Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines

Abstract

An auxotrophic mutant which requires a metabolite not available in vertebrate tissues should be unable to grow in such tissue and thus be non-virulent. Most mutations to auxotrophy do not affect virulence^1–3, presumably because the required metabolites are available at sufficient concentration. However, Bacon et al.^1,2 noted that a Salmonella typhi mutant requiring p-amino-benzoic acid (p AB) was less virulent (for mice) than its parent. Salmonella, unlike vertebrates, cannot assimilate exogenous folate and must synthesize it from p AB; the virtual absence of p AB from vertebrate tissues is shown by the efficacy of sulphonamide chemotherapy. Yancey et al.⁴ reported reduced virulence for a S. typhimurium mutant with a requirement for 2,3-dihydroxybenzoate (DHB), the precursor of the bacterial iron-acquisition compound, enterochelin. As DHB is not a known vertebrate metabolite, it would be expected to be absent from mouse tissues. Salmonella synthesize p AB and DHB from chorismate, the final product of the aromatic biosynthetic (aro) pathway. A complete block at any step of this pathway should make S. typhimurium auxotrophic for two compounds not available in vertebrate tissues, and thus non-virulent. We describe here the use of a tetracycline-resistance transposon, Tn10 (refs 5, 6), inserted in gene aroA to produce non-reverting, aromatic-requiring derivatives of virulent S. typhimurium strains. These derivatives were virtually non-virulent; their use as live vaccines conferred excellent protection against challenge with a virulent strain.