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.     

Challenges for the development of vaccines against Haemophilus influenzae and Neisseria meningitidis

The development of protein-polysaccharide conjugate vaccines has had a major impact on Haemophilus influenzae type b disease. The application of this technology to Neisseria meningitidis is also striking, particularly for serogroup C. However, significant challenges exist for the development of vaccines against non-typeable H. influenzae and against N. meningitidis serogroup B. Issues such as non-vaccine-strain replacement and correlates of protection need to be addressed as well as the longer-term implications of vaccination against what are essentially 'normal' microflora.     

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.     

Adherence of Neisseria meningitidis to human epithelial cells.

Carrier strains of Neisseria meningitidis are recovered almost solely from the posterior pharynx and they are often nongroupable or rough. Invasive strains can be serogrouped (encapsulated). We studied adherence of both carrier- and patient-derived serogroupable and nongroupable meningococci to buccal epithelial and posterior pharyngeal cells. Fresh meningococcal isolates attached significantly better to pharyngeal than to buccal cells (P = 0.01). Strains that could be serogrouped adhered less than nongroupable strains (P less than 0.05). Meningococci passed in vitro became hemagglutinin negative and nonpiliated. Hemagglutinin-negative meningococci always adhered less to both epithelial cell types than the hemagglutinin-positive variants of the same strain. These results indicate that meningococcal pili probably mediate attachment to oropharyngeal cells, but encapsulation may reduce adherence. Localization of meningococci in the posterior pharynx is in part explained by the receptivity of the epithelial cells in this area for meningococci.     

Preparation of human hyperimmune globulin to Haemophilus influenzae b, Streptococcus pneumoniae, and Neisseria meningitidis

As a first step in exploring the feasibility of passive antibody prophylaxis and therapy of serious infections caused by common encapsulated bacteria, we have immunized healthy adults with Haemophilus influenzae type b vaccine, 14-valent pneumococcal vaccine, and meningococcal group A and C vaccine; collected plasma by repeated pheresis; and purified a hyperimmune globulin termed bacterial polysaccharide immune globulin by the cold-ethanol fractionation method of Cohn and Oncley. Specific antibacterial antibody concentrations were measured in individual donors before and after immunization. In addition, antibody concentrations were measured in plasma pools prepared from immunized donors and from unimmunized controls and in the immunoglobulin-containing Cohn-Oncley fractions II and III derived from the respective plasma pools. A comparison of Cohn-Oncley fractions II, which contain primarily immunoglobulin G and which are used therapeutically as immune globulin, revealed that antibody to H. influenzae type b was enriched 15.3-fold and that antibody to meningococcal serogroups and pneumococcal types was enriched a mean of 4.4-fold (range, 1.2- to 9.9-fold). Enrichment of antibacterial antibody in Cohn fraction III, which contains substantial amounts of immunoglobulin M and immunoglobulin A in addition to immunoglobulin G, closely paralleled that in fraction II. Only antibodies to pneumococcal types 1 and 7 were increased disproportionately in fraction III. Based on the clinical experience that conventional immune serum globulin at a dose of 100 mg/kg protects agammaglobulinemic patients for ca. 1 month, we estimate that bacterial polysaccharide immune globulin, in similar dosage, will provide protection from systemic H. influenzae type b infection for 4 to 6 months and from pneumococcal and meningococcal infections for 3 to 4 months.     

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.     

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.     

Antigenic diversity of lipooligosaccharides of nontypable Haemophilus influenzae.

The lipooligosaccharides (LOS) of nontypable Haemophilus influenzae are an antigenically heterogeneous group of macromolecules. Immunodiffusion and enzyme-linked immunosorbent assay inhibition studies with phenol-water-extracted LOS and absorbed antisera specific for the oligosaccharide portion of the LOS identified six LOS strain-specific antigens. To facilitate screening large numbers of strains to search for LOS antigenic heterogeneity, a system utilizing proteinase K whole cell digests in Western blots was developed. Seventy-two nontypable H. influenzae LOS extracts were analyzed in this Western blot assay. Thirty-seven of these extracts could be segregated into 10 antigenically distinct LOS groups based on immunologic recognition by one or more of the rabbit antisera. Thirty-five of the strains did not contain these LOS antigens. These results demonstrate that antigenic differences exist among the LOS of nontypable H. influenzae strains, and this heterogeneity has the potential to be used to establish an LOS-based serogrouping system.     

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.     

Common antigenic domains in transferrin-binding protein 2 of Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae type b.

There is now considerable evidence to show that in the Neisseria and Haemophilus species, membrane receptors specific for either transferrin or lactoferrin are involved in the acquisition of iron from these glycoproteins. In Neisseria meningitidis, the transferrin receptor appears to consist of two proteins, one of which (TBP 1) has an M(r) of 95,000 and the other of which (TBP 2) has an M(r) ranging from 68,000 to 85,000, depending on the strain; TBP 2 binds transferrin after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting, but TBP 1 does not do so. The relative contributions of these two proteins to the binding reaction observed with intact cells and to iron uptake are presently unknown. However, they are being considered as potential components of a group B meningococcal vaccine. Analogous higher- and lower-molecular-weight proteins associated with transferrin binding have been found in N. gonorrhoeae and Haemophilus influenzae. Previous work with polyclonal antibodies raised in mice with whole cells of iron-restricted N. meningitidis showed that the meningococcal TBP 2 exhibits considerable antigenic heterogeneity. Here, we report that antiserum against purified TBP 2 from one strain of N. meningitidis cross-reacts on immunoblotting with the TBP 2 of all meningococcal isolates examined, as well as with the TBP 2 of N. gonorrhoeae. This antiserum also cross-reacted with the TBP 2 of several strains of H. influenzae type b, thus showing the presence of common antigenic domains among these functionally equivalent proteins in different pathogens; no cross-reaction was detected with a purified sample of the human transferrin receptor.     

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.     

Sialylation of lipooligosaccharides promotes biofilm formation by nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract infections, including otitis media and bronchitis. The persistence of NTHi in vivo is thought to involve bacterial persistence in a biofilm community. Therefore, there is a need for further definition of bacterial factors contributing to biofilm formation by NTHi. Like other bacteria inhabiting host mucosal surfaces, NTHi has on its surface a diverse array of lipooligosaccharides (LOS) that influence host-bacterial interactions. In this study, we show that LOS containing sialic (N-acetyl-neuraminic) acid promotes biofilm formation by NTHi in vitro and bacterial persistence within the middle ear or lung in vivo. LOS from NTHi in biofilms was sialylated, as determined by comparison of electrophoretic mobilities and immunochemical reactivities before and after neuraminidase treatment. Biofilm formation was significantly reduced in media lacking sialic acid, and a siaB (CMP-sialic acid synthetase) mutant was deficient in biofilm formation in three different in vitro model systems. The persistence of an asialylated siaB mutant was attenuated in a gerbil middle ear infection model system, as well as in a rat pulmonary challenge model system. These data show that sialylated LOS glycoforms promote biofilm formation by NTHi and persistence in vivo.     

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.     

Use of Dorset Egg Medium for Maintenance and Transport of Neisseria meningitidis and Haemophilus influenzae Type b

Studies of bacterial meningitis are hampered by the inability to maintain the viability of etiological agents during transport to reference laboratories. The long-term survival rate of 20 isolates of Neisseria meningitidis and Haemophilus influenzae type b (Hib) on Dorset egg medium, supplemented Columbia agar base medium, chocolate agar, and Amies medium was compared with that on 70% GC agar (chocolate) transport medium. N. meningitidis isolates were also inoculated onto 5% horse blood agar, and Hib was inoculated onto Haemophilus test medium. All of the N. meningitidis isolates remained viable on Dorset egg medium for 21 days; viability on the other media was poor after only 7 days. Recovery rates of Hib isolates were similar on Dorset egg and Haemophilus test media (100% after 21 days) and significantly better than on the other media. Dorset egg medium is inexpensive and easy to make and may be invaluable for studies of bacterial meningitis in developing countries.     

Failure of adherence to buccal cells and surface hydrophobicity as virulence markers in Neisseria meningitidis

Surface hydrophobicity and adherence to buccal epithelial cells were studied in 33 carrier and 34 invasive Neisseria meningitidis strains. It was found that hydrophobicity is statistically similar in both groups (P = 0.0507) although it could be considered that carrier strains are slightly more hydrophobic than invasive ones. Adherence was similar in both groups although more homogeneous in the carrier strains. No correlation could be demonstrated between these two properties nor can they be considered relevant as markers of the carrier or invasive status of this bacterium, indicating that at least in N. meningitidis they are not good properties to discriminate virulent strains.     

TREM-2 binds to lipooligosaccharides of Neisseria gonorrhoeae and is expressed on reproductive tract epithelial cells

Triggering receptor expressed on myeloid cells-2 (TREM-2) is an innate immune receptor that initiates cellular activation upon ligation. In this study, we examined the interaction of TREM-2 with Neisseria gonorrhoeae using murine TREM-2A, as it has been reported to recognize bacterial ligands. Using a whole-bacteria enzyme-linked immunosorbent assay (ELISA), TREM-2A bound to all six strains in variable degrees. Far-western blots of gonococcal outer membranes revealed TREM-2A binding to lipooligosaccharide (LOS) and opacity (Opa) protein, with predominant binding to LOS. Binding of TREM-2A to LOS was confirmed by ELISA and surface plasmon resonance. O-deacylation of the lipid A significantly reduced binding. Flow cytometry and reporter cell assays showed that gonococci bound to TREM-2A-transfected cells and induced transmembrane signaling. In humans, TREM-2 was constitutively expressed by genitourinary and fallopian tube epithelial cells, both of which are primary targets of gonococcal invasion. Ligation of TREM-2 by LOS induced interleukin-6 production in HeLa cervical carcinoma cells. To our knowledge, this is the first report of the expression of human TREM-2 by cells deriving from a non-myeloid lineage. We conclude that gonococci can interact with TREM-2 receptors through binding to LOS and Opa protein and initiate cell signaling and cytokine production.     

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.     

International circumpolar surveillance interlaboratory quality control program for serotyping Haemophilus influenzae and serogrouping Neisseria meningitidis, 2005 to 2009

The International Circumpolar Surveillance (ICS) program was initiated in 1999 to conduct population-based surveillance for invasive pneumococcal disease in select regions of the Arctic. The program was expanded to include the surveillance of invasive diseases caused by Neisseria meningitidis and Haemophilus influenzae. An interlaboratory quality control (QC) program to monitor laboratory proficiencies in the serogrouping of N. meningitidis and serotyping of H. influenzae strains was codeveloped by the Arctic Investigations Program (Anchorage, AK) and the Public Health Agency of Canada National Microbiology Laboratory (Winnipeg, Manitoba, Canada) and introduced into the ICS program in 2005. Other participating laboratories included the Provincial Laboratory for Public Health (Edmonton, Alberta, Canada), Laboratoire Santé Publique du Québec (Sainte-Anne-de-Bellevue, Québec, Canada), and Statens Serum Institut (Copenhagen, Denmark). From 2005 through 2009, 50 isolates (24 N. meningitidis and 26 H. influenzae isolates) were distributed among the five participating laboratories. The overall serogroup concordance for N. meningitidis strains was 92.3% (96/104), without including three isolates that were found to express both serogroup Y and W135 specificities. Concordant results were obtained for serogroups A, B, C, and Y among all laboratories. Discrepancies were observed most frequently for serogroups W135, X, Z, and 29E. The overall serotype concordance for H. influenzae was 98% (125/127 attempts). The two discrepant results involved a serotype c strain and a serotype e strain, and in both cases, the serotypeable H. influenzae isolates were misidentified as being nontypeable. These data demonstrate a high degree of concordance for serogroup and serotype determinations of N. meningitidis and H. influenzae isolates, respectively, among the five laboratories participating in this quality control program.     

Nucleotide sequence homology between the immunoglobulin A1 protease genes of Neisseria gonorrhoeae, Neisseria meningitidis, and Haemophilus influenzae.

Isolated DNA fragments encoding the immunoglobulin A1 (IgA1) protease of Neisseria gonorrhoeae were used as hybridization probes to search for homologous sequences in whole cell DNA from Neisseria meningitidis and Haemophilus influenzae. Significant homology was detected. That the detected homology represented IgA1 protease-specific sequences was confirmed by the cloning of these sequences in Escherichia coli HB101 and demonstrating the expression of IgA1 protease by these transformed cells. Molecular probing of commensal Neisseria and Haemophilus species, which do not elaborate IgA1 protease activity, revealed that they were devoid of sequence homology with the cloned IgA1 protease gene DNA.     

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.