Binding of Haemophilus influenzae to purified mucins from the human respiratory tract.

Mucins are high-molecular-weight glycoproteins and major constituents of the mucus layer which covers the airway surface. We have studied the interactions between bacteria, mucins, and epithelial cells from the human respiratory tract. Nontypeable strains of Haemophilus influenzae were found to bind to purified airway mucins in suspension and on solid phase. Mucins in suspension inhibited the attachment of these strains to nasopharyngeal epithelial cells, while mucin coating of the cells enhanced their binding. In contrast, strains of Streptococcus pneumoniae and encapsulated and other nontypeable H. influenzae strains failed to interact with mucins. These H. influenzae strains used other strategies for adherence to epithelial cells. The type b strain 770235 attached via fimbriae but also expressed a subcapsular adhesin that was detected in a capsule- and fimbria-defective mutant. Mucin pretreatment of these bacteria did not inhibit adherence, but mucin pretreatment of epithelial cells inhibited adherence, probably by shielding of the receptors for these adhesins. Non-mucin-binding nontypeable and encapsulated H. influenzae strains would, therefore, adhere only after disruption of the mucus layer and exposure of cellular receptors. Differences in tissue toxicity and invasiveness among H. influenzae strains may also be influenced by the mucin interactions of the strains.     

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.     

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.     

Adhesion of nontypeable Haemophilus influenzae from blood and sputum to human tracheobronchial mucins and lactoferrin.

Nontypeable Haemophilus influenzae strains are the most common pathogens encountered in patients with chronic bronchitis. These organisms chronically colonize the airways of patients and occasionally cause bacteremia. Nontypeable H. influenzae strains have been demonstrated microscopically to bind to mucus, but quantitative studies of adhesion have not been published to date. We have therefore developed a reproducible microtiter plate assay to study mucin binding and have examined the adhesion of sputum and blood strains of nontypeable H. influenzae. The assay is similar to that described for Pseudomonas aeruginosa (S. Vishwanath and R. Ramphal, Infect. Immun. 45:197-202, 1984), but notably 2% Tween 20 is used to desorb bacteria from the wells to quantitate bacterial binding. Using a standard strain, we have established that 1 h of incubation is optimum with an inoculum of < or = 5 x 10(8) CFU/ml. The standard strain binds to bronchitic and cystic fibrosis mucins equally well but binds less to bronchiectasis mucins. It does not bind to bovine serum albumin or fetuin. We have also examined the levels of adhesion of freshly isolated sputum and bacteremia strains and find very significant differences in adhesion. Blood strains bound six to seven times less than sputum strains ([13.8 +/- 7] x 10(2) per well versus [102 +/- 43] x 10(2); P < 0.001). Studies with adhesion to lactoferrin, another glycosylated protein, revealed variable binding of respiratory strains but marked binding of blood strains compared with mucin. An isogenic pair of respiratory and blood isolates was examined by electron microscopy but did not show surface differences. We speculate that bacteremic strains studied may have masked, lost, or downregulated adhesin production to allow them to escape from mucins or upregulated adhesins for lactoferrin to invade the bloodstream.     

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.     

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.     

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.     

Pilus-mediated adherence of Escherichia coli K1 to human oral epithelial cells.

We examined the effect of host age and health status on the adherence of mannose-sensitive piliated Escherichia coli K1 to human oral epithelial cells. Mannose-sensitive piliated bacteria adhered in comparable numbers to newborn, older infant, and adult cells (125 +/- 61, 198 +/- 54, and 139 +/- 69 bacteria per cell, respectively). Prematurity and serious illness did not alter adherence in newborns. The increased susceptibility of premature newborns to E. coli K1 cannot be explained by enhanced epithelial cell adherence.     

Haemophilus influenzae in respiratory pathology in adults

Fifty patients were diagnosed bronchopulmonary Haemophilus infections, because of the production of a purulent sputum, containing at least 10(8) Haemophilus influenzae per ml. Among them were 36 males (average 52 years old) and 14 females (average 58 years old). There was a high percentage (64%) of smokers (over 30 packs/year) within this population, which also included heavy drinkers. The top incidence occurred in winter and spring. Most cases were related to an acute infection in a chronic bronchitis (26 cases). The other cases included 6 cancers, 6 sequelae of tuberculosis, 4 bronchiectasis, 4 asthma, and only 3 pulmonary consolidations. There usually was a low grade fever (only 8 cases reached or went beyond 38 degrees, while in 29 cases the body temperature kept below 38 degrees). The return to a normal temperature was obtained after 4 to 10 days of ampicillin therapy, with no fatal case within this series. The 50 strains were studied by the microbiology laboratory. The minimum inhibitory concentrations showed a peculiar response to ampicillin and erythromycin, and a less dramatic response to chloramphenicol and tetracyclin. Some strains were proved resistant (MIC over 4 micrograms per ml) to cefoxitine and cefamandole.     

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.     

Pilus-mediated binding of bovine enterotoxigenic Escherichia coli to calf small intestinal mucins.

In this study we show that the adhesion to mucus of the enterotoxigenic Escherichia coli strains responsible for diarrhea in calves involves a bacterium-mucin recognition phenomenon in which the bacterial pili and specific mucus receptors carried by the glycoproteins (2,000 to 400 kilodalton) play a major role. An adhesion maximum was observed at a pH of less than 6 (4.75 to 5.25). The sialic acids and galactose appeared to be at least partly responsible for the attachment of K99 pili, whereas F41 pili preferentially recognized desialylated receptors. The attachment of different strains of E. coli characterized by the presence of the three main pili, K99, F41, and FY, known to be responsible for the binding of enterotoxigenic E. coli to the intestinal epithelium of the calf, was studied using Scatchard and Hill analyses. The attachment mechanism of bacteria carrying K99 pili showed positive cooperativity. FY and F41 pili recognized independent receptor sites, the first on sialylated mucus and the second on sialidase-treated mucus. Moreover, F41 pili were found to bind the native mucus according to a negative cooperativity phenomenon. Finally, the recognition sites carried by bacterial pilins may be saturated by some animal glycoprotein glycans which are therefore adhesion inhibitors.     

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.     

Antibodies to Haemophilus influenzae type b polysaccharide affect bacterial adherence and multiplication.

Since immunization of infants with conjugated Haemophilus influenzae type b (Hib) capsular polysaccharide (PS) vaccines results in a reduction of colonization, we determined the inhibitory effect of anti-Hib PS on two steps in the colonization, i.e., adherence of H. influenzae to nasopharyngeal epithelium and bacterial growth. Monoclonal antibody (MAb) E117-5 specific for Hib PS inhibited at a concentration of at least 80 microg/ml in vitro the adherence of Hib strain 770235f+b+ to oropharyngeal epithelial cells by 50% (P <, 0.02), but this MAb and sera from children immunized with Hib PS conjugate vaccine (n = 10) were not inhibitory in final dilutions containing up to 20 microg of anti-Hib PS per ml. The growth of Hib strain 770235f+b+ did completely stop in the presence of 5 microg of anti-Hib PS MAb E117-5 per ml and human sera with an anti-Hib PS concentration of 2 microg/ml or more, in contrast to the growth of the nonencapsular variant strain 770235f+b0.     

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.     

Contribution of the major and minor subunits to fimbria-mediated adherence of Haemophilus influenzae to human epithelial cells and erythrocytes.

Fimbriae are colonization factors of the human pathogen Haemophilus influenzae in that they mediate bacterial adherence to human eukaryotic cells. The contribution of the major (HifA) and putative minor (HifD and HifE) subunits of H. influenzae fimbriae to fimbria-specific adherence was studied by using mutants that were inactivated in distinct fimbrial genes. Both the major and minor subunits were required for adherence of H. influenzae to oropharyngeal epithelial cells and human erythrocytes carrying the AnWj antigen. Cloning of defined H. influenzae fimbrial genes in an Escherichia coli strain with type 1 fimbriae yielded recombinants expressing high amounts of HifA-containing H. influenzae fimbriae either with or without coexpression of both H. influenzae minor subunits. Both clones exhibited the specific adherence properties of H. influenzae fimbriae, implying that the minor H. influenzae subunits are dispensable for adherence and that the adhesive domain resides in the major subunit, HifA. In H. influenzae itself, the minor subunits probably affect adherence by raising the number of fimbriae above the minimal level required to establish adherence.     

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.     

Frequency of fimbriation of nontypable Haemophilus influenzae and its ability to adhere to chinchilla and human respiratory epithelium.

To date, we have examined nearly 60 clinical isolates of nontypable Haemophilus influenzae (26 nasopharyngeal, 33 from middle ear effusions) and have found that 100% were fimbriated. The percentage of cells bearing fimbriae within each isolate varied from less than 10 to 100%, with fimbriae being either peritrichous or bipolar in distribution. Fimbriae were approximately 2.4 to 3.6 nm in width; however, there was a high degree of variability in both length and number of fimbriae per individual bacterial cell among these isolates. All isolates tested adhered to both human oropharyngeal cells and chinchilla tracheal epithelium regardless of the degree to which the particular isolate was fimbriate. The level or degree of fimbriation did not correlate with either site of isolation, biotype, strength of hemagglutination reaction, or type of effusion present in the ear. These appendages appear to be quite different from those described for type b H. influenzae in which the ability to adhere and strength of ability to hemagglutinate correlated strongly with degree of fimbriation.     

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.     

A comparison of the adherence of fimbriated and nonfimbriated Haemophilus influenzae type b to human adenoids in organ culture.

Adherence of fimbriated and nonfimbriated variants of a single strain of Haemophilus influenzae type b to organ cultures of human adenoidal tissue was measured by three assays, two of which were quantitative. In one assay, the adherence of radioactively labeled bacteria was measured; the numbers of CFU of bacteria per gram of adenoidal tissue were 16.0 +/- 6.7 for fimbriated bacteria and 10.2 +/- 4.0 for nonfimbriated bacteria (P less than 0.05). In the second assay, adherent CFU were determined directly; the results were 23.4 +/- 17.2 CFU/g of tissue for fimbriated bacteria and 5.1 +/- 2.2 CFU/g for the nonfimbriated variant (P less than 0.02). By combining data from the two assays it appears that fimbriated and nonfimbriated bacteria do not compete for the same site on the tissue, and that the adherent bacteria do not change their state of fimbriation under the assay conditions used. In contrast, the third assay, scanning electron microscopy, showed very poor adherence of nonfimbriated bacteria. Fimbriated bacteria, on the other hand, adhered in clusters to nonciliated epithelial cells. Overall, the data indicate that fimbriae enhance adherence of H. influenzae type b to a type of tissue that is a normal site of human colonization and that nonfimbriated bacteria adhere by a distinctly different mechanism.