Simultaneous prevention of glutamine synthesis and high-affinity transport attenuates Salmonella typhimurium virulence

In Salmonella typhimurium, transcription of the glnA gene (encoding glutamine synthetase) is under the control of the nitrogen-regulatory (ntr) system comprising the alternate sigma factor σ⁵⁴ (NtrA) and the two- component sensor-transcriptional activator pair NtrB and NtrC. The glnA, ntrB, and ntrC genes form an operon. We measured the virulence of S. typhimurium strains with nitrogen-regulatory mutations after intraperitoneal (i.p.) or oral inoculations of BALB/c mice. Strains with single mutations in glnA, ntrA, ntrB, or ntrC had i.p. 50% lethal doses (LD₅₀s) of <10 bacteria, similar to the wild-type strain. However, a strain with a Δ(glnA- ntrC) operon deletion had an i.p. LD₅₀ of > 10⁵ bacteria, as did ΔglnA ntrA and ΔglnA ntrC strains, suggesting that glnA strains require an ntr- transcribed gene for full virulence. High-level transcription of the glutamine transport operon (glnHPQ) is dependent upon both ntrA and ntrC, as determined by glnHp-lacZ fusion measurements. Moreover, ΔglnA glnH and ΔglnA glnQ strains are attenuated, similar to ΔglnA ntrA and ΔglnA ntrC strains. These results reveal that access of S. typhimurium to host glutamine depends on the ntr system, which apparently is required for the transcription of the glutamine transport genes. The Δ(glnA-ntrC) strain exhibited a reduced ability to survive within the macrophage cell line J774, identifying a potential host environment with low levels of glutamine. Finally, the Δ(glnA-ntrC) strain, when inoculated at doses as low as 10 organisms, provided mice with protective immunity against challenge by the wild-type strain, demonstrating its potential use as a live vaccine.