The SPI2-encoded SseA chaperone has discrete domains required for SseB stabilization and export, and binds within the C-terminus of SseB and SseD

• Published in: Microbiology (Society for General Microbiology) 2004-07, Vol.150 (7), p.2055-2068
• Main Authors: Zurawski, Daniel V, Stein, Murry A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial plant pathogens; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; HeLa Cells; Humans; Microbiology; Miscellaneous; Molecular Chaperones - chemistry; Molecular Chaperones - genetics; Molecular Chaperones - metabolism; Molecular Sequence Data; Mutation; Phytopathology. Animal pests. Plant and forest protection; Protein Interaction Mapping; Protein Transport; Salmonella; Salmonella typhimurium - pathogenicity; Two-Hybrid System Techniques; Virulence
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.26997-0

Department of Microbiology and Molecular Genetics and the Department of Animal Sciences, Markey Center for Molecular Genetics, University of Vermont, 95 Carrigan Drive, Room 118, Stafford Hall, Burlington, VT 05405, USA Correspondence Murry A. Stein mastein{at}uvm.edu SseA, a key Salmonella virulence determinant, is a small, basic pI protein encoded within the Salmonella pathogenicity island 2 and serves as a type III secretion system chaperone for SseB and SseD. Both SseA partners are subunits of the surface-localized translocon module that delivers effectors into the host cell; SseB is predicted to compose the translocon sheath and SseD is a putative translocon pore subunit. In this study, SseA molecular interactions with its partners were characterized further. Yeast two-hybrid screens indicate that SseA binding requires a C-terminal domain within both partners. An additional central domain within SseD was found to influence binding. The SseA-binding region within SseB was found to encompass a predicted amphipathic helix of a type participating in coiled-coil interactions that are implicated in the assembly of translocon sheaths. Deletions that impinge upon this putative coiled-coiled domain prevent SseA binding, suggesting that SseA occupies a portion of the coiled-coil. SseA occupancy of this motif is envisioned to be sufficient to prevent premature SseB self-association inside bacteria. Domain mapping on the chaperone was also performed. A deletion of the SseA N-terminus, or site-directed mutations within this region, allowed stabilization of SseB, but its export was disrupted. Therefore, the N-terminus of SseA provides a function that is essential for SseB export, but dispensable for partner binding and stabilization. Abbreviations: EPEC, enteropathogenic E. coli ; GST, glutathione S -transferase; LEE, locus for enterocyte effacement; MCS, multiple cloning site; SPI2, Salmonella pathogenicity island 2; TTS, type III secretion