Evolutionary and functional relationships among the nontypeable Haemophilus influenzae HMW family of adhesins

Abstract

Nontypeable Haemophilus influenzae (NTHi) is a common cause of localized respiratory tract disease and initiates infection by colonizing the nasopharynx. Approximately 75 to 80% of NTHi clinical isolates produce proteins that belong to the HMW family of adhesins, which are believed to facilitate colonization. The prototype HMW adhesins are designated HMW1 and HMW2 and were identified in NTHi strain 12. HMW1 and HMW2 are 71% identical and 80% similar overall, yet display differing cellular binding specificities. In the present study we set out to define more clearly the relationships between HMW1 and HMW2 and other members of the HMW family of adhesins. PCR analysis of 49 epidemiologically distinct isolates revealed that all strains possessing hmw genes as determined by Southern analysis contain two hmw loci in conserved, unlinked physical locations on the chromosome. Functional analysis of the HMW adhesins produced by three unrelated strains demonstrated that each isolate possesses one protein with HMW1-like adherence properties and another with HMW2-like adherence properties. These findings suggest that the hmw1 and hmw2 loci may have arisen via a gene duplication event in an ancestral strain. In addition, they support the hypothesis that the distinct binding specificities of HMW1 and HMW2 emerged early and have persisted over time, suggesting an ongoing selective advantage.

FIG. 1.

Schematic representation of PCR primer location used for analysis of numbers and chromosomal locations of hmw loci from 49 epidemiologically and genetically distinct NTHi clinical isolates. Diagram is based on locations and organization of the hmw1 and hmw2 loci in NTHi strain 12. Small arrows represent primers used in PCR analysis. Arrowheads designate direction of ORF transcription. HI1679 and HI1598 are designations based on the published sequence of H. influenzae strain Rd.

FIG. 2.

Expression and surface localization of HMW adhesins expressed in DH5α. (A) Western blot of whole-cell sonicates of DH5α expressing 12-1679hmw, 12-1598hmw, 5-1679hmw, 5-1598hmw, 15-1679hmw, or 15-1598hmw coexpressed with hmw1BC were separated via SDS-PAGE, transferred to nitrocellulose, and probed with GP75 (polyclonal antiserum raised against HMW1 from strain 12). The blot shows the fully processed, mature adhesins. (B) Surface localization of the adhesins is shown via whole-cell dot immunoblotting of the strains depicted in panel A, probing with GP75.

FIG. 3.

Adherence properties of HMW adhesins from NTHi strains 12, 5, and 15 expressed in DH5α. Graphs represent adherence, as a percentage of the inoculum, to Chang cells (A), HEp-2 cells (B), HaCaT cells (C), and NCI-H292 cells (D). Values represent the averages ± standard errors of three experiments, each performed in triplicate.

FIG. 4.

Inhibition of adherence with MAA. Monolayers of Chang cells (A) and HaCaT cells (B) were untreated (hatched bars) or preincubated with 5 μg of MAA/ml (shaded bars). Adherence of DH5α expressing HMW adhesins from NTHi strains 12, 5, and 15 was assessed in a 30-min adherence assay. Adherence is expressed as a percentage of the inoculum. Values represent the averages ± standard errors from three experiments, each performed in triplicate.

FIG. 5.

Western blot of NTHi isogenic derivatives. Whole-cell sonicates of the parent strain (wt) and 1598hmw, 1679hmw, and 1598hmw 1679hmw derivatives of NTHi strains 12, 5, and 15 were separated via SDS-7.5% PAGE, transferred to nitrocellulose, and probed with GP75 (polyclonal antiserum raised against HMW1 from strain 12). The blot shows the fully processed, mature adhesins.

FIG. 6.

Adherence properties of isogenic hmw derivatives in NTHi strains 12, 5, and 15. Graphs represent adherence of isogenic derivatives of strain 12 (open bars), strain 5 (shaded bars) and strain 15 (hatched bars), as a percent of the inoculum, to Chang cells (A), HEp-2 cells (B), HaCaT cells (C), and NCI-H292 cells (D). Values represent the averages ± standard errors of three experiments each performed in triplicate. hmw2, hmw1, and hmw1 hmw2 designations are based on phenotypes of the adhesins as established for Fig. 3 and 4.

FIG. 7.

Phylogenetic analysis of HMW mature and binding domain amino acid sequences from NTHi strains 12, 5, and 15. Midpoint rooted phylogenetic trees were constructed with MEGA version 2.1 (21) using the neighbor joining method with Poisson-corrected distances. Bootstrap confidence values are shown at the branches. Scale bars represent the number of amino acid substitutions per site. (A) Phylogenetic tree based on the mature HMW amino acid sequences as defined in Table 2. (B) Phylogenetic tree based on the binding domain of the HMW adhesins from strains 12, 5, and 15 as defined in Table 2.