Fimbria-mediated adherence and hemagglutination of Haemophilus influenzae

The gram-negative bacterium Haemophilus influenzae expresses morphologically and functionally distinct types of fimbriae, of which the LKP fimbriae mediate hemagglutination and adherence to human epithelial cells but hamper mucosal invasion. Therefore, the both in vivo and in vitro observed fimbrial phase variation may contribute to the pathogenesis of the infection. The existence of greater than 14 LKP serotypes hampers vaccine development based on fimbriae, since a monovalent fimbria vaccine confers protection against only the homologous strain. Cloning of the fimbrial genes in Escherichia coli results in the expression of morphologically intact fimbriae. Analysis of the cloned DNA indicates that a fimbrial gene cluster is necessary for formation of complete fimbriae and for fimbria-mediated adherence. The gene encoding the subunit is highly conserved among H. influenzae and belongs to the family of E. coli fimbriae. The phase variation is transcriptionally regulated by variation of the length of the reiterated sequence that forms the promoter region of the subunit gen

In vivo and in vitro expression of outer membrane components of Haemophilus influenzae

The outer membrane protein composition of Haemophilus influenzae grown under a variety of culture conditions including growth in sputum and serum, and intraperitoneally in rats was analyzed. The pattern of the major outer membrane proteins, a, b,c, d, e and P6 remained very similar under all these conditions. Outer membrane proteins expressed during iron limitation were also expressed in bacteria growing in rats, in serum or in sputum. To determine the expression of the major outer membrane proteins and lipopolysaccharide in patient materials (sputum, cerebrospinal fluid, postmortem tissue) monoclonal antibodies specific for the outer membrane proteins a, b,c, d and P6 as well as lipopolysaccharide were used in immunoblotting. They showed the same reaction patterns with bacteria in the patient materials as with the bacteria isolated from these specimens. We conclude that the major outer membrane components expressed under in vitro conditions are also expressed in various clinical materials during infectio

Relationship between secretion of the Anton blood group antigen in saliva and adherence of Haemophilus influenzae to oropharynx epithelial cells

Inhibition of adherence of bacteria to epithelial cells contributes to a reduction of infections by these bacteria. We have shown that the Anton blood group antigen, the erythrocyte receptor for Haemophilus influenzae (van Alphen et al. 1986, FEMS Microbiol. Lett. 37, 69-71), occurs in saliva, that the occurrence is not related to the secretor state of the donor of the saliva and that saliva containing Anton antigen could not inhibit the adherence of H. influenzae to oropharynx epithelial cells. Anton antigen was detected in saliva samples of 14 donors by immunoblotting with two different anti-Anton sera. The amount of Anton antigen correlated with the ability of H. influenzae to adhere to the epithelial cells of the donor of the saliva: 4.1 +/- 0.1 Anton antigen units for donors with more than 50 H. influenzae per cell and 1.6 +/- 0.5 units for donors with less adhering epithelial cells. No correlation between the amount of Anton antigen in saliva and secretor status of the donor was observed. Adherence of H. influenzae to epithelial cells was not inhibited by saliva of secretors (N = 11) or non-secretors (N = 3). The same saliva did not inhibit the interaction of the bacteria with Anton antigen bearing erythrocytes as measured by haemagglutination inhibition. This indicates that the amount of Anton antigen in saliva is probably too low to interfere with the interaction of H. influenzae with oropharynx epithelial cells and erythrocyte

The fimbrial gene cluster of Haemophilus influenzae type b

Haemophilus influenzae infections are preceded by airway colonization, a process facilitated by fimbriae. Here, we identified the complete fimbrial gene cluster of H. influenzae type b. HifA forms the major subunit. HifB, a periplasmic chaperone, and HifC, an outer membrane usher, are typical assembly genes; their inactivation abolished fimbriae formation. HifD and HifE are putative minor subunits, both participating in fimbriae biogenesis. Inactivation of either one drastically reduced fimbriae expression. HifD represents a novel type of fimbrial subunit with lipoprotein characteristics, pointing to a membrane-associated function of HifD. Transcription of all fimbrial genes is coregulated through two clustered promoters. The flanking of the fimbrial gene cluster by repetitive extragenic palindromic sequences together with a partial duplication of an adjacent unrelated operon indicated that the cluster was once inserted in the H. influenzae genome as a mobile virulence uni

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

Phase variation of H. influenzae fimbriae: transcriptional control of two divergent genes through a variable combined promoter region

The expression of H. influenzae fimbriae is subject to reversible phase variation between three expression levels. This phenomenon is controlled at the transcriptional level of two divergently orientated genes, hifA and hifB, encoding the major fimbrial subunit and the fimbrial chaperone, respectively. The hifA and hifB promoter regions were found to be clustered through an almost complete divergent overlap with a variable DNA backbone of repetitive TA units. Variation in the number of units changes the normally strictly constrained spacing between the -35 and -10 sequences and controls the bidirectional transcription initiation, thus forming a novel mechanism directing multiple gene transcriptio

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

The Fimbria Gene Cluster of Nonencapsulated Haemophilus influenzae

The occurrence of fimbria gene clusters in nonencapsulated Haemophilus influenzae strains from chronic bronchitis patients (n = 58), patients with acute otitis media (n = 13), and healthy carriers (n = 12) was determined by DNA hybridization and PCR, based on sequences of fimbriate H. influenzae type b. Although an average of 18% of all nonencapsulated strains had a fimbria gene cluster consisting of hifA to hifE inserted in the chromosome between purE and pepN, differences in the frequency of fimbria cluster-positive strains were observed, depending on the source of isolates. The compositions of the fimbria gene clusters of seven strains from chronic bronchitis patients and one strain from an otitis media patient were analyzed in more detail. After enrichment for fimbria expression, the promoter of the gene cluster contained 10 TA repeats (n = 2), leading to optimal positioning between the −10 and −35 promoter regions. The promoter regions of five fimbria-negative strains were sequenced; four were found to have nine TA repeats, and one had only four TA repeats. The protein sequence of three ganglioside GM1-specific HifA adhesins consisted of conserved regions intermingled with regions of sequence diversity. hifA appeared to be flanked by intergenic regions that varied between strains and contained both direct and inverted DNA repeats. Since noncoding DNA between hifA and purE has not been found in H. influenzae type b, these DNA sequences are probably not essential for fimbria expression. An analysis of strains lacking the gene cluster revealed the presence of similar sequences in 13 of 15 strains from chronic bronchitis patients, 5 of 5 strains from otitis media patients, and 3 of 5 strains from healthy carriers. The lengths of these intergenic regions were the same for multiple isolates of strains obtained during persistent infections. The presence or absence and the composition of the fimbria gene cluster and other sequences between the flanking genes purE and pepN suggest that the fimbria gene cluster was originally contained on a mobile element

Cloning and expression in Escherichia coli of Haemophilus influenzae fimbrial genes establishes adherence to oropharyngeal epithelial cells.

In this report the first example of functional expression of a fimbrial gene cluster of a non-enteric human pathogen in Escherichia coli is described. This is shown for Haemophilus influenzae fimbriae which mediate adherence to oropharyngeal epithelial cells. A genomic library of H.influenzae type b, strain 770235f+bo, was constructed using a cosmid vector and screened with a synthetic oligonucleotide probe derived from the N-terminal sequence of the fimbrial subunit of H.influenzae. Four cosmid clones were found which hybridized to this oligonucleotide probe. Escherichia coli strains harbouring these clones expressed the H.influenzae fimbriae at their cell surface, as was demonstrated in a whole-cell ELISA and by immunogold electron microscopy using a monoclonal antibody specific for the H.influenzae fimbriae. Surface expression could be maintained during subcloning until a minimal H.influenzae DNA insert of approximately 8.1 kb was obtained. Escherichia coli strains harbouring the 8.1 kb H. influenzae DNA were able to cause a mannose-resistant adherence to oropharyngeal epithelial cells and a mannose-resistant haemagglutination of human AnWj-positive erythrocytes. The nucleotide sequence of hifA, the gene encoding the major fimbrial subunit, was determined. The predicted amino acid sequence shows a significant homology with a number of E.coli fimbrial subunits