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Journal of Bacteriology, November 2009, p. 6843-6854, Vol. 191, No. 22
0021-9193/09/$08.00+0     doi:10.1128/JB.00863-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Functional Characterization of SsaE, a Novel Chaperone Protein of the Type III Secretion System Encoded by Salmonella Pathogenicity Island 2{triangledown}

Tsuyoshi Miki,1 Yoshio Shibagaki,2 Hirofumi Danbara,1 and Nobuhiko Okada1*

Department of Microbiology,1 Department of Biochemistry, School of Pharmacy, Kitasato University, Minato-ku, Tokyo 108-8641, Japan2

Received 1 July 2009/ Accepted 9 September 2009

The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI-2) is involved in systemic infection and intracellular replication of Salmonella enterica serovar Typhimurium. In this study, we investigated the function of SsaE, a small cytoplasmic protein encoded within the SPI-2 locus, which shows structural similarity to the T3SS class V chaperones. An S. enterica serovar Typhimurium ssaE mutant failed to secrete SPI-2 translocator SseB and SPI-2-dependent effector PipB proteins. Coimmunoprecipitation and mass spectrometry analyses using an SsaE-FLAG fusion protein indicated that SsaE interacts with SseB and a putative T3SS-associated ATPase, SsaN. A series of deleted and point-mutated SsaE-FLAG fusion proteins revealed that the C-terminal coiled-coil domain of SsaE is critical for protein-protein interactions. Although SseA was reported to be a chaperone for SseB and to be required for its secretion and stability in the bacterial cytoplasm, an sseA deletion mutant was able to secrete the SseB in vitro when plasmid-derived SseB was overexpressed. In contrast, ssaE mutant strains could not transport SseB extracellularly under the same assay conditions. In addition, an ssaE(I55G) point-mutated strain that expresses the SsaE derivative lacking the ability to form a C-terminal coiled-coil structure showed attenuated virulence comparable to that of an SPI-2 T3SS null mutant, suggesting that the coiled-coil interaction of SsaE is absolutely essential for the functional SPI-2 T3SS and for Salmonella virulence. Based on these findings, we propose that SsaE recognizes translocator SseB and controls its secretion via SPI-2 type III secretion machinery.

* Corresponding author. Mailing address: Department of Microbiology, School of Pharmacy, Kitasato University, Minato-ku, Tokyo 108-8641, Japan. Phone: 81-3-5791-6256. Fax: 81-3-3444-4831. E-mail: okadan{at}pharm.kitasato-u.ac.jp

{triangledown} Published ahead of print on 18 September 2009.

Journal of Bacteriology, November 2009, p. 6843-6854, Vol. 191, No. 22
0021-9193/09/$08.00+0     doi:10.1128/JB.00863-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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