Conserved Salt Bridges Facilitate Assembly of the Helical Core Export Apparatus of a Salmonella enterica Type III Secretion System |
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| Institution: | 1. Interfaculty Institute of Microbiology and Infection Medicine (IMIT), University of Tübingen, Tübingen, Germany;2. University Hospital Tübingen, Department of Internal Medicine VIII, Tübingen, Germany;3. School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, 70211 Kuopio, Finland;4. Excellence Cluster “Controlling Microbes to Fight Infections” (CMFI), Tübingen, Germany;5. Partner-site Tübingen, German Center for Infection Research (DZIF), Tübingen, Germany;6. Proteome Center Tübingen, University of Tübingen, Tübingen, Germany;1. Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada;2. Centre for Blood Research, University of British Columbia, Vancouver, BC V6T 1Z3, Canada;3. HRMEM facility, University of British Columbia, Vancouver, BC V6T 1Z3, Canada;4. Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4, Canada;5. Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada;1. Department of Biochemistry and Molecular Biology, The University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada;2. Centre for Blood Research, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada;3. National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA;4. Department of Biochemistry, Department of Genome Sciences, and Howard Hughes Medical Institute, University of Washington, 4000 15th Avenue NE, Seattle, WA 98195, USA;5. Department of Chemistry, University of British Columbia, 2350 Health Sciences Mall, Vancouver BC, V6T 1Z3, Canada;3. University of Tübingen, Interfaculty Institute of Microbiology and Infection Medicine (IMIT), Section of Cellular and Molecular Microbiology, Elfriede-Aulhorn-Str. 6, 72076 Tübingen, Germany;4. University of Tübingen, Proteome Center Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany;5. University of Tübingen, Center for Plant Molecular Biology (ZMBP), Auf der Morgenstelle 32, 72076 Tübingen, Germany;6. Yale University School of Medicine, Department of Microbial Pathogenesis, 295 Congress Ave, New Haven, CT;1. Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030, USA;2. Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06536, USA;3. Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville, FL 32608, USA |
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| Abstract: | Virulence-associated type III secretion systems (T3SS) are utilized by Gram negative bacterial pathogens for injection of effector proteins into eukaryotic host cells. The transmembrane export apparatus at the core of T3SS is composed of a unique helical complex of the hydrophobic proteins SctR, SctS, SctT, and SctU. These components comprise a number of highly conserved charged residues within their hydrophobic domains. The structure of the closed state of the core complex SctR5S4T1 revealed that several of these residues form inter- and intramolecular salt bridges, some of which have to be broken for pore opening. Mutagenesis of individual residues was shown to compromise assembly or secretion of both, the virulence-associated and the related flagellar T3SS. However, the exact role of these conserved charged residues in the assembly and function of T3SS remains elusive. Here we performed an in-depth mutagenesis analysis of these residues in the T3SS of Salmonella Typhimurium, coupled to blue native PAGE, in vivo photocrosslinking and luciferase-based secretion assays. Our data show that these conserved salt bridges are not critical for assembly of the respective protein but rather facilitate the incorporation of the following subunit into the assembling complex. Our data also indicate that these conserved charged residues are critical for type III-dependent secretion and reveal a functional link between SctSE44 and SctTR204 and the cytoplasmic domain of SctU in gating the T3SS injectisome. Overall, our analysis provides an unprecedented insight into the delicate requirements for the assembly and function of the machinery at the core of T3SS. |
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| Keywords: | type III secretion system protein secretion export apparatus injectisome |
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