Expression of peptidoglycan-associated lipoprotein is required for virulence in the human model of Haemophilus ducreyi infection

Haemophilus ducreyi expresses a peptidoglycan-associated lipoprotein (PAL) that exhibits extensive homology to Haemophilus influenzae protein 6, We co nstructed an isogenic PAL mutant (35000HP-SMS4) by the use of a suicide vec tor that contains lacZ as a counterselectable marker. H. ducreyi 35000HP-SM S4 and its parent, 35000HP, had similar growth rates in broth and similar l ipooligosaccharide profiles. 35000HP-SMS4 formed smaller, more transparent colonies than 35000HP and, unlike its parent, was hypersensitive to antibio tics, Complementation of the mutant in trans restored the parental phenotyp es. To test whether expression of PAL is required for virulence, nine human volunteers were experimentally infected. Each subject was inoculated with two doses (41 to 89 CFU) of live 35000HP and one dose of heat-killed bacter ia on one arm and with three doses (ranging from 28 to 800 CFU) of live 350 00HP-SMS4 on the other arm. Papules developed at similar rates at sites ino culated with the mutant or parent but were significantly smaller at mutant- inoculated sites than at parent-inoculated sites. The pustule formation rat e was 72% (95% confidence interval [CI], 46.5 to 90.3%) at 18 parent sites and 11% (95% CI,2.4 to 29.2%) at 27 mutant sites (P < 0.0001). The rates of recovery of H. ducreyi from surface cultures were 8% (n = 130; 95% CI, 4.3 to 14.6%) for parent-inoculated sites and 0% (n = 120; 95% CI, 0.0 to 2.5% ) for mutant-inoculated sites (P < 0.001). H. ducreyi was recovered from si x of seven biopsied parent-inoculated sites and from one of three biopsied mutant-inoculated sites. Confocal microscopy confirmed that the bacteria pr esent in a mutant inoculation site pustule lacked a PAL-specific epitope. A lthough biosafety regulations precluded our testing the complemented mutant in humans, these results suggest that expression of PAL facilitates the ab ility of H. ducreyi to progress to the pustular stage of disease.