Supplemental text

LOS biosynthesis

The H. ducreyi 35000HP genome contained unique genes (not found in H. influenzae Rd or H. somnus 129Pt) that could be involved in LOS biosynthesis. These genes included a possible beta galactoside alpha-2,6-sialyltransferase (HD0053), probable glycosyltransferases (HD0375, HD0884), and a cluster of genes (HD1833-HD1843) corresponding to the wec(rff) locus of E. coli, which is involved in enterobacterial common antigen biosynthesis (13, 31).

H. influenzae Rd and H. somnus 129Pt contained the licABCD genes, which encode the enzymes necessary for adding phosphocholine to LOS. However it appears that in H. somnus 129Pt licA is non-functional due to interruption of the gene with an insertion sequence at its 5’ end. H. somnus 129Pt had HS_0727, an extra copy of licD, which contained a frameshift. H. ducreyi 35000HP had HD1021, which corresponded to the C-terminal region of licA, and did not have the licBCD genes. The licA genes of 129Pt and H. influenzae Rd did not have CAAT repeats in the coding sequence, as does the licA gene from pathogenic H. influenzae strains (21). Instead, the region upstream of the H. influenzae Rd gene (HI1537) contained an alternative start followed by 17 CAAT repeats; this region was out of frame with the actual gene start. The H. somnus 129Pt licA gene (and the upstream region) had no repeats at all.

H. ducreyi 35000HP contains two sialyltransferase genes, neuA (HD0685) and lst (HD0686) (Bozue et al, 1999). neuA was also present in H. somnus 129Pt (HS_0706) and H. influenzae Rd (HI1279). We found that the H. somnus 129Pt neuA gene was truncated, as described previously (M. D. Howard, A. J. Duncan, A. D. Cox, W.Wakarchuk, and T. J. Inzana, Abstr.103rd Gen. Meeting Am. Soc. Microbiol., abstr. Z-003, 2003). H. influenzae Rd contains a gene (HI0871) (6) that had 45% amino acid identity to the H. ducreyi lst gene, but H. somnus 129Pt did not have this gene.

Bacteria may gain access to these iron sources by secreting hemolysin (44) or cytotoxin (37) to lyse the host cells and release the heme-containing compounds. H. somnus 129Pt, H. ducreyi 35000HP and H. influenzae Rd had three hemolysin genes in common (HS_0237/HD1145/HI0301; HS_0737/HD0800/HI1658; HS_1429/HD0770/HI0107; Table S14). In addition, H. ducreyi 35000HP had two genes (HD1326 hhdB, pore-forming component, and HD1327 hhdA, secreted hemolysin) that were not present in H. somnus 129Pt or H. influenzae. Only H. ducreyi has genes encoding cytolethal distending toxin (HD0902 - HD0904), which is not found in other Haemophilus species (37). H. somnus 129Pt also had a gene (HS_0842), originally identified in H. somnus 8025 (48), encoding a bi-functional protein that has hemolysin activity in the N-terminal part, while the C-terminal is similar to OmpA of H. influenzae and H. ducreyi. However, among these three organisms, only H. ducreyi is reported to be hemolytic (24, 33, 44).