Haemophilus influenzae - human specific bacteria.

Haemophilus influenzae is both a commensal and a pathogen specific to humans. Here we review this bacterium with special emphasis on characteristics that may be involved in virulence.

Evidence for donor strand complementation in the biogenesis of Haemophilus influenzae haemagglutinating pili.

Haemophilus influenzae haemagglutinating pili are surface appendages that promote attachment to host cells and facilitate respiratory tract colonization, an essential step in the pathogenesis of disease. In contrast to other well-characterized forms of pili, H. influenzae haemagglutinating pili are two-stranded helical structures. Nevertheless, haemagglutinating pili are assembled by a pathway that involves a periplasmic chaperone and an outer membrane usher, analogous to the prototype pathway involved in the biogenesis of Escherichia coli P pili. In this study, we performed site-directed mutagenesis of the H. influenzae HifB chaperone and HifA major pilus subunit at positions homologous to sites important for chaperone-subunit interactions and subunit oligomerization in P pili. Mutations at putative subunit binding pocket residues in HifB or at the penultimate tyrosine in HifA abolished formation of HifB-HifA periplasmic complexes, whereas mutations at the -14 glycine in HifA had no effect on HifB-HifA interactions but abrogated HifA oligomerization. To define further the constraints of the interaction between HifA and HifB, we examined the interchangeability of pilus gene cluster components from H. influenzae type b strain Eagan (hifA-hifEEag) and the related H. influenzae biogroup aegyptius strain F3031 (hifA-hifEF3031). Functional pili were assembled both with HifAEag and the strain F3031 gene cluster and with HifAF3031 and the strain Eagan gene cluster, underscoring the flexibility of the H. influenzae chaperone/usher pathway in incorporating HifA subunits with significant sequence diversity. To gain additional insight into the interactive surfaces of HifA and HifB, we aligned HifA sequences from 20 different strains and then modelled the HifA structure based on the recently crystallized PapD-PapK complex. Analysis of the resulting structure revealed high levels of sequence conservation in regions predicted to interact with HifB, and maximal sequence diversity in regions potentially exposed on the surface of assembled pili. These results suggest broad applicability of structure-function relationships identified in studies of P pili, including the concepts of donor strand complementation and donor strand exchange. In addition, they provide insight into the structure of HifA and suggest a basis for antigenic variation in H. influenzae haemagglutinating pili.

Molecular determinants of the pathogenesis of disease due to non-typable Haemophilus influenzae.

Non-typable Haemophilus influenzae is a common commensal organism in the human upper respiratory tract and an important cause of localized respiratory tract disease. The pathogenesis of disease begins with bacterial colonization of the nasopharynx, a process that involves establishment on the mucosal surface and evasion of local immune mechanisms. Under the proper circumstances, the organism spreads contiguously to the middle ear, the sinuses, or the lungs, and then stimulates a brisk inflammatory response, producing symptomatic infection. In this review, we summarize our present understanding of the molecular determinants of this sequence of events. Continued investigation of the molecular mechanism of non-typable H. influenzae pathogenicity should facilitate development of novel approaches to the treatment and prevention of H. influenzae disease.

Adhesin expression in matched nasopharyngeal and middle ear isolates of nontypeable Haemophilus influenzae from children with acute otitis media.

The HMW1 and HMW2 proteins, Hia, and hemagglutinating pili are important adherence factors in nontypeable Haemophilus influenzae. To gain insight into the relative importance of these adhesins in nasopharyngeal colonization and localized respiratory tract disease, we assessed their expression in matched nasopharyngeal and middle ear isolates of nontypeable H. influenzae from 17 children with acute otitis media. In all patients, including 11 with bilateral disease, the matched isolates were isogenic based on total protein profiles and genomic fingerprints. Of the nasopharyngeal isolates, 14 expressed only HMW1/HMW2-like proteins, 1 expressed only Hia, 1 expressed only pili, and 1 expressed both Hia and pili. Further analysis revealed concordance between nasopharyngeal isolates and the matched middle ear isolates for expression of the HMW1/HMW2-like proteins and Hia. In contrast, in the two children whose nasopharynges were colonized by piliated organisms, the corresponding middle ear isolates were nonpiliated and could not be enriched for piliation. Nevertheless, Southern analysis revealed that these two middle ear isolates contained all five hif genes required for pilus biogenesis and had no evidence of major genetic rearrangement. In summary, the vast majority of isolates of nontypeable H. influenzae associated with acute otitis media express HMW1/HMW2-like proteins, with expression present in both the nasopharynx and the middle ear. A smaller fraction of nasopharyngeal isolates express pili, while isogenic strains recovered from the middle ear are often refractory to enrichment for piliation. We speculate that the HMW adhesins and Hia are important at multiple steps in the pathogenesis of otitis media while pili contribute to early colonization and then become dispensable.

Haemophilus influenzae pili are composite structures assembled via the HifB chaperone.

Haemophilus influenzae is a Gram-negative bacterium that represents a common cause of human disease. Disease due to this organism begins with colonization of the upper respiratory mucosa, a process facilitated by adhesive fibers called pili. In the present study, we investigated the structure and assembly of H. influenzae pili. Examination of pili by electron microscopy using quick-freeze, deep-etch and immunogold techniques revealed the presence of two distinct subassemblies, including a flexible two-stranded helical rod comprised of HifA and a short, thin, distal tip structure containing HifD. Genetic and biochemical studies demonstrated that the biogenesis of H. influenzae pili is dependent on a periplasmic chaperone called HifB, which belongs to the PapD family of immunoglobulin-like chaperones. HifB bound directly to HifA and HifD, forming HifB-HifA and HifB-HifD complexes, which were purified from periplasmic extracts by ion-exchange chromatography. Continued investigation of the biogenesis of H. influenzae pili should provide general insights into organelle development and may suggest novel strategies for disease prevention.