A LETHAL ROLE FOR LIPID-A IN SALMONELLA INFECTIONS

Salmonella infections in naturally susceptible mice grow rapidly, with death occurring only after bacterial numbers in vivo have reached a h igh threshold level, commonly called the lethal load. Despite much spe culation, no direct evidence has been available to substantiate a role for any candidate bacterial components in causing death. One of the m ost likely candidates for the lethal toxin in salmonellosis is endotox in, specifically the lipid A domain of the lipopolysaccharide (LPS) mo lecule. Consequently, we have constructed a Salmonella mutant with a d eletion-insertion in its waaN gene, which encodes the enzyme that cata lyses one of the two secondary acylation reactions that complete lipid A biosynthesis, The mutant biosynthesizes a lipid A molecule racking a single fatty acyl chain and is consequently less able to induce cyto kine and inducible nitric oxide synthase (iNOS) responses both in vivo and in vitro. The mutant bacteria appear healthy, are not sensitive t o 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 s almonellae grew at the expected rate of approximately 10-fold per day in livers and spleens and caused the death of the infected mice when l ethal loads of approximately 10(8) were attained in these organs. Some what unexpectedly, waaN mutant bacteria grew at exactly the same rate as wild-type bacteria in BALB/c mice but, when counts reached 10(8) pe r organ, mice infected with mutant bacteria survived. Bacterial growth continued until unprecedentedly high counts of 10(9) per organ were a ttained, when approximately 10% of the mice died. Most of the animals carrying these high bacterial loads survived, and the bacteria were sl owly cleared from the organs. These experiments provide the first dire ct evidence that death in a mouse typhoid infection is directly depend ent on the toxicity of lipid A and Suggest that this may be mediated v ia proinflammatory cytokine and/or iNOS responses.