ml) was added. Tissue culture plates were incubated for another 2 h, rinsed twice with PBS, and then treated with trypsin-EDTA (0.05% trypsin, 0.5 mM EDTA) in PBS. Well
H. INFLUENZAE ADHERENCE AND INVASION
4037
contents were agitated, and dilutions were plated to quantitate the number of internalized bacteria per monolayer. Outer membrane protein analysis. Outer membrane fractions were prepared from samples of radiolabeled bacteria by the microprocedure of Carlone et al. (2), with minor modifications. Bacteria were initially pelleted by centrifugation at 5,000 x g and 4°C for 10 min. Pellets were washed once with 1 ml of cold 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, pH 7.4, and then resuspended in 1 ml of the same buffer in preparation for sonication. Samples were sonicated on ice with eight 10-s bursts at maximal output with a microsonicator and a 2.3-mm probe (Kontes). Intact cells were removed by centrifugation at 15,600 x g and 4°C for 2 min in a microcentrifuge (Brinkmann). Supernatants were decanted into fresh tubes and centrifuged at 15,600 x g and 4°C for 30 min to pellet bacterial membranes. Pellets were resuspended in 0.2 ml of 10 mM HEPES buffer (pH 7.4). Following the addition of 0.2 ml of 2%Y sarcosyl (N-lauroyl sarcosine) in 10 mM HEPES buffer (pH 7.4) to solubilize cytoplasmic membranes, samples were incubated at room temperature for 30 min with intermittent mixing. Insoluble fractions containing outer membranes were collected by centrifugation at 15,600 x g and 4°C for 30 min. The outer membranes were suspended in equal quantities of distilled water and Laemmli buffer (14) and were stored at -20°C. Samples were heated at 100°C for 5 min, and 100,000 cpm was loaded onto sodium dodecyl sulfate-polyacrylamide gels. Electrophoresis was performed at a constant current of 25 mA with stacking and resolving gels that contained 5 and 10% polyacrylamide, respectively (14). Gels were stained with Coomassie blue for 30 min and were destained overnight. Autoradiograms were made with XAR-5 film. Microscopy. (i) Transmission electron microscopy. Chang epithelial cells were seeded into Contur Permanox tissue culture dishes (35 by 10 mm; Miles Scientific, Naperville, lll.), and bacteria were inoculated onto monolayers as described above. Following the appropriate incubation period, monolayers were rinsed four times with PBS and fixed with 2% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4, at 4°C for 2 h. After being washed with 0.1 M sodium phosphate buffer, samples were postfixed with 1% OS04 in sodium phosphate buffer for 90 min and then incubated in buffer overnight. Samples were next washed with distilled water and stained with 0.25% aqueous uranyl acetate for 1 h. They were dehydrated with a series of graded ethanol solutions and embedded in a firm Spurr resin. Samples were sectioned, stained with uranyl acetate and lead citrate, and examined in a Phillips 201c electron microscope. (ii) Scanning electron microscopy. Monolayers were prepared on glass cover slips as described for radiolabeled adherence assays. They were inoculated with bacteria, incubated for variable periods of time, and then rinsed four times with PBS. Samples were fixed with 2% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4, for 2 h and then dehydrated in a critical-point apparatus (Polaron). After a gold evaporation step, samples were examined with a JEOL scanning electron microscope. (iii) Light microscopy. Monolayers were prepared on glass cover slips as described above. After inoculation of bacteria and incubation for the desired period of time, monolayers were rinsed four times with PBS. Samples were fixed with high-performance liquid chromatography-grade methanol for 15 min and were then stained with Giemsa stain diluted 1:20 for 30 min. They were rinsed four times with distilled water
4038
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and were mounted on glass slides for viewing by light microscopy.
INFECT. IMMUN.
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RESULTS Adherence. Using radiolabeled bacteria and measuring adherence as a percentage of the original radioactivity, we noted minimal adherence by strain SUl after 1 h of incubation with the epithelial monolayer. However, adherence continued to increase over time, peaking about 10-fold higher by 4 to 6 h. In contrast, there was minimal nonspecific adherence with no significant change over time. E. coli K-12 demonstrated negligible adherence that failed to change appreciably over the course of an assay. Adherence by E. coli was measured for up to 4 h; at later time points, the monolayer began to lose integrity. Figure 1A depicts results from a representative experiment. Adherence by H. influenzae SUl gradually increased from 0.5% at 1 h to greater than 8% by 6 h. Because of bacterial replication that occurred during the course of the assay, the actual number of adherent bacteria per eucaryotic cell increased more than 100-fold between hours 1 and 6 (Fig. 1B). The kinetics of adherence were similar for inocula between 106 and 108 CFU (data not shown). Examination of Giemsa-stained samples and results from the viable-count adherence assay confirmed this gradual increase in adherence over time with a peak at 4 to 6 h (data not shown). This increment in adherence with time suggested a dynamic interaction between infecting H. influenzae and the epithelial monolayer. To begin to explore the nature of this interaction, we examined adherence to a monolayer that had been chemically fixed for 2 h with 2% glutaraldehyde. Fixation virtually abolished bacterial attachment (Fig. 2), implying the importance of viable epithelial cells or a receptor cross-linked by glutaraldehyde or both. In contrast, treatment of monolayers with emetine dihydrochloride (25), a selective inhibitor of eucaryotic protein synthesis (9), had no effect on adherence. In initial studies of bacterial events occurring during the course of the bacterium-epithelial cell interaction, we pretreated bacteria with the bactericidal antibiotic gentamicin and then added these nonviable bacteria to the cell monolayer. Adherence by nonviable bacteria was markedly reduced compared with that by viable organisms (Fig. 3). By 6 h, there was an average of two bacteria per eucaryotic cell. Incubation at 4°C produced a similar inhibition of adherence. Addition of a bacteriostatic concentration of tetracycline (6 ,ug/ml) to the monolayers just prior to inoculation of viable bacteria again produced a marked inhibition of adherence, indicating the need for protein synthesis at some point following initial exposure to the epithelial cell monolayer. Examination by light microscopy confirmed the results depicted in Fig. 2 and 3. The above evidence demonstrating a requirement for bacterial de novo protein synthesis suggested that an adhesin might be induced during incubation. Bacteria sampled at 1, 2, 4, and 6 h were unable to agglutinate human erythrocytes, arguing against pili as the putative adhesin. Similarly, negative-staining electron microscopy of bacteria examined after incubation for 6 h revealed no piluslike structures. Identification of candidate adhesin molecules. To identify proteins that might be mediating the process of adherence, we compared profiles of outer membrane proteins synthesized during incubation with epithelial cells for adherent versus nonadherent populations of bacteria. In this experiment, methionine-free Dulbecco modified Eagle medium was substituted for the usual tissue culture medium and
A
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Time (hours post Inoculation)
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U S D11 0 1. U 0 a 0 OX 3
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100
50
0 0
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3
4
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Time (hours post Inoculation) FIG. 1. Adherence by radiolabeled H. influenzae SUl to Chang epithelial cells. Values represent the means and standard errors of the mean of triplicate measurements from a representative experiment. (A) Adherence values are expressed as the percentage of total counts inoculated that remained associated with the monolayer. The time course of adherence is plotted for strain SUl incubated with epithelial monolayers (A) or glass cover slips alone (0). Adherence to monolayers is also plotted for E. coli K-12 (l). (B) Adherence values are expressed as the average number of adherent bacteria per epithelial cell.
emetine dihydrochloride was added to monolayers to inhibit selectively eucaryotic cell protein synthesis. Bacteria were then inoculated onto monolayers and were radiolabeled with [35S]methionine from hours 2 to 4 of incubation. Following the period of radiolabeling, we separated bacteria remaining in the supernatant (nonadherent) from those associated with the epithelial monolayer (adherent). As a control, we simultaneously radiolabeled bacteria incubated in tissue culture medium alone (i.e., in the absence of a monolayer). Subsequently we isolated detergent-insoluble proteins from adherent bacteria, supernatant bacteria, and bacteria labeled in medium alone. To confirm that emetine had completely inhibited protein synthesis by the epithelial monolayer, we performed the same procedure on an uninfected monolayer
H. INFLUENZAE ADHERENCE AND INVASION
VOL. 58, 1990
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Vol. 58, No. 12
Haemophilus influenzae Adheres to and Enters Cultured Human Epithelial Cells JOSEPH W. ST. GEME III1* AND STANLEY FALKOW12 Department of Microbiology and Immunology, Stanford University, Stanford, California 94305-5402,1 and
National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, Montana 598402 Received 20 June 1990/Accepted 27 September 1990
Haemophilus influenzae is a common commensal organism of the human respiratory tract that initiates infection by colonizing the nasopharyngeal epithelium. In some individuals, colonization is followed by localized respiratory tract or systemic disease. To gain insight into the mechanisms by which H. influenzae attaches to and persists within the nasopharynx, we examined the interactions between a nonpiliated clinical isolate of H. influenzae and human epithelial cells. We noted substantial adherence that occurred independently of pili and required viable bacteria capable of de novo protein synthesis. Comparison of profiles of outer membrane proteins synthesized during incubation with epithelial cells for adherent and nonadherent bacteria identified several candidate adhesin molecules. In addition, a small number of adherent bacteria were capable of entering epithelial cells in a process that was inhibited by cytochalasin D and colchicine. The suggestion from our studies is that one or more of several newly synthesized nonpilus bacterial proteins are required for maximal in vitro adherence and invasion. We speculate that H. influenzae entry into epithelial cells may provide a mechanism for evasion of host defenses, thereby allowing persistence in the nasopharynx.
nasopharynx, we examined the interactions between a clinical isolate of H. influenzae and tissue culture cells derived from normal human conjunctival epithelium. We noted substantial adherence that occurred independently of pili and required viable bacteria capable of de novo protein synthesis. In addition, we observed that a small percentage of adherent organisms were capable of epithelial cell entry in a process that involved actin filaments and microtubules. (This work was presented in part at the Society for Pediatric Research Meeting, Anaheim, Calif., 1990.)
Haemophilus influenzae is a common commensal organism of the human respiratory tract. In addition, this bacterium is an important cause of both localized respiratory tract and systemic (invasive) disease (27). Nonencapsulated, nontypeable strains account for the majority of local disease, including otitis media, sinusitis, bronchitis, and conjunctivitis. In contrast, serotype b strains are responsible for over 95% of cases of H. influenzae invasive disease (26). It is widely believed that disease due to H. influenzae begins with colonization of the nasopharyngeal epithelium, followed by either contiguous spread within the respiratory tract or invasion of the bloodstream (19). Despite the importance of nasopharyngeal colonization, characteristics of H. influenzae that promote its survival in the nasopharynx remain poorly defined. In 1982, Guerina et al. (10) and Pichichero et al. (21) independently reported the presence of pili in selected isolates of H. influenzae type b. These investigators noted a correlation between piliation and increased adherence to human oropharyngeal cells and erythrocytes, suggesting a role for pili as adhesins. More recently, several investigators have presented evidence that H. influenzae may possess nonpilus adhesins as well. Farley et al. noted that nonpiliated strains of H. influenzae were capable of mucosal attachment to nasopharyngeal tissue in organ culture (6, 7). In a comparison of piliated and nonpiliated strains of H. influenzae b, Sable and co-workers found that adherence to HEp-2 cells was greater by nonpiliated strains (24). Bakaletz et al. recently studied a series of clinical isolates of nontypeable H. influenzae and observed no correlation between the degree of piliation and the ability to adhere to human oropharyngeal cells or chinchilla tracheal epithelium in organ culture; all isolates adhered regardless of their state of piliation (1). In an effort to gain additional insight into the mechanisms by which H. influenzae attaches to and persists within the *
MATERIALS AND METHODS Bacterial strains and culture conditions. H. influenzae SUl is a clinical isolate that was obtained from the Clinical Microbiology Laboratory at Stanford University Hospital. It is nonencapsulated and appears to be a nontypeable strain, on the basis of multilocus enzyme electrophoresis (kindly performed by J. Musser, University of Pennsylvania) and outer membrane protein analysis. Negative-staining electron microscopy demonstrated that this strain is nonpiliated (data not shown). Furthermore, erythrocyte selection failed to enrich for a piliated variant (4). SUl was stored frozen at -80°C in brain heart infusion broth with 20% glycerol. For all assays, bacteria were streaked from frozen aliquots onto fresh chocolate agar supplemented with 1% Supplement VX (Difco Laboratories, Detroit, Mich.) and were grown at 37°C in a 5% CO2 incubator overnight. For growth in broth, bacteria were suspended from an 18-h chocolate agar plate in brain heart infusion broth supplemented with hemin (10 ,xg/ml) and NAD (3.5 jig/ml). Escherichia coli K-12 was obtained from the Stanford University collection and is the Stanford wild-type prototrophic strain (ATCC 10798) (15). It was stored frozen at -80°C in LB broth with 50% glycerol and was grown either on LB agar plates or in LB broth (GIBCO, Grand Island, N.Y.). Tissue culture cells. Chang epithelial cells (Wong-Kilbourne derivative, clone 1-5c-4 [human conjunctival) were
Corresponding author. 4036
VOL. 58, 1990
obtained from the American Type Culture Collection (ATCC CCL 20.2). Cells were maintained in modified Eagle medium with Earle salts and nonessential amino acids (Irvine Scientific) supplemented with 10%c heat-inactivated fetal calf serum (GIBCO) and 2.0 mM L-glutamine. They were subcultivated every 2 to 5 days and were used between passages 3 and 50. For adherence and invasion assays, approximately 1.5 x 105 cells suspended in 0.5 ml of tissue culture medium were seeded into each well of 24-well tissue culture plates (Becton Dickinson Labware), which were then incubated at 37°C in 5% CO, for 18 to 20 h. In the case of radiolabeled adherence assays, cells were seeded onto 12-mm-diameter glass cover slips placed on the bottom of each well of tissue culture plates. Adherence assays. (i) Radiolabeled assay. Bacteria were suspended in supplemented brain heart infusion broth and grown at 37°C with aeration to late log phase (109 CFU/ml). They were pelleted, washed once with phosphate-buffered saline (PBS), pH 7.0, and resuspended in methionine assay medium (Difco). After incubation at 37°C with aeration for 15 min, 50 pCi of [3-5SJmethionine (Amersham, Arlington Heights, Ill.) was added to the suspension along with hemin and NAD, and incubation was continued for another 45 min. While there was minimal bacterial growth during the period of radiolabeling, on average the level of incorporation of radioactivity was 0.01 cpm per bacterium. Radiolabeled bacteria were washed three times with PBS, resuspended in PBS, and inoculated (10 ,ul, 1 x 107 to 2 x 107 CFU) onto monolayers. Tissue culture plates were incubated at 37°C (5% CO) for various times. After the desired incubation period, the infected monolayers were rinsed three times with PBS with gentle rocking to remove nonadherent bacteria. Nonspecific adherence was determined by inoculating radiolabeled bacteria onto cover slips without epithelial cells, followed by rinsing with PBS. Cover slips were transferred to vials containing 5 ml of aqueous counting scintillant (ACS II; Amersham), and counts were determined and compared with those of the inoculum. The expression of adherence as a percentage of the original radioactivity provided a measure of bacterial attachment which was independent of later bacterial events, such as replication. Calculations of the average number of adherent bacteria per eucaryotic cell accounted for bacterial replication and assumed no epithelial cell division during the course of an assay. (ii) Viable-count assay. Bacteria were grown to log phase as for the radiolabeled adherence assay. Approximately 1 x 107 to 2 x 107 CFU (10 RI) was inoculated directly from the broth culture onto monolayers, and tissue culture plates were incubated at 37°C in 5% CO,. Following the appropriate incubation period, monolayers were rinsed four times with PBS and then treated with trypsin-EDTA (0.05% trypsin, 0.5 mM EDTA) in PBS to release them from the plastic support. Well contents were agitated, and dilutions were plated on supplemented chocolate agar for H. inflienzae or LB agar for E. coli, yielding the number of adherent bacteria per monolayer. Invasion assay. To determine the number of bacteria entering the cells of an epithelial monolayer, we adapted the assay of Isberg and Falkow (12). Briefly, bacteria were grown and monolayers were infected as outlined above for the viable-count adherence assay. After the desired incubation, monolayers were rinsed three times with PBS, and fresh tissue culture medium containing gentamicin (100 ,ug/ml) was added. Tissue culture plates were incubated for another 2 h, rinsed twice with PBS, and then treated with trypsin-EDTA (0.05% trypsin, 0.5 mM EDTA) in PBS. Well
H. INFLUENZAE ADHERENCE AND INVASION
4037
contents were agitated, and dilutions were plated to quantitate the number of internalized bacteria per monolayer. Outer membrane protein analysis. Outer membrane fractions were prepared from samples of radiolabeled bacteria by the microprocedure of Carlone et al. (2), with minor modifications. Bacteria were initially pelleted by centrifugation at 5,000 x g and 4°C for 10 min. Pellets were washed once with 1 ml of cold 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, pH 7.4, and then resuspended in 1 ml of the same buffer in preparation for sonication. Samples were sonicated on ice with eight 10-s bursts at maximal output with a microsonicator and a 2.3-mm probe (Kontes). Intact cells were removed by centrifugation at 15,600 x g and 4°C for 2 min in a microcentrifuge (Brinkmann). Supernatants were decanted into fresh tubes and centrifuged at 15,600 x g and 4°C for 30 min to pellet bacterial membranes. Pellets were resuspended in 0.2 ml of 10 mM HEPES buffer (pH 7.4). Following the addition of 0.2 ml of 2%Y sarcosyl (N-lauroyl sarcosine) in 10 mM HEPES buffer (pH 7.4) to solubilize cytoplasmic membranes, samples were incubated at room temperature for 30 min with intermittent mixing. Insoluble fractions containing outer membranes were collected by centrifugation at 15,600 x g and 4°C for 30 min. The outer membranes were suspended in equal quantities of distilled water and Laemmli buffer (14) and were stored at -20°C. Samples were heated at 100°C for 5 min, and 100,000 cpm was loaded onto sodium dodecyl sulfate-polyacrylamide gels. Electrophoresis was performed at a constant current of 25 mA with stacking and resolving gels that contained 5 and 10% polyacrylamide, respectively (14). Gels were stained with Coomassie blue for 30 min and were destained overnight. Autoradiograms were made with XAR-5 film. Microscopy. (i) Transmission electron microscopy. Chang epithelial cells were seeded into Contur Permanox tissue culture dishes (35 by 10 mm; Miles Scientific, Naperville, lll.), and bacteria were inoculated onto monolayers as described above. Following the appropriate incubation period, monolayers were rinsed four times with PBS and fixed with 2% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4, at 4°C for 2 h. After being washed with 0.1 M sodium phosphate buffer, samples were postfixed with 1% OS04 in sodium phosphate buffer for 90 min and then incubated in buffer overnight. Samples were next washed with distilled water and stained with 0.25% aqueous uranyl acetate for 1 h. They were dehydrated with a series of graded ethanol solutions and embedded in a firm Spurr resin. Samples were sectioned, stained with uranyl acetate and lead citrate, and examined in a Phillips 201c electron microscope. (ii) Scanning electron microscopy. Monolayers were prepared on glass cover slips as described for radiolabeled adherence assays. They were inoculated with bacteria, incubated for variable periods of time, and then rinsed four times with PBS. Samples were fixed with 2% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4, for 2 h and then dehydrated in a critical-point apparatus (Polaron). After a gold evaporation step, samples were examined with a JEOL scanning electron microscope. (iii) Light microscopy. Monolayers were prepared on glass cover slips as described above. After inoculation of bacteria and incubation for the desired period of time, monolayers were rinsed four times with PBS. Samples were fixed with high-performance liquid chromatography-grade methanol for 15 min and were then stained with Giemsa stain diluted 1:20 for 30 min. They were rinsed four times with distilled water
4038
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and were mounted on glass slides for viewing by light microscopy.
INFECT. IMMUN.
a U
RESULTS Adherence. Using radiolabeled bacteria and measuring adherence as a percentage of the original radioactivity, we noted minimal adherence by strain SUl after 1 h of incubation with the epithelial monolayer. However, adherence continued to increase over time, peaking about 10-fold higher by 4 to 6 h. In contrast, there was minimal nonspecific adherence with no significant change over time. E. coli K-12 demonstrated negligible adherence that failed to change appreciably over the course of an assay. Adherence by E. coli was measured for up to 4 h; at later time points, the monolayer began to lose integrity. Figure 1A depicts results from a representative experiment. Adherence by H. influenzae SUl gradually increased from 0.5% at 1 h to greater than 8% by 6 h. Because of bacterial replication that occurred during the course of the assay, the actual number of adherent bacteria per eucaryotic cell increased more than 100-fold between hours 1 and 6 (Fig. 1B). The kinetics of adherence were similar for inocula between 106 and 108 CFU (data not shown). Examination of Giemsa-stained samples and results from the viable-count adherence assay confirmed this gradual increase in adherence over time with a peak at 4 to 6 h (data not shown). This increment in adherence with time suggested a dynamic interaction between infecting H. influenzae and the epithelial monolayer. To begin to explore the nature of this interaction, we examined adherence to a monolayer that had been chemically fixed for 2 h with 2% glutaraldehyde. Fixation virtually abolished bacterial attachment (Fig. 2), implying the importance of viable epithelial cells or a receptor cross-linked by glutaraldehyde or both. In contrast, treatment of monolayers with emetine dihydrochloride (25), a selective inhibitor of eucaryotic protein synthesis (9), had no effect on adherence. In initial studies of bacterial events occurring during the course of the bacterium-epithelial cell interaction, we pretreated bacteria with the bactericidal antibiotic gentamicin and then added these nonviable bacteria to the cell monolayer. Adherence by nonviable bacteria was markedly reduced compared with that by viable organisms (Fig. 3). By 6 h, there was an average of two bacteria per eucaryotic cell. Incubation at 4°C produced a similar inhibition of adherence. Addition of a bacteriostatic concentration of tetracycline (6 ,ug/ml) to the monolayers just prior to inoculation of viable bacteria again produced a marked inhibition of adherence, indicating the need for protein synthesis at some point following initial exposure to the epithelial cell monolayer. Examination by light microscopy confirmed the results depicted in Fig. 2 and 3. The above evidence demonstrating a requirement for bacterial de novo protein synthesis suggested that an adhesin might be induced during incubation. Bacteria sampled at 1, 2, 4, and 6 h were unable to agglutinate human erythrocytes, arguing against pili as the putative adhesin. Similarly, negative-staining electron microscopy of bacteria examined after incubation for 6 h revealed no piluslike structures. Identification of candidate adhesin molecules. To identify proteins that might be mediating the process of adherence, we compared profiles of outer membrane proteins synthesized during incubation with epithelial cells for adherent versus nonadherent populations of bacteria. In this experiment, methionine-free Dulbecco modified Eagle medium was substituted for the usual tissue culture medium and
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Time (hours post Inoculation)
U0 .0 =
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U S D11 0 1. U 0 a 0 OX 3
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0 0
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Time (hours post Inoculation) FIG. 1. Adherence by radiolabeled H. influenzae SUl to Chang epithelial cells. Values represent the means and standard errors of the mean of triplicate measurements from a representative experiment. (A) Adherence values are expressed as the percentage of total counts inoculated that remained associated with the monolayer. The time course of adherence is plotted for strain SUl incubated with epithelial monolayers (A) or glass cover slips alone (0). Adherence to monolayers is also plotted for E. coli K-12 (l). (B) Adherence values are expressed as the average number of adherent bacteria per epithelial cell.
emetine dihydrochloride was added to monolayers to inhibit selectively eucaryotic cell protein synthesis. Bacteria were then inoculated onto monolayers and were radiolabeled with [35S]methionine from hours 2 to 4 of incubation. Following the period of radiolabeling, we separated bacteria remaining in the supernatant (nonadherent) from those associated with the epithelial monolayer (adherent). As a control, we simultaneously radiolabeled bacteria incubated in tissue culture medium alone (i.e., in the absence of a monolayer). Subsequently we isolated detergent-insoluble proteins from adherent bacteria, supernatant bacteria, and bacteria labeled in medium alone. To confirm that emetine had completely inhibited protein synthesis by the epithelial monolayer, we performed the same procedure on an uninfected monolayer
H. INFLUENZAE ADHERENCE AND INVASION
VOL. 58, 1990
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