Fortney K R, Young R S, Bauer M E, Katz B P, Hood A F, Munson R S, Spinola S M
Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202-5124, USA.
Infect Immun. 2000 Nov;68(11):6441-8. doi: 10.1128/IAI.68.11.6441-6448.2000.
Haemophilus ducreyi expresses a peptidoglycan-associated lipoprotein (PAL) that exhibits extensive homology to Haemophilus influenzae protein 6. We constructed an isogenic PAL mutant (35000HP-SMS4) by the use of a suicide vector that contains lacZ as a counterselectable marker. H. ducreyi 35000HP-SMS4 and its parent, 35000HP, had similar growth rates in broth and similar lipooligosaccharide profiles. 35000HP-SMS4 formed smaller, more transparent colonies than 35000HP and, unlike its parent, was hypersensitive to antibiotics. Complementation of the mutant in trans restored the parental phenotypes. 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 bacteria on one arm and with three doses (ranging from 28 to 800 CFU) of live 35000HP-SMS4 on the other arm. Papules developed at similar rates at sites inoculated with the mutant or parent but were significantly smaller at mutant-inoculated sites than at parent-inoculated sites. The pustule formation rate 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 six of seven biopsied parent-inoculated sites and from one of three biopsied mutant-inoculated sites. Confocal microscopy confirmed that the bacteria present in a mutant inoculation site pustule lacked a PAL-specific epitope. Although biosafety regulations precluded our testing the complemented mutant in humans, these results suggest that expression of PAL facilitates the ability of H. ducreyi to progress to the pustular stage of disease.
杜克雷嗜血杆菌表达一种与肽聚糖相关的脂蛋白（PAL），该蛋白与流感嗜血杆菌蛋白6具有广泛的同源性。我们使用含有lacZ作为反选择标记的自杀载体构建了一个同基因PAL突变体（35000HP - SMS4）。杜克雷嗜血杆菌35000HP - SMS4及其亲本35000HP在肉汤中的生长速率相似，脂寡糖谱也相似。35000HP - SMS4形成的菌落比35000HP更小、更透明，并且与其亲本不同，它对抗生素高度敏感。该突变体的反式互补恢复了亲本的表型。为了测试PAL的表达是否是毒力所必需的，对9名人类志愿者进行了实验性感染。每个受试者在一只手臂上接种两剂（41至89 CFU）活的35000HP和一剂热灭活细菌，在另一只手臂上接种三剂（28至800 CFU）活的35000HP - SMS4。丘疹在接种突变体或亲本的部位以相似的速率出现，但在接种突变体的部位明显小于接种亲本的部位。脓疱形成率在18个接种亲本的部位为72%（95%置信区间[CI]，46.5至90.3%），在27个接种突变体的部位为11%（95%CI，2.4至29.2%）（P < 0.0001）。从表面培养物中回收杜克雷嗜血杆菌的比率在接种亲本的部位为8%（n = 130；95%CI，4.3至14.6%），在接种突变体的部位为0%（n = 120；95%CI，0.0至2.5%）（P < 0.001）。在7个活检的接种亲本的部位中有6个部位回收了杜克雷嗜血杆菌，在3个活检的接种突变体的部位中有1个部位回收了该菌。共聚焦显微镜检查证实，突变体接种部位脓疱中的细菌缺乏PAL特异性表位。尽管生物安全规定使我们无法在人体中测试互补突变体，但这些结果表明PAL的表达促进了杜克雷嗜血杆菌发展到疾病脓疱阶段的能力。
