Comparative proteomic analysis of Salmonella enterica serovar Typhimurium ppGpp-deficient mutant to identify a novel virulence protein required for intracellular survival in macrophages

<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>The global ppGpp-mediated stringent response in pathogenic bacteria plays an important role in the pathogenesis of bacterial infections. In <jats:italic>Salmonella enterica</jats:italic> serovar Typhimurium (<jats:italic>S</jats:italic>. Typhimurium), several genes, including virulence genes, are regulated by ppGpp when bacteria are under the stringent response. To understand the control of virulence genes by ppGpp in <jats:italic>S</jats:italic>. Typhimurium, agarose 2-dimensional electrophoresis (2-DE) combined with mass spectrometry was used and a comprehensive 2-DE reference map of amino acid-starved <jats:italic>S</jats:italic>. Typhimurium strain SH100, a derivative of ATCC 14028, was established.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>Of the 366 examined spots, 269 proteins were successfully identified. The comparative analysis of the wild-type and ppGpp<jats:sup>0</jats:sup> mutant strains revealed 55 proteins, the expression patterns of which were affected by ppGpp. Using a mouse infection model, we further identified a novel virulence-associated factor, STM3169, from the ppGpp-regulated and <jats:italic>Salmonella</jats:italic>-specific proteins. In addition, <jats:italic>Salmonella</jats:italic> strains carrying mutations in the gene encoding STM3169 showed growth defects and impaired growth within macrophage-like RAW264.7 cells. Furthermore, we found that expression of <jats:italic>stm3169</jats:italic> was controlled by ppGpp and SsrB, a response regulator of the two-component system located on <jats:italic>Salmonella</jats:italic> pathogenicity island 2.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>A proteomic approach using a 2-DE reference map can prove a powerful tool for analyzing virulence factors and the regulatory network involved in <jats:italic>Salmonella</jats:italic> pathogenesis. Our results also provide evidence of a global response mediated by ppGpp in <jats:italic>S. enterica</jats:italic>.</jats:p> </jats:sec>