Analysis of pilus adhesins from Haemophilus influenzae biotype IV strains

A subset of nontypeable Haemophilus influenzae (NTHI) biotype IV isolates from the human genital tract or from infected newborn infants forms a cryptic genospecies characterized by, among other features, the presence of peritrichous pili. The objective of this study was to determine the similarity of these pili to hemagglutinating, HifA- and HifE-containing pili expressed by respiratory H. influenzae isolates. For this analysis, the presence of hifA and hifE and their gene products in NTHI biotype IV strains was assessed, the binding of H. influenzae biotype IV strains to human epithelial cells was characterized, possible genital tissue tropism of these isolates was explored, and the role of HifA- and HifE-possessing pili in the adhesion of NTHI biotype IV strains to human epithelial cells was determined. None of the six biotype IV NTHI isolates tested agglutinated human red blood cells, nor could they be enriched for hemagglutinating variants. Although hifA, which encodes the major structural subunit of hemagglutinating pili, and hifE, which encodes the tip adhesin of hemagglutinating pili, were detected by PCR from six and five, respectively, of the six biotype IV strains tested, neither HifA nor HifE (the gene products of hifA and hifE) were detected in any of these strains by Western blot analysis using antisera that recognize HifA and HifE of respiratory strains. Transmission electron microscopy showed no surface pili on the two biotype IV H. influenzae isolates examined; strain 4162 containing an insertional mutation in hifA also showed no surface pili, whereas strain 1595 containing an insertional mutation in hifB showed pilus-like structures that were shorter and thicker than hemagglutinating pili of the respiratory strains AAr176 and M43. In enzyme-linked immunosorbent assays, biotype IV strains adhered to 16HBE14o(-) and HEp-2 cells of respiratory origin as well as to ME180 and HeLa cells of genital origin. This adherence was not pilus specific, however, as GM-1, a known pilus receptor analog, did not inhibit binding of biotype IV strains to ME180, HEp-2, or HeLa cells, and GM-1 inhibition of binding to 16HBE14o(-) cells did not correlate with the presence of hifE. While both nonpiliated variants and hifA and hifB (encoding the pilus chaperone) mutants of respiratory strain AAr176 showed reduced binding (64 to 87% of that of piliated AAr176) to 16HBE14o(-) and ME180 cells, hifA and hifB mutants of the biotype IV strains showed minimal reduction in binding to these cell lines (91 to 98% of that of wild-type strains). Thus, although biotype IV H. influenzae isolates of the cryptic genospecies possess the genes that code for HifA- and HifE-containing hemagglutinating pili, epithelial cell adherence exhibited by these strains is not mediated by expression of hemagglutinating pili.     

Comparison of hemagglutinating pili of Haemophilus influenzae type b with similar structures of nontypeable H. influenzae.

Thirty-eight clinical isolates of nontypeable Haemophilus influenzae were tested for the presence of hemagglutinating pili similar to those of H. influenzae type b (Hib) that mediate buccal epithelial cell adherence. Four endogenously hemagglutinating (HA+) strains were identified, and eight additional HA+ variants were obtained from HA- strains by erythrocyte enrichment. All 12 HA+ nontypeable H. influenzae isolates bound antisera directed against denatured pilins of Hib, but none bound antisera against assembled native pili of Hib. In erythrocyte- and buccal-cell-binding assays, HA+ nontypeable H. influenzae binding was reduced compared with HA+ Hib binding and was not significantly different from HA- nontypeable H. influenzae binding. Both HA- and HA+ nontypeable H. influenzae binding was increased over binding of HA- Hib. HA+ nontypeable H. influenzae strains agglutinated adult erythrocytes that possess the Anton antigen, which is thought to be the receptor for Hib pili, and did not agglutinate cord or Lu(a-b-) dominant erythrocytes, which lack the Anton antigen. Electron microscopy of HA- and HA+ variants of three nontypeable H. influenzae strains showed few or no surface appendages on the HA- organisms, but piluslike structures were seen on many organisms from two HA+ nontypeable H. influenzae strains and on a few organisms from one strain. Thus, nontypeable H. influenzae appears to possess structures that are immunologically similar to the pilins that make up the hemagglutinating pili of Hib. However, nontypeable H. influenzae appears to also possess mechanisms for erythrocyte and buccal cell adherence that are not directly correlated with the presence of a hemagglutinating pilus.     

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.     

Comparative Analysis of Haemophilus influenzae hifA (Pilin) Genes

Adherence of Haemophilus influenzae to epithelial cells plays a central role in colonization and is the first step in infection with this organism. Pili, which are large polymorphic surface proteins, have been shown to mediate the binding of H. influenzae to cells of the human respiratory tract. Earlier experiments have demonstrated that the major epitopes of H. influenzae pili are highly conformational and immunologically heterogenous; their subunit pilins are, however, immunologically homogenous. To define the extent of structural variation in pilins, which polymerize to form pili, the pilin genes (hifA) of 26 type a to f and 16 nontypeable strains of H. influenzae were amplified by PCR and subjected to restriction fragment length polymorphism (RFLP) analysis with AluI and RsaI. Six different RFLP patterns were identified. Four further RFLP patterns were identified from published hifA sequences from five nontypeable H. influenzae strains. Two patterns contained only nontypeable isolates; one of these contained H. influenzae biotype aegyptius strains F3031 and F3037. Another pattern contained predominantly H. influenzae type f strains. All other patterns were displayed by a variety of capsular and noncapsular types. Sequence analysis of selected hifA genes confirmed the 10 RFLP patterns and showed strong identity among representatives displaying the same RFLP patterns. In addition, the immunologic reactivity of pili with antipilus antisera correlated with the groupings of strains based on hifA RFLP patterns. Those strains that show greater reactivity with antiserum directed against H. influenzae type b strain M43 pili tend to fall into one RFLP pattern (pattern 3); while those strains that show equal or greater reactivity with antiserum directed against H. influenzae type b strain Eagan pili tend to fall in a different RFLP pattern (pattern 1). Sequence analysis of representative HifA pilins from typeable and nontypeable H. influenzae identified several highly conserved regions that play a role in bacterial pilus assembly and other regions with considerable amino acid heterogeneity. These regions of HifA amino acid sequence heterogeneity may explain the immunologic diversity seen in intact pili.     

Protein D of Haemophilus influenzae is not a universal immunoglobulin D-binding protein.

Haemophilus influenzae type b and nontypeable H. influenzae have been reported to bind human immunoglobulin D (IgD). IgD myeloma sera from five patients were tested for the ability of IgD to bind to H. influenzae. Serotype b strains bound human IgD in four of the five sera tested. IgD in the fifth serum bound strongly to type b strain MinnA but poorly to other type b strains. Additionally, IgD binding was not observed when nontypeable strains were tested. The gene for protein D, the putative IgD-binding protein, was cloned from the IgD-binding H. influenzae type b strain MinnA and expressed in Escherichia coli. IgD binding to E. coli expressing protein D was not demonstrable. Recombinant protein D was purified, and antisera were generated in rabbits. Using these rabbit sera, we detected protein D in nontypeable as well as serotype b strains by Western blotting (immunoblotting). In contrast, IgD myeloma protein 4490, which was previously reported to bind to protein D by Ruan and coworkers (M. Ruan, M. Akkoyunlu, A. Grubb, and A. Forsgren, J. Immunol. 145:3379-3384), bound strongly to both type b and nontypeable H. influenzae as well as to E. coli expressing protein D. Thus, IgD binding is a general property of H. influenzae type b strains but not a general property of nontypeable strains, although both type b and nontypeable strains produce protein D. With the exception of IgD myeloma protein 4490 binding, we have no evidence for a role of protein D in IgD binding to H. influenzae.     

Structural and serological relatedness of Haemophilus influenzae type b pili.

The structural and serological relatedness of the pilus proteins of several isolates of Haemophilus influenzae type b cultured from patients with invasive disease and from different anatomic sites within the same patient was examined. Epithelial cell-adherent variants of 25 nonadherent parent isolates were obtained by selection for organisms that adhered to human erythrocytes. Outer membrane protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of an additional 24- to 24.5-kilodalton protein among all adherent variants but absent from all nonadherent parent isolates. Polyclonal rabbit antiserum against the intact native pilus protein of H. influenzae M43 cross-reacted with 20 of 25 adherent H. influenzae in both immunodot and slide-agglutination assays. No differences in reactivity among isolates cultured from more than one anatomic site in the same patient were noted. Anti-M43 pilus antiserum had bactericidal activity against both the homologous strain and a heterologous strain that demonstrated serologic identity in the immunodot and slide agglutination assays. The adherence of these strains to human epithelial cells in vitro was inhibited by Fab fragments purified from the antipilus antiserum. These data indicate that a remarkable degree of homogeneity in pilin subunit size exists among the pili of H. influenzae type b and that major antigenic determinants are shared among most of these pili. Also, antibodies directed against H. influenzae pilus proteins may be able to contribute to host defenses through serum bactericidal activity and by blocking the adherence of this bacterium to host epithelial cells.     

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.     

Limited diversity of the protein D gene (hpd) among encapsulated and nonencapsulated Haemophilus influenzae strains.

Protein D is a surface-exposed lipoprotein of the gram-negative bacterium Haemophilus influenzae with affinity for human immunoglobulin D myeloma protein. The gene encoding protein D (hpd) in a serotype b strain of H. influenzae was cloned. Escherichia coli carrying the hpd gene bound human myeloma immunoglobulin D. Nucleotide sequence analysis identified an 1,092-bp open reading frame that was more than 99% identical to the hpd gene from a nontypeable H. influenzae strain. In the deduced amino acid sequences for protein D, only 2 of 364 amino acid residues differed. The restriction fragment length polymorphism of the hpd region in different strains was analyzed by Southern blot analyses of PstI- or EcoRI-digested genomic DNA from 100 H. influenzae strains. The analysis was performed by using isolated fragments of the cloned hpd gene, originating from the nontypeable H. influenzae 772, as probes. All strains tested had DNA sequences with a high degree of homology to the hpd probes. The analysis also showed that restriction endonuclease sites within the gene were more conserved than sites adjacent to the hpd gene. An interesting difference between type b strains and unencapsulated strains was observed. The majority of type b strains seem to have a 1.4-kbp DNA fragment upstream of the hpd gene that is absent in nontypeable strains. On the basis of the high degree of conservation of the hpd gene among H. influenzae strains, we conclude that protein D is a possible vaccine candidate.     

Identification of hifD and hifE in the pilus gene cluster of Haemophilus influenzae type b strain Eagan.

Haemophilus influenzae produces surface structures called pili that promote adherence to human cells. Three genes encoding the major pilus structural component (pilin), chaperone, and usher proteins (designated hifA, -B, and -C, respectively) have been identified previously. In this study, transposon mutagenesis and DNA sequence analysis identified two open reading frames (ORFs) downstream of, and in the same orientation as, hifC. These genes have been designated hifD and hifE. Both genes have predicted C-terminal amino acid homology to HifA, and mutations in either gene resulted in the loss of morphologic and functional pili, indicating that hifD and hifE encode pilus structural components and are required for pilus expression. Another ORF, identified immediately downstream of hifE, has a predicted amino acid sequence that is 70% identical to an aminopeptidase of Escherichia coli called PepN, and a mutation within this ORF did not alter pilus expression. These data indicate that the pepN homolog is not required for pilus biogenesis and that one end of the pilus gene cluster has been defined.     

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.     

Prevalence and distribution of adhesins in invasive non-type b encapsulated Haemophilus influenzae

Adhesion to the respiratory epithelium plays an important role in Haemophilus influenzae infection. The distribution of H. influenzae adhesins in type b and nontypeable strains has been characterized, but little is known about the prevalence of these factors in non-type b encapsulated strains. We analyzed 53 invasive type a, type e, and type f strains for the presence of hap, hia, hmw, and hif genes; Hap, Hia, and HMW1/2 adhesins; and hemagglutinating pili. The hap gene was ubiquitous, and homologs of hmw and hia were present in 7 of 53 (13.2%) and 45 of 53 (84.9%) strains, respectively. Hap was detected in 28 of 45 (62.2%) hap(+) strains, HMW1/2 was detected in 5 of 7 (71.4%) hmw(+) strains, and Hia was detected in 31 of 45 (68.8%) hia(+) strains. The hif gene cluster was present in 26 of 53 strains (49.1%), and 21 of 26 hif(+) strains (80.8%) agglutinated (HA) red blood cells. Nine isolates exhibited HA but lacked the hif gene cluster. The distribution of adhesin genes correlated with the genetic relatedness of the strains. Strains belonging to one type a clonotype and the major type e clonotype possessed hia but lacked the hif cluster. Strains belonging to the second type a clonotype possessed both hia and hif genes. All type f strains belonging to the major type f clonotype possessed hia and lacked hifB. Although the specific complement of adhesin genes in non-type b encapsulated H. influenzae varies, most invasive strains express Hap and Hia, suggesting these adhesins may be especially important to the virulence of these organisms.     

The gene encoding protein D (hpd) is highly conserved among Haemophilus influenzae type b and nontypeable strains.

The molecular conservation of a surface-exposed lipoprotein, protein D, of Haemophilus influenzae was studied by cloning and sequencing of the gene encoding protein D from three encapsulated type b strains and three nontypeable strains of H. influenzae. These nucleotide sequences were analyzed with previously reported sequences from one type b strain and one nontypeable strain. The nucleotide sequences and the deduced amino acid sequences for protein D were highly conserved. The deduced amino acid sequence (364 amino acids) of protein D from six strains differed only in two amino acids near the C-terminal end. The remaining two strains, one type b and one nontypeable, differed from the consensus sequence in 7 amino acids each. Protein D is 64 and 36% identical and 77 and 56% similar to the glycerophosphodiester phosphodiesterases (GlpQ) of Escherichia coli and Bacillus subtilis.     

Opacity-Associated Protein A Contributes to the Binding of Haemophilus influenzae to Chang Epithelial Cells

Opacity-associated protein A (OapA), which is responsible for the transparent-colony phenotype of Haemophilus influenzae, has been implicated in the colonization of the nasopharynx in an infant rat model of carriage. In this report, we show that OapA mediates attachment to Chang epithelial cells examined by using genetically defined type b and nontypeable H. influenzae strains with or without OapA. We also showed that OapA was conserved among H. influenzae strains by comparing deduced amino acid sequences. Both recombinant OapA and polyclonal anti-OapA antiserum blocked the binding of H. influenzae to Chang epithelial cells, suggesting that the interaction of H. influenzae is specific to OapA. Moreover, the binding of recombinant OapA to epithelial cells further provided evidence that OapA can promote attachment of H. influenzae. Expression of oapA gene in a nonadherent Escherichia coli strain significantly increased the binding to Chang epithelial cells, and disruption of the oapA gene with kanamycin resistance cassette insertion resulted in a significant loss of binding. These findings demonstrate that OapA plays a role in H. influenzae binding to human conjunctival epithelial cells.     

Nontypeable Haemophilus influenzae strains with the capsule-associated insertion element IS1016 may mimic encapsulated strains

With the elimination of Haemophilus influenzae type b through vaccination, it has been suggested that other types of H. influenzae strains might acquire virulence traits and emerge as important pathogens. The gene sequence IS1016 has been associated with an increased capacity to cause severe infections. It is usually present in encapsulated strains but is sometimes harbored by nontypeable H. influenzae strains. To explore this further, 118 H. influenzae isolates, collected from both patients and healthy carriers, were investigated with PCR with reference to this gene sequence. Isolates positive for the insertion element were bio- and serotyped. The presence of hmw genes for adherence, the genetic profile, and the ability to form biofilm in vitro were investigated. A total of 15 isolates were IS1016-positive, whereof 12 were nontypeable. All 12 nontypeable isolates were obtained from healthy carriers, and 92% of the isolates were biotype I. They cross-reacted to some extent with type-specific antisera or exhibited a restricted genetic diversity like encapsulated strains. Furthermore, they lacked hmw-genes, and their ability to form biofilms was comparable with a capsule-deficient type b strain. Although this subset of strains mimicked traits usually exhibited by encapsulated strains, the isolation frequency did not seem to have been affected by vaccination.     

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.     

Identification of a gene essential for piliation in Haemophilus influenzae type b with homology to the pilus assembly platform genes of gram-negative bacteria.

Haemophilus influenzae type b (Hib) pili are complex filamentous surface structures consisting predominantly of pilin protein subunits. The gene encoding the major pilin protein subunit of Hib adherence pili has been cloned and its nucleotide sequence has been determined. In order to identify specific accessory genes involved in pilus expression and assembly, we constructed isogenic Hib mutants containing insertional chromosomal mutations in the DNA flanking the pilin structural gene. These mutants were screened for pilin production, pilus expression, and hemagglutination. Pili and pilin production were assessed by immunoassays with polyclonal antisera specific for pilin and pili of Hib strain Eagan. Hemagglutination was semiquantitatively evaluated in a microtiter plate assay. Six Hib mutants produced proteins immunoreactive with antipilin antiserum but no longer produced structures reactive with antipilus antiserum. In addition, the mutants were unable to agglutinate human erythrocytes. Nucleotide sequence analysis localized the insertion sites in the six mutants to 2.5-kb open reading frame upstream of the pilin structural gene and immediately downstream of an Hib pilin chaperone gene. The amino acid sequence encoded by this open reading frame has significant homology to members of the pilus assembly platform protein family, including FhaA of Bordetella pertussis, MrkC of Klebsiella pneumoniae, and the Escherichia coli assembly platform proteins FimD and PapC. This open reading frame, designated hifC, appears to represent a gene essential to Hib pilus biogenesis that has genetic and functional similarity to the pilus platform assembly genes of other gram-negative rods.     

Prevalence of the hifBC, hmw1A, hmw2A, hmwC, and hia Genes in Haemophilus influenzae Isolates

Adherence of Haemophilus influenzae to respiratory epithelial cells is the first step in the pathogenesis of H. influenzae infection and is facilitated by the action of several adhesins located on the surface of the bacteria. In this study, prevalences of hifBC, which represent the pilus gene cluster; hmw1A, hmw2A, and hmwC, which represent high-molecular-weight (HMW) adhesin genes; and hia, which represents H. influenzae adhesin (Hia) genes were determined among clinical isolates of encapsulated type b (Hib) and nonencapsulated (NTHi) H. influenzae. hifBC genes were detected in 109 of 170 (64%) Hib strains and in 46 of 162 (28%) NTHi isolates (P = 0.0001) and were more prevalent among the invasive type b strains than invasive NTHi strains (P = 0.00003). Furthermore, hifBC genes were significantly more prevalent (P = 0.0398) among NTHi throat isolates than NTHi middle ear isolates. hmw1A, hmw2A, hmwC, and hia genes were not detected in Hib strains. Among NTHi isolates, the prevalence of hmw1A was 51%, the prevalence of hmw2A was 23%, the prevalence of hmwC was 48%, and the prevalence of hia was 33%. The hmw genes were significantly more prevalent among middle ear than throat isolates, while hia did not segregate with a respiratory tract site. These results show the variability of the presence of adhesin genes among clinical H. influenzae isolates and suggest that hemagglutinating pili may play a larger role in H. influenzae nasopharyngeal colonization than in acute otitis media whereas the HMW adhesins may be virulence factors for acute otitis media.     

Immunologic and Structural Relationships of the Minor Pilus Subunits among Haemophilus influenzae Isolates

Two proteins, HifD and HifE, have been identified as structural components of Haemophilus influenzae pili. Both are localized at the pilus tip, and HifE appears to mediate pilus adherence to host cells. In this study we examined the immunologic and structural diversity of these pilus subunits among type b H. influenzae (Hib) and nontypeable H. influenzae (NTHI) strains. Western immunoblot analysis revealed that antibodies directed against the C terminus of HifD and HifE from Hib strain Eagan bound to HifD and HifE proteins, respectively, of all piliated Hib and NTHI strains tested. Whole-cell enzyme-linked immunosorbent assays showed that antibodies specific for native HifD or HifE of strain Eagan also bound to all piliated Hib strains but did not bind to the piliated NTHI strains. Antibodies against HifE of strain Eagan inhibited the binding of Hib to human erythrocytes but did not inhibit the binding of NTHI strains. Restriction fragment length polymorphism (RFLP) analysis was used to determine strain-to-strain structural differences within hifD and hifE genes, either by PCR or by nucleotide sequence analysis. DNA and derived amino acid sequence analyses of HifD and HifE confirmed the uniqueness of the RFLP types. The hifD and hifE genes of all type b strains showed identical restriction patterns. Analysis of hifD and hifE genes from the NTHI strains, however, revealed seven unique RFLP patterns, suggesting that these genes encode proteins with diverse primary structures. These results indicate that HifD and HifE are immunologically and structurally similar among the Hib strains but vary among the NTHI strains.     

Demonstration of Type IV pilus expression and a twitching phenotype by Haemophilus influenzae

Haemophilus influenzae is considered a nonmotile organism that expresses neither flagella nor type IV pili, although H. influenzae strain Rd possesses a cryptic pilus locus. We demonstrate here that the homologous gene cluster pilABCD in an otitis media isolate of nontypeable H. influenzae strain 86-028NP encodes a surface appendage that is highly similar, structurally and functionally, to the well-characterized subgroup of bacterial pili known as type IV pili. This gene cluster includes a gene (pilA) that likely encodes the major subunit of the heretofore uncharacterized H. influenzae-expressed type IV pilus, a gene with homology to a type IV prepilin peptidase (pilD) as well as two additional uncharacterized genes (pilB and pilC). A second gene cluster (comABCDEF) was also identified by homology to other pil or type II secretion system genes. When grown in chemically defined medium at an alkaline pH, strain 86-028NP produces approximately 7-nm-diameter structures that are near polar in location. Importantly, these organisms exhibit twitching motility. A mutation in the pilA gene abolishes both expression of the pilus structure and the twitching phenotype, whereas a mutant lacking ComE, a Pseudomonas PilQ homologue, produced large appendages that appeared to be membrane bound and terminated in a slightly bulbous tip. These latter structures often showed a regular pattern of areas of constriction and expansion. The recognition that H. influenzae possesses a mechanism for twitching motility will likely profoundly influence our understanding of H. influenzae-induced diseases of the respiratory tract and their sequelae.     

Comparison and analysis of the nucleotide sequences of pilin genes from Haemophilus influenzae type b strains Eagan and M43.

Previous studies have demonstrated antigenic differences among the pili expressed by various strains of Haemophilus influenzae type b (Hib). In order to understand the molecular basis for these differences, the structural gene for pilin was cloned from Hib strain Eagan (p+) and the nucleotide sequence was compared to those of strains M43 (p+) and 770235 b0f+, which had been previously determined. The pilin gene of Hib strain Eagan (p+) had a 648-bp open reading frame that encoded a 20-amino-acid leader sequence followed by the 196 amino acids found in mature pilin. The translated sequence was three amino acids larger than pilins of strains M43 (p+) and 770235 b0f+ and was 78% identical and 95% homologous when conservative amino acid substitutions were considered. Differences between the amino acid sequences were not localized to any one region but rather were distributed throughout the proteins. Comparison of protein hydrophilicity profiles showed several hydrophilic regions with sequences that were conserved between strain Eagan (p+) and pilins of other Hib strains, and these regions represent potentially conserved antigenic domains. Southern blot analyses using an intragenic probe from the pilin gene of strain Eagan (p+) showed that the pilin gene was conserved among all type b and nontypeable strains of H. influenzae examined, and only a single copy was present in these strains. Homologous genes were not present in the phylogenetically related species Pasteurella multocida, Pasteurella haemolytica, and Actinobacillus pleuropneumoniae. These data indicate that the pilin gene was highly conserved among different strains of H. influenzae and that small differences in the pilin amino acid sequences account for the observed antigenic differences of assembled pili from these strains.