Khan S A, Everest P, Servos S, Foxwell N, Zähringer U, Brade H, Rietschel E T, Dougan G, Charles I G, Maskell D J
The Centre for Veterinary Science, Department of Clinical Veterinary Medicine, University of Cambridge, UK.
Mol Microbiol. 1998 Jul;29(2):571-9. doi: 10.1046/j.1365-2958.1998.00952.x.
Salmonella infections in naturally susceptible mice grow rapidly, with death occurring only after bacterial numbers in vivo have reached a high threshold level, commonly called the lethal load. Despite much speculation, no direct evidence has been available to substantiate a role for any candidate bacterial components in causing death. One of the most likely candidates for the lethal toxin in salmonellosis is endotoxin, specifically the lipid A domain of the lipopolysaccharide (LPS) molecule. Consequently, we have constructed a Salmonella mutant with a deletion-insertion in its waaN gene, which encodes the enzyme that catalyses one of the two secondary acylation reactions that complete lipid A biosynthesis. The mutant biosynthesizes a lipid A molecule lacking a single fatty acyl chain and is consequently less able to induce cytokine and inducible nitric oxide synthase (iNOS) responses both in vivo and in vitro. The mutant bacteria appear healthy, are not sensitive to increased growth temperature and synthesize a full-length O-antigen-containing LPS molecule lacking only the expected secondary acyl chain. On intravenous inoculation into susceptible BALB/c mice, wild-type salmonellae grew at the expected rate of approximately 10-fold per day in livers and spleens and caused the death of the infected mice when lethal loads of approximately 10(8) were attained in these organs. Somewhat unexpectedly, waaN mutant bacteria grew at exactly the same rate as wild-type bacteria in BALB/c mice but, when counts reached 10(8) per organ, mice infected with mutant bacteria survived. Bacterial growth continued until unprecedentedly high counts of 10(9) per organ were attained, when approximately 10% of the mice died. Most of the animals carrying these high bacterial loads survived, and the bacteria were slowly cleared from the organs. These experiments provide the first direct evidence that death in a mouse typhoid infection is directly dependent on the toxicity of lipid A and suggest that this may be mediated via pro-inflammatory cytokine and/or iNOS responses.
在天然易感小鼠中，沙门氏菌感染迅速发展，只有在体内细菌数量达到高阈值水平（通常称为致死量）后才会导致死亡。尽管有很多推测，但一直没有直接证据证实任何候选细菌成分在导致死亡中所起的作用。沙门氏菌病致死毒素最可能的候选成分之一是内毒素，特别是脂多糖（LPS）分子的脂质A结构域。因此，我们构建了一个在waaN基因中有缺失插入的沙门氏菌突变体，该基因编码的酶催化完成脂质A生物合成的两个二级酰化反应之一。该突变体生物合成的脂质A分子缺少一条脂肪酰链，因此在体内和体外诱导细胞因子和诱导型一氧化氮合酶（iNOS）反应的能力都较弱。突变细菌看起来健康，对升高的生长温度不敏感，并且合成全长含O抗原的LPS分子，只是缺少预期的二级酰链。将野生型沙门氏菌静脉接种到易感的BALB/c小鼠中，其在肝脏和脾脏中以每天约10倍的预期速率生长，当这些器官中的细菌数量达到约10⁸的致死量时，会导致感染小鼠死亡。有点出乎意料的是，waaN突变细菌在BALB/c小鼠中的生长速率与野生型细菌完全相同，但是当每个器官的细菌数量达到10⁸时，感染突变细菌的小鼠存活下来。细菌继续生长，直到每个器官达到前所未有的10⁹高数量，此时约10%的小鼠死亡。大多数携带这些高细菌载量的动物存活下来，并且细菌从器官中缓慢清除。这些实验提供了首个直接证据，表明小鼠伤寒感染中的死亡直接取决于脂质A的毒性，并表明这可能是通过促炎细胞因子和/或iNOS反应介导的。
