Protection Elicited by Glutamine Auxotroph of Yersinia pestis

<b>Background/Objectives:</b><i>Yersinia pestis</i> is an important zoonotic pathogen responsible for the rare but deadly disease of people with bubonic, septic, or pneumonic forms of plague. The emergence of multidrug-resistant <i>Y. pestis</i> strains has attracted more and more researchers' attention to the search for molecular targets for antivirulence therapy, including anti-nutritional-virulence therapy. The <i>glnALG</i> operon plays a crucial role in regulating the nitrogen content within a bacterial cell. This operon codes for three genes: the structural gene <i>glnA</i> and the two regulatory genes <i>glnL</i> and <i>glnG</i>. In this study, we tested the effect of the deletion of glnA and <i>glnALG</i> on the pathogenic properties of <i>Y. pestis</i>. <b>Methods:</b> To assess the contribution of nitrogen metabolism to <i>Y. pestis</i> virulence, knockout mutants Δ<i>glnA</i> and Δ<i>glnALG</i> were constructed. The former was unable to synthesize glutamine, while the latter was not only defective in glutamine synthesis but also lacked the two-component sensor-transcriptional activator pair GlnL and GlnG, which could partially compensate for the decrease in intracellular glutamine concentrations by transporting it from the host or by catabolic reactions. For vaccine studies, immunized mice and guinea pigs were injected s.c. with 200 LD₁₀₀ of the wild-type <i>Y. pestis</i> strain. <b>Results:</b> A single knockout mutation in the <i>glnA</i> gene did not affect the virulence of <i>Y. pestis</i> in mice and guinea pigs. Knockout of the entire <i>glnALG</i> gene cluster was required for attenuation in these animals. The Δ<i>glnALG</i> strain of <i>Y. pestis</i> did not cause death in mice (LD₅₀ > 10⁵ CFU) and guinea pigs (LD₅₀ > 10⁷ CFU) when administered subcutaneously and provided 100% protection of animals when subsequently infected with 200 LD₁₀₀ of the <i>Y. pestis</i> virulent wild-type strain 231. <b>Conclusions:</b><i>Y. pestis</i>, defective in both the glutamine synthetase GlnA and the two-component sensor-transcriptional activator pair GlnL-GlnG, completely lost virulence and provided potent protective immunity to mice and guinea pigs subsequently challenged with a wild-type <i>Y. pestis</i> strain, demonstrating the potential use of the <i>glnALG</i> operon as a new molecular target for developing a safe and efficient live plague vaccine.