Copy number of pilus gene clusters in Haemophilus influenzae and variation in the hifE pilin gene.

Haemophilus influenzae is a gram-negative bacterium that colonizes the human nasopharynx. Strains with the serotype b capsule are highly virulent in nonimmune children, whereas nonencapsulated (nontypeable H. influenzae [NTHI]) bacteria are an increasingly prevalent cause of localized respiratory tract infections in the adult population. Many H. influenzae strains express pili that enable the bacteria to agglutinate human erythrocytes (9). The primary role of the pili is believed to be initiation of contact between the bacterium and human cell during infection. Analysis of the chromosomal region containing the pilus determinants of H. influenzae serotype b (Hib) strains Eagan and M43 revealed five genes, hifA to hifE, necessary for pilus production (13, 16, 38, 40). The H. influenzae genes have sequence homology with, and have similarities in organization to, the Pap and type I pilus genes (8, 9, 37). HifA is the major pilus antigen, HifB is a periplasmic chaperone, HifC acts as an outer membrane assembly platform, and HifD is believed to be minor pilus component (33). Recent evidence suggests that the fifth gene in the cluster, hifE, encodes a pilus tip-associated adhesin responsible for binding to gangliosides on the surface of mammalian cells (3, 9). Mutations in hifE do not affect the stability of the other pilins but severely reduce assembly of the whole pilus (16, 17, 24, 38). For Hib strain Eagan, McCrea et al. (17) have shown that anti-HifE antiserum binds to pilus tips and blocks pilus-mediated hemagglutination. Conversely, van Ham et al. (36) reported that the major pilus subunit HifA and not the minor pilins HifD or HifE effect binding to erythrocytes and human oropharyngeal epithelial cells. These conflicting results might be due to the slight (20%) difference in primary sequences between the HifA proteins studied or because of differential expression of the recombinant H. influenzae genes in the Escherichia coli DH5α background used in the second study. Pilus expression by H. influenzae oscillates between phase off and phase on at a rate of about 10−4/generation (4). This phase variation is likely due to slip-strand mispairing in a poly(TA) dinucleotide tract separating the divergent, bidirectional hifA and hifBCDE promoters altering the spacing upstream of the transcriptional start sites (39). In 1984, a unique and lethal condition caused by H. influenzae emerged in central Brazil. The disease called Brazilian purpuric fever (BPF), afflicted young children who had recovered earlier from conjunctivitis, causing an array of symptoms including acute fever, petichiae, and vascular collapse (1). The etiologic agent of BPF was found to be a clonal strain of H. influenzae biogroup aegyptius, nonencapsulated organisms in biotype III that had previously only been known to cause self-limited conjunctivitis. The BPF-associated reference strain, F3031, had several characteristics not encountered in other NTHI strains, such as endothelial cell toxicity, resistance to human serum, and the ability to cause bacteremia in infant rats (15, 20, 22). One possible factor in the unusual pathogenicity of F3031 is the immunologically distinctive hemagglutinating pilus elaborated by the strain (41). The pilus appears to be necessary for invasion of primary epithelial cells by F3031 (5). Further, BPF clone strains isolated from systemic sources are piliated more often than is Hib (15). In a recent study we cloned and characterized genetically the F3031 pilus determinants (24). F3031 has two independently phase-variable fimbrial clusters, one of which, hif1, is in a chromosomal location not previously described for H. influenzae (Fig. ​(Fig.1).1). Therefore, pilus production occurs when one or both sets of genes are in the on phase, perhaps explaining the abundant piliation of the strain. We originally designated the F3031 genes hafA to hafE (24), but because they are functionally similar to the H. influenzae hif cluster, we will henceforth refer to them as hifF3031 alleles. While the BPF-associated HifA to HifD gene products share 72 to 96% identity with Hib proteins, the HifEF3031 putative pilus tip adhesin has only 40% identity with its Hib homolog. FIG. 1 Schematic diagram of hif1 and hif2 clusters of BPF-associated strain F3031 (24). The five pilus genes of each cluster are indicated by open arrows, while the flanking H. influenzae genes are indicated by gray arrows. hif1 and hif2 are flanked by direct ... The purpose of this study at its outset was to determine whether the possession of duplicated pilus clusters and the distinctive HifE adhesin were unique features of BPF-associated H. influenzae strains. To further this aim we examined a collection of H. influenzae biogroup aegyptius, NTHI, and capsulated H. influenzae by Southern DNA hybridization, PCR, and sequencing of variant hifE genes.