Structural and functional analyses of a new form of bacterial type I secretion

• Applicants: Dr. Roman Gerlach; Professor Dr. Michael Hensel; Professor Dr. Yves André Muller
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens ; Structural Biology
• Term: from 2008 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 56379207

Penetration of polarized epithelial cell barriers by the gastrointestinal pathogen Salmonella enterica requires the cooperation of two protein secretion systems encoded by the Salmonella Pathogenicity Islands (SPI) 1 and 4. SiiE is the only secreted substrate of the SPI4-encoded Type I Secretion System (T1SS) which is required for efficient invasion of polarized epithelial cells. SiiE is a highly repetitive protein containing 53 bacterial immunoglobulin (BIg) domains. Its structural analysis indicated that the molecule is rectified by Ca2+ complexing by two distinct binding sites in BIg domains. SiiE is synthesized and secreted into culture media during the growth phase in which SPI1-T3SS is active. However in this phase, SiiE is also located on the surface of a proportion of cells, and these cells are likely to be productive in adhesion to polarized cells and subsequent invasion. We found that deletions in the N-terminal part of SiiE affect retention and release of SiiE. Our preparatory work also showed that SiiA and SiiB, two accessory proteins of the SPI4-T1SS, are involved in control of SiiE surface expression.This project is directed to unravel the molecular and structural mechanisms of how the T1SS components SiiC, SiiD and SiiF together with SiiAB can control surface retention and release of SiiE. Based on local sequence similarities to Mot, Exb and Tol systems, we propose that SiiA and SiiB form a proton channel in the inner membrane. The activity of this channel might signal changes in the extracellular environment or factors such as host cell contact, to the SPI4-T1SS and SiiE. We want to understand the structure-function relationship of these novel regulatory subunits with the T1SS core components and SiiE. Our specific aims are: (i) to test experimentally if SiiA and SiiB form a proton-conducting channel; (ii) to understand the structural and functional link of the SiiAB subcomplex to the SPI4-T1SS and SiiE; (iii) to characterize the unique properties of T1SS components SiiD and SiiF, and their role in control of SiiE surface expression; and (iv) to understand how SiiE retention and release is controlled.We anticipate a novel mechanism of protein secretion with implications for a growing family of large repetitive proteins of Gram-negative bacteria. Furthermore, our findings should significantly contribute to the mechanistic understanding of ABC transporters in general and T1SS in particular. Although widely distributed amongst pro- and eukaryotic organisms, surprisingly little is known about the exact mechanisms involved. Compared to E. coli Hly system, the SPI4-T1SS represents an alternative model system with additional layers of complexity and significant importance for pathogenicity. This interdisciplinary approach combines the expertise of three groups in molecular biology/in vivo protein-protein interactions, cellular microbiology/imaging and structural biology/biochemistry to analyze the SPI4-T1SS.

• DFG Programme: Research Grants