Expression of sialylated or paragloboside-like lipooligosaccharides are not required for pustule formation by Haemophilus ducreyi in human volunteers

Abstract

The lipooligosaccharide (LOS) of Haemophilus ducreyi, the etiologic agent of chancroid, chemically and immunologically resembles human glycosphingolipid antigens. To test whether LOS that contains paragloboside-like structures was required for pustule formation, an isogenic mutant (35000HP-RSM2) was constructed in losB, which encodes D-glycero-D-manno-heptosyltransferase. 35000HP-RSM2 produces a truncated LOS whose major glycoform terminates in a single glucose attached to a heptose trisaccharide core and 2-keto-3-deoxyoctulosonic acid. Five human subjects were inoculated with 35000HP and 35000HP-RSM2 in a dose-response trial. For estimated delivered doses (EDDs) of >/=25 CFU, the pustule formation rates were 80% for 35000HP and 58% for 35000HP-RSM2. Preliminary data indicated that a previously described Tn916 losB mutant made a minor glycoform that does not require DD-heptose to form the terminal N-acetyllactosamine. If 35000HP-RSM2 made this glycoform, then 35000HP-RSM2 could theoretically make a sialylated glycoform. To test whether sialylated LOS was required for pustule formation, a second trial comparing an isogenic sialyltransferase mutant (35000HP-RSM203) to 35000HP was performed in five additional subjects. For EDDs of >/=25 CFU, the pustule formation rates were 30% for both 35000HP and 35000HP-RSM203. The histopathology and recovery rates of H. ducreyi from surface cultures and biopsies obtained from mutant and parent sites in both trials were similar. These results indicate that neither the expression of a major glycoform resembling paragloboside nor sialylated LOS is required for pustule formation by H. ducreyi in humans.

FIG. 1

Silver stain of an SDS–16% PAGE gel of LOS preparations from the parent and mutant strains of H. ducreyi. Lane 1, LOS from strain 35000HP; lane 2, LOS from the sialyltransferase mutant 35000HP-RSM203; lane 3, LOS from the dd-heptosyltransferase mutant 35000HP-RSM2. The sialic acid-containing glycoform in the parent strain is designated by the arrow.

FIG. 2

Hematoxylin-eosin-stained sections (×25) obtained from sites inoculated with 35000HP (A), 35000HP-RSM2 (B), 35000HP (C), and 35000HP-RSM203 (D). Tissues shown in panels A and B were obtained from subject 88, and those in panels C and D were from subject 119. Note that all specimens contain subepidermal or intraepidermal pustules.