Prevalence of genetic differences in phosphorylcholine expression between nontypeable Haemophilus influenzae and Haemophilus haemolyticus

Abstract

Background: Although non-typeable (NT) Haemophilus influenzae and Haemophilus haemolyticus are closely related human commensals, H. haemolyticus is non-pathogenic while NT H. influenzae is an important cause of respiratory tract infections. Phase-variable phosphorylcholine (ChoP) modification of lipooligosaccharide (LOS) is a NT H. influenzae virulence factor that, paradoxically, may also promote complement activation by binding C-reactive protein (CRP). CRP is known to bind more to ChoP positioned distally than proximally in LOS, and the position of ChoP within LOS is dictated by specific licD alleles (designated here as licDI, licDIII, and licDIV) that are present in a lic1 locus. The lic1 locus contains the licA-licD genes, and ChoP-host interactions may also be influenced by a second lic1 locus that allows for dual ChoP substitutions in the same strain, or by the number of licA gene tetranucleotide repeats (5'-CAAT-3') that reflect phase-variation mutation rates.

Results: Using dot-blot hybridization, 92% of 88 NT H. influenzae and 42.6% of 109 H. haemolyticus strains possessed a lic1 locus. Eight percent of NT H. influenzae and none of the H. haemolyticus strains possessed dual copies of lic1. The licDIII and licDIV gene alleles were distributed similarly (18-22%) among the NT H. influenzae and H. haemolyticus strains while licDI alleles were present in 45.5% of NT H. influenzae but in less than 1% of H. haemolyticus strains (P < .0001). NT H. influenzae had an average of 26.8 tetranucleotide repeats in licA compared to 14.8 repeats in H. haemolyticus (P < .05). In addition, NT H. influenzae strains that possessed a licDIII allele had increased numbers of repeats compared to NT H. influenzae with other licD alleles (P < .05).

Conclusions: These data demonstrate that genetic similarities and differences of ChoP expression exist between NT H. influenzae and H. haemolyticus and strengthen the hypothesis that, at the population level, these differences may, in part, provide an advantage in the virulence of NT H. influenzae.

Figure 1

LOS profiles and TEPC-15 mAb reactivity in H. haemolyticus. H. influenzae and H. haemolyticus whole-cell lysates were run on tricine SDS-PAGE and silver stained to visualize LOS migration (upper panel) or transferred to nitrocellulose membrane for reactivity with the ChoP-specific mAb, TEPC-15 (lower panel). Lanes 1-3, H. influenzae ChoP phase-on variant strains (E1a, Rd, and Mr15); lanes 4-9, H. haemolyticus strains hybridizing with a licA gene probe (M07-22, 60P3H1, 7P24 H, 3P41H5, C03-22, and H01-21); and lanes 10-14, H. haemolyticus strains not hybridizing with a licA gene probe (ATCC 33390, 3P18H1, 24P4 H, 26428, 26322)

Figure 2

Clustering of H. influenzae and H. haemolyticus LicD alleles. The major clusters of H. influenzae (blue dots) and H. haemolyticus (red dots) strains are labeled by their predicted allele (LicD_I, LicD_III, and LicD_IV) and prototype LicD alleles from H. influenzae strains are shown for each cluster (black dots, E1a is partially hidden). The LicD protein of N. lactamica is the out-group for the analysis (green triangle).

Figure 3

Distribution of NT H. influenzae and H. haemolyticus strains with various numbers of CAAT repeats. Percent of lic1-positive NT H. influenzae and H. haemolyticus strains based on the number of CAAT repeats they contain. NT H. influenzae and H. haemolyticus are labeled in blue and red, respectively.