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

Abstract

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.

Identification of Vibrio cholerae Type III Secretion System Effector Proteins

“…CesA is a chaperone in EPEC that has a unique structure, consisting of helices shaped like a hairpin, and has a pI of 9.5 (61). CesA is sometimes referred to as a class IV chaperone (39). Class V chaperones interact with the needle subunit in Yersinia and Pseudomonas and also with a translocator in Salmonella (39).…”

Salmonella enterica Serovar Typhimurium Skills To Succeed in the Host: Virulence and Regulation

“…Strikingly, SPI-3 and SPI-5 play a dual role in pathogenesis, since the two islands encode proteins involved in both invasion and intracellular survival. This dual role is particularly evident in the case of SPI-5, since it encodes effectors secreted via T3SS-1 and T3SS-2 (95,96,239,338). Additional ORFs are located on each of these two islands, although their function has not been completely elucidated, making understanding of their overall role and transcription difficult.…”

Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa

“…Because the internal diameter of the needle is on the order of 25 Å, it is believed that effectors must travel in a semi-unfolded state (2, 3) due to energy provided by an ATPase located at the base of the system (4 -6). Introduction of point mutations into key proteins of the T3SS have shown to be highly deleterious for the cytotoxicity potential of certain pathogens (7)(8)(9)(10)(11), and T3SS-deficient strains display attenuated infectivity in animal models (12)(13)(14), indicating that an understanding of T3SS formation and regulation mechanisms could lead to the development of strategies for infection control.…”