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Open Access Highly Accessed Research article

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

Takeshi Haneda1, Mariko Sugimoto1, Yukie Yoshida-Ohta1, Yoshio Kodera2, Masamichi Oh-Ishi2, Tadakazu Maeda2, Satomi Shimizu-Izumi1, Tsuyoshi Miki1, Yoshinori Kumagai1, Hirofumi Danbara1 and Nobuhiko Okada1*

Author Affiliations

1 Department of Microbiology, School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan

2 Laboratory of Biomolecular Dynamics, Department of Physics, Kitasato University School of Science, 1-15-1 Kitasato, Minami-ku, Sagamihara City, Kanagawa 252-0373, Japan

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BMC Microbiology 2010, 10:324  doi:10.1186/1471-2180-10-324

Published: 21 December 2010

Abstract

Background

The global ppGpp-mediated stringent response in pathogenic bacteria plays an important role in the pathogenesis of bacterial infections. In Salmonella enterica serovar Typhimurium (S. 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 S. Typhimurium, agarose 2-dimensional electrophoresis (2-DE) combined with mass spectrometry was used and a comprehensive 2-DE reference map of amino acid-starved S. Typhimurium strain SH100, a derivative of ATCC 14028, was established.

Results

Of the 366 examined spots, 269 proteins were successfully identified. The comparative analysis of the wild-type and ppGpp0 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 Salmonella-specific proteins. In addition, Salmonella 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 stm3169 was controlled by ppGpp and SsrB, a response regulator of the two-component system located on Salmonella pathogenicity island 2.

Conclusions

A proteomic approach using a 2-DE reference map can prove a powerful tool for analyzing virulence factors and the regulatory network involved in Salmonella pathogenesis. Our results also provide evidence of a global response mediated by ppGpp in S. enterica.