Characterization of adherence of nontypeable Haemophilus influenzae to human epithelial cells

The adherence of 58 nontypeable Haemophilus influenzae isolates obtained from patients with otitis media or chronic obstructive pulmonary disease (COPD) and obtained from the throats of healthy individuals to Chang and NCI-H292 epithelial cells was compared. Otitis media isolates, but not COPD isolates, adhered significantly more to both cell lines than did throat isolates. Since high-molecular-weight (HMW) proteins are major adhesins of nontypeable H. influenzae, the isolates were screened for HMW protein expression by Western blotting with two polyclonal sera and PCR with hmw-specific primers. Twenty-three of the 32 adhering isolates (72%) and only 1 of the 26 nonadherent strains were HMW protein or hmw gene positive. Among the 32 isolates adhering to either cell line, 5 different adherence patterns were distinguished based on the inhibiting effect of dextran sulfate. Using H. influenzae strain 12 expressing two well-defined HMW proteins (HMW1 and HMW2) and its isogenic mutants as a reference, we observed HMW1-like adherence to both cell lines for 16 of the 32 adherent isolates. Four others showed HMW2-like adherence to NCI-H292. Of the three other patterns of adherence, one probably also involved HMW protein. Screening of the isolates with six HMW-specific monoclonal antibodies in a whole-cell enzyme-linked immunosorbent assay showed that the HMW proteins of COPD isolates and carrier isolates were more distinct from the HMW proteins from H. influenzae strain 12 than those from otitis media isolates. Characterization of the HMW protein of a COPD isolate by adherence and DNA sequence analysis showed that despite large sequence diversity in the hmwA gene, probably resulting in the antigenic differences, the HMW protein mediated the HMW2-like adherence of this strain.     

Blocking of fimbria-mediated adherence of Haemophilus influenzae by sialyl gangliosides.

The structure of the receptor for the fimbriae of Haemophilus influenzae on human oropharyngeal epithelial cells and erythrocytes was determined in inhibition experiments with various sugars, glycolipids, and glycoproteins. Of 30 monosaccharides and disaccharides at a concentration of 0.1 M and of 3 polysaccharides at a concentration of 1 mg/ml, none inhibited fimbria-specific adherence and hemagglutination. Inhibition was obtained with gangliosides GM1, GM2, GM3, and GD1a in nanomolar concentrations, whereas the asialo derivative of GM1, sialyl-lactose, and sialoglycoproteins were poor inhibitors. These findings indicate that sialyl-lactosylceramide (GM3) is the minimal structure for the fimbria-dependent binding of H. influenzae to its receptor on oropharyngeal epithelial cells and erythrocytes. As is the case with GM2, substitution of GM3 with N-acetylgalactosamine makes the molecule a 10-fold-better receptor analog.     

Specific binding of Haemophilus influenzae to minor gangliosides of human respiratory epithelial cells.

Gangliosides are sialylated glycosphingolipids that serve as receptors for various bacteria. To investigate endogenous gangliosides of human respiratory epithelial cells as potential receptors for Haemophilus influenzae, three strains, including nontypeable H. influenzae (NTHI) 1479, and isogenic fimbriated (f+) and nonfimbriated (f0) H. influenzae type b 770235, were 3H labeled and overlaid on two-dimensional thin-layer chromatography (TLC) plates containing either purified HEp-2 gangliosides or murine brain gangliosides. NTHI 1479 bound exclusively to two distinct minor ganglioside doublets, with mobilities near that of GM1. These minor gangliosides comprised only 14.2 and 9.4% of the total, respectively. NTHI 1479 also bound to a distinct ganglioside of human macrophages whose chromatographic mobilities closely resemble those of one of the NTHI-binding gangliosides of HEp-2 cells. H. influenzae type b 770235 f+ and f0 each bound to a different minor HEp-2 ganglioside doublet, with proportionately weaker affinity for a major ganglioside doublet. Remarkably, none of the three strains bound to any murine brain gangliosides. Moreover, when 80 to 90% of sialic acid residues were enzymatically removed from HEp-2 gangliosides, NTHI 1479 binding was proportionately impaired, compared with untreated controls. Our findings support a role for specific gangliosides of specific cells as receptors for H. influenzae strains. Our findings further demonstrate that individual minor gangliosides possess unique biological properties.     

Relationship of Haemophilus influenzae type b pilus structure and adherence to human erythrocytes.

Six strains of Haemophilus influenzae type b, some expressing immunologically different pili, showed identical patterns of binding to erythrocytes that were characterized for 38 blood group antigens. All six strains appeared to bind to the Anton antigen, as they agglutinated all erythrocytes tested except cord erythrocytes and those characterized as Lu(a-b-), dominant type, including Anton-negative cells.     

Role of capsule in adherence of Haemophilus influenzae type b to human buccal epithelial cells.

The role of capsule in the adherence of Haemophilus influenzae type b to human epithelial cells in vitro was examined. A group of 30 nonadherent isolates did not differ in degree of encapsulation compared with their respective adherent variants. Furthermore, capsule-deficient mutants of both nonadherent and adherent variants did not differ significantly in degree of adherence compared with their respective encapsulated parents. These data indicate that capsule does not significantly influence the adherence of H. influenzae type b to human epithelial cells.     

Haemophilus influenzae adheres to and enters cultured human epithelial cells.

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.     

Variable adherence of fimbriated Haemophilus influenzae type b to human cells.

The attachment of isogenic fimbriated and nonfimbriated Haemophilus influenzae type b variants to human cells was studied by using a radioactive assay and an indirect immunofluorescent assay. As described previously, fimbriated H. influenzae variants adhered to a greater extent than nonfimbriated variants to human buccal epithelial cells (2.1 and 0.29 bacteria per cell, respectively, as determined by the radioactive assay [P less than 0.05]; 7.6 and 1.6 bacteria per cell, respectively, as determined by the immunofluorescent assay [P less than 0.01]). As the concentration of fimbriated bacteria was increased, so were the numbers of adherent bacteria; in contrast, increasing the bacterial concentration had a much smaller effect on adherence of nonfimbriated H. influenzae type b. The distribution of bacteria on the buccal cells also differed. Whereas 37% of the buccal cells failed to bind nonfimbriated H. influenzae type b, failure to bind was observed for only 4% of the buccal cells exposed to fimbriated H. influenzae. In contrast, adherence to human foreskin fibroblasts was low regardless of the presence of fimbriae. On the other hand, fimbriated H. influenzae type b adhered less well than nonfimbriated variants to HEp-2 cells (1.6 and 3.8 bacteria per cell, respectively, as determined by the radioactive assay [P less than 0.05]; 1.3 and 4.8 bacteria per cell, respectively, as determined by the immunofluorescent assay [P less than 0.02]). Whereas adherence to HEp-2 cells increased considerably as the concentration of nonfimbriated bacteria was increased, there was only a small enhancement of adherence with an increase in the concentration of fimbriated H. influenzae type b. Furthermore, only 16% of the HEp-2 cells failed to bind nonfimbriated H. influenzae type b, whereas 50% failed to bind fimbriated H. influenzae type b. These data indicate that H. influenzae type b may contain two adhesins. One is associated with fimbriae and enables adherence to buccal cells, whereas the other is nonfimbrial and is associated with adherence to HEp-2 cells. It is not known whether either of these adhesins plays a role in pathogenesis.     

Adherence of Haemophilus influenzae to buccal epithelial cells.

The role of adherence of Haemophilus influenzae to epithelial surfaces in the pathogenesis of infection is unknown. Fluorescent-antibody and radiolabeled adherence methods were adapted to study H. influenzae adherence to human buccal epithelial cells. By the fluorescent-antibody method, 19 of 21 (90%) nontypable H. influenzae strains were found to be adherent compared with 2 of 42 (5%) type b strains (P less than 0.0001). Using a radiolabeled adherence method, we found that 9 of 12 (75%) nontypable H. influenzae strains were adherent to buccal epithelial cells whereas only 3 of 32 (9%) type b strains were adherent (P = 0.001). Results of H. influenzae adherence examined by both methods correlated significantly (P = 0.01). H. influenzae adherence to adult pharyngeal, nasal, and buccal epithelial cells was comparable. Type b H. influenzae did not adhere to the buccal epithelial cells of well children, children with H. influenzae type b disease, or children with upper respiratory infections. In contrast, nontypable H. influenzae did adhere to the buccal epithelial cells of well children and children with upper respiratory infections. These observed in vitro differences in adherence between nontypable and type b H. influenzae strains may explain differences in colonization, pathogenesis, and types of infection due to nontypable and type b H. influenzae.     

Pilus-mediated adherence of Haemophilus influenzae to human respiratory mucins

Haemophilus influenzae, especially the nontypeable strains, are among the most common pathogens encountered in patients with chronic lung disease and otitis media. We and others have demonstrated that respiratory isolates of nontypeable H. influenzae bind to human mucins, but the mechanism of binding is not entirely clear. We have therefore examined the role of pili in the adherence of both type b and nontypeable H. influenzae to human respiratory mucins. We used isogenic H. influenzae strains with a mutation in the structural gene for pilin (hifA), a laboratory H. influenzae strain transformed with a type b pilus gene cluster (from strain C54), antibodies raised against H. influenzae HifA, and Escherichia coli strains carrying a cloned type b pilus gene cluster (from strain AM30) in these studies. All bacteria lacking HifA or the pilus gene cluster had decreased adherence of piliated H. influenzae to mucins, and Fab fragments of anti-HifA antibodies inhibited the adherence. E. coli strains carrying the cloned type b pilus gene cluster were six to seven times more adhesive than strains carrying the vector. The role of other putative adhesins was not examined and thus cannot be excluded, but these studies support a role for pili in the binding of H. influenzae to human respiratory mucins.     

Interaction of Haemophilus influenzae with human erythrocytes and oropharyngeal epithelial cells is mediated by a common fimbrial epitope.

The role of fimbriae in the adherence of Haemophilus influenzae to oropharyngeal epithelial cells and the hemagglutination (HA) of human Anton-positive erythrocytes was examined. HA of bacteria was lost after shearing. Fimbriae purified from the extracellular fluid caused HA and bound to oropharyngeal epithelial cells, as analyzed with immunoperoxidase staining, in a way which was similar to the adherence of bacteria to these cells: binding was over the entire surface of the cells and showed cell-to-cell variation. The specific role of fimbriae in HA and adherence was further examined by inhibition experiments with monoclonal antibodies elicited against the isolated fimbriae. These monoclonal antibodies bound along the entire length of the fimbriae, as seen by immunogold electron microscopy. The monoclonal antibodies and their Fab fragments inhibited HA (reduction in titer from 1:512 to 1:128 and 1:64, respectively) and inhibited the adherence of the homologous H. influenzae strain and of three of eight heterologous H. influenzae strains to oropharyngeal epithelial cells. These results indicate that fimbriae are involved in adherence and HA and that the binding site for the monoclonal antibodies on the fimbriae is not common on all strains.     

Involvement of lipooligosaccharides of Haemophilus influenzae and Neisseria meningitidis in defensin-enhanced bacterial adherence to epithelial cells

Stimulated neutrophils release a variety of antimicrobial peptides, including neutrophil defensins (HNP1-4). We have previously reported that neutrophil defensins enhanced the adherence of Haemophilus influenzae and Neisseria meningitidis to cultured respiratory epithelial cells. In this study, the effect of defensins on the adherence of H. influenzae and N. meningitidis lipooligosaccharide (LOS) mutants to epithelial cells was tested. Neutrophil defensins enhanced the adherence of the oligosaccharide mutants of H. influenzae and N. meningitidis, whilst the adherence of the lipid A mutants B29 of H. influenzae and lpxL1 and lpxL2 of N. meningitidis was not or only moderately stimulated by neutrophil defensins. The adherence of the N. meningitidis LOS negative mutant lpxA was not enhanced by defensins. These findings suggested that the secondary fatty acids of lipid A were involved in the defensin-enhanced adherence. LOS from strain H44/76 or HNP-LOS complexes did not affect or stimulate the adherence of N. meningitidis, although the defensin-enhanced adherence is specific for certain bacterial species having LOS in their outer membrane. These results indicated that LOS is involved in the defensin-enhanced adherence. However, the mechanism by which defensins and LOS interact with epithelial cells to promote bacterial adherence remains to be resolved.     

Method for testing adherence ofHaemophilus influenzae to human buccal epithelial cells

A method for testing adherence ofHaemophilus influenzae strains to buccal mucosal cells is described. Bacteria grown in broth for 4 h were mixed with buccal mucosal cells. After elimination of unattached bacteria by repeated cycles of centrifugation and resuspension in PBS, the number of attached bacteria was counted microscopically. Optimal results were obtained with an early log-phase bacterial culture at a concentration of 10⁹ bacteria/ml mixed with 2×10⁴ cells/ml and incubated at 37 °C for 60 min. This assay showed an at least ten times higher rate of adherence forHaemophilus influenzae than previous studies. Nontypeable strains attached in higher numbers than strains with the type b capsule. Adherence was related to the frequency of nontypeable strains rather than to the site of isolation or type of infection. Thus all the isolates from middle ear fluid were nontypeable, and all but one adhered. The results suggest a difference in virulence mechanisms between type b and nontypeableHaemophilus influenzae strains.     

Evidence of Haemophilus ducreyi adherence to and cytotoxin destruction of human epithelial cells

The adherence of ten different Haemophilus ducreyi strains to cultured human epithelial cells and the subsequent destruction of these cells was investigated in vitro using H Ep-2 and HeLa cells. Bacterial adherence was measured with two assays, one employing viable bacteria and the other radiolabeled bacteria. In addition, the capacity of H. ducreyi to invade/penetrate the H Ep-2 cells was examined. Differential interference contrast and transmission electron microscopy techniques were also used. In both cell lines, all ten strains of H. ducreyi manifested substantial adherence (the rates being 4-20% of the inoculum), irrespective of whether the bacteria were cultivated on solid or liquid media. Bacterial adherence reached a peak after about 2-3 h of incubation, though it was already manifest after only 15 min, a finding suggesting constitutive rather than inducible properties of H. ducreyi adhesins to be involved. The adherence capacity was diminished, but not totally abolished, when bacteria were heat-treated at 100°C for 30 min, indicating the adhesins to be fairly stable. On the other hand, treatment of H Ep-2 cells with methanol, glutaraldehyde and emetine dichloride significantly reduced the adherence, indicating viable eukaryotic cells with native surface structures to be involved in bacterial adherence. This capacity of H. ducreyi to adhere to H Ep-2 cells was confirmed both by electron microscopy and by differential interference microscopy. Some adherent bacteria were also capable of penetrating epithelial cells, as observed with an invasion assay and confirmed by transmission electron microscopy. Further incubation of the cell monolayers with the ten strains resulted in the cell-death and total damage of monolayers for seven cytotoxin-producing strains, indicating cytotoxin action to be responsible for the destruction of the monolayer. All strains manifested capacity to survive and multiply on the cell monolayer. We propose the first step in the pathogenesis of chancroid to be the adherence of bacteria to epithelial cells, followed by the action of cytotoxin and further bacterial proliferation. This sequence of events is suggested to result in the production of genital ulcers by H. ducreyi organisms.     

The effect of influenza virus on the adherence of Haemophilus influenzae to human cells in tissue culture.

The adherence of eleven strains of Haemophilus influenzae to MRC5 cells was studied and compared with adherence of the same eleven strains to MRC5 cells infected with influenza A/NWS/33 virus. Per cent Adhesion (the proportion of cells to which more than two bacteria were adhering) was estimated. Organisms grown on solid media adhered better than those grown in liquid media though the difference was not statistically significant (t test for independent means). A wide range of % Adhesion values for organisms grown on solid media to control cells was exhibited (1-88%). Ten of eleven strains grown on solid media or in broth showed increased adherence to influenza virus infected cells; this difference was significant (P less than 0.05, t test for independent means). The effect of virus infection in increasing % Adhesion was inversely proportional to the adhesiveness of the strain in question to uninfected cells. Strains that adhered most efficiently to control cells showed little increase in % Adhesion following virus infection, while strains that adhered poorly to control cells showed large increases in % Adhesion following virus infection.     

Adherence of Haemophilus influenzae to human buccal and pharyngeal epithelial cells: relationship to pilation

An improved understanding of the role of pili in adherence of Haemophilus influenzae type b to human epithelial cells (EC) would enhance knowledge of the pathogenesis of H. influenzae b infections. In this study a highly sensitive in-vitro assay allowed the quantitative assessment of H. influenzae b adherence to EC. The degree of adherence was influenced by incubation time, temperature, bacteria/EC ratio, EC type and the growth phase of the bacteria. Most serially subcultured (SC) capsular type-b strains originally isolated from cerebrospinal fluid, blood, nasopharynx or throat gave similar low degrees of adherence, as did representative single strains of capsular types a, c, d, e and f. SC non-capsulated H. influenzae strains adhered in significantly greater numbers than most SC capsulated strains (p less than 0.001). One SC type-b strain isolated from a throat, with stable piliation, adhered in very high numbers despite capsulation. Piliated subpopulations selected from type-b capsulated strains adhered in greater numbers than did their parent strains. These data suggest that capsulation of H. influenzae is a deterrent to adherence of the bacteria to EC. However, the presence of pili may allow type-b organisms to overcome the effects of capsulation.     

Pneumococcal adherence to human epithelial cells.

Evidence is presented that pneumococci adhere poorly to oropharyngeal cells in vitro and that the capsule may interfere with adherence. A brief survey indicated that pneumococci may also adhere poorly in vivo.     

Protein D expression promotes the adherence and internalization of non-typeable Haemophilus influenzae into human monocytic cells

Protein D, having a glycerol-3-phosphodiester phosphodiesterase activity, is found at the surface of all Haemophilus influenzae strains and is a possible virulence factor. In the present study, the involvement of protein D in the entry of NTHi into human monocytic cells is reported. Primary monocytes and the monocytic cell lines U-937 and THP-1 were infected with NTHi strain 772 and the mutant 772 Delta hpd 1 (lacking the gene for protein D). NTHi 772 adhered to and entered monocytic cells up to four-fold more efficiently compared to 772 Delta hpd 1. When an Escherichia coli transformant expressing protein D was incubated with monocytic cells, the number of intracellular bacteria increased 1.6-fold compared to protein D-deficient controls. Any correlation between internalization and phosphorylcholine expression was not detected. In conclusion, our data suggest that surface-expressed protein D promotes the adherence of NTHi to human monocytes leading to a higher number of internalized bacteria.     

Loss of capsule expression by Haemophilus influenzae type b results in enhanced adherence to and invasion of human cells.

Haemophilus influenzae type b is a common cause of systemic bacterial disease in children, and the serotype b capsule is a major determinant of virulence. Nevertheless, as a consequence of the genetic configuration of the capb locus, type b strains become capsule deficient at a high frequency. To investigate the potential biological relevance of the predisposition to capsule loss, we compared the adherent and invasive abilities of several strains of H. influenzae type b and their isogenic capsule-deficient mutants by using cultured human epithelial cells. In all cases the capsule-deficient mutant demonstrated significantly greater adherence and invasion than the encapsulated parent. Transformation of one capsule-deficient mutant to restore encapsulation resulted in a marked decrease in adherence and invasion. All strains were capable of adherence and invasion by a pilus-independent mechanism. We conclude that capsule loss by H. influenzae type b results in enhanced in vitro adherence and invasion, properties that may be relevant to colonization of the nasopharynx and persistence within the respiratory tract. These observations suggest an explanation for the evolution of the capb locus as directly repeated segments of DNA with a consequent predisposition to recombination resulting in capsule loss.     

Adhesive properties of Haemophilus influenzae to different human cells

The adhesion of 19 nontypable strains and 3 typable (type b) Haemophilus influenzae to human cells was examined using buccal epithelial cells (BEC), the continuous HEp-2 cell line and human 0 erythrocytes. The strains were classified into three phenotypes, according to their adhesive properties. Phenotype 1 consists of strains that adhere to both buccal epithelial cells and HEp-2 cells. Phenotype 2 consists of strains that adhere to both buccal epithelial cells and erythrocytes and strains belonging to phenotype 3 adhere to none of the three cell types used. Among 22 strains studied, 18 (81.8%) belonged to phenotype 1, 2 (9.1%) to phenotype 2 and 2 (9.1%) to phenotype 3. Fimbriae were observed for 11 (61%) among the 18 adherent strains belonging to phenotype 1. The 7 nonpiliated strains adhered with a significant adhesion index, thus this results would indicate that a non fimbrial adhesin exists.