Strukturelle und funktionelle Analyse einer neuen Form bakterieller Typ-I-Sekretion
Strukturbiologie
Zusammenfassung der Projektergebnisse
Salmonella enterica is a versatile zoonotic pathogen able to cause diseases in human ranging from mild gastroenteritis to severe systemic infections. Adhesion to host cells is a crucial step in the pathogenesis of facultative intracellular Salmonella. The initial contact between Salmonella and polarized epithelial cells is established via the Salmonella pathogenicity island 4 (SPI-4)-encoded type I secretion system (T1SS). The SPI-4-located sii operon encodes for six proteins: SiiABCDEF. SiiCDF form a canonical T1SS, i.e. SiiC corresponds to the outer membrane (OM) pore protein, SiiD to the periplasmic adapter protein and SiiF to the inner membrane (IM) ATP-binding cassette (ABC) protein. The 595 kDa SiiE is a giant non-fimbrial adhesin and the only substrate secreted by the SPI-4-encoded T1SS. The function of the two remaining proteins SiiA and SiiB was at the beginning of our study only poorly understood. The goal of this collaborative project was to better understand Salmonella infectivity and to derive structure-function relationships for the proteins of the SPI-4-encoded T1SS. We succeeded in producing various T1SS proteins, namely a folded domain of SiiA (SiiA-PD), three variants of SiiD, two variants of the SiiE N-terminal region and one variant of the SiiF N-terminal domain. At the same time, protocols for the recombinant production of the integral membrane protein complex SiiAB were established that resulted in initial crystallization and electron microscopy trials. For SiiE, the contribution of two different types of Ca2+-binding sites that are present in almost all 53 BIg domains to the function of this giant adhesin was analyzed. After deletion of type I and/or type II Ca2+-binding sites, we interrogated the SiiE variants for structural integrity, secretion, surface expression, and function as adhesin for interaction with polarized epithelial cells. Type I Ca2+-binding sites were critical for efficient secretion of SiiE and a decreasing number of type I sites correlated with reduced secretion. Type II sites were less important for secretion, stability and surface expression of SiiE, however integrity of type II sites in the C-terminal portion was required for the function of SiiE in mediating adhesion and invasion. We also investigated in detail the structure of SiiA and its contribution to the function of the SPI-4- encoded T1SS. The structure of SiiA-PD was solved at 1.9 Å resolution and displays homology to that of MotB and other peptidoglycan (PG)-binding domains. SiiA-PD binds PG in vitro, albeit at an acidic pH only. Mutation of Arg162 impedes PG-binding of SiiA and, at the same time, reduces Salmonella invasion efficacy. SiiA forms a complex with SiiB, and the observed SiiA-MotB homology is paralleled by a predicted SiiB-MotA homology. We showed that, similar to MotAB, the SiiAB complex translocates protons (H+) across the IM. While MotAB uses the proton motive force (PMF) to propel the bacterial flagellum, the link from SiiAB-harvested PMF to T1SS function remained to be shown. To gain more insight in SiiAB function, we identified protein interaction partners as potential regulators. Surprisingly, the methyl-accepting chemotaxis protein (MCP) CheM was found to interact with SiiA and SiiB. Using a set of MCP mutants, the presence of CheM decreased invasion of non-polarized HeLa cells, while the phenotype was reversed for polarized MDCK. With addition of a derivative of the CheM attractant L-aspartate, elevated Salmonella invasion was observed, which was independent of the chemotaxis signaling cascade and motility. Furthermore, CheM attractant shifted SiiE localization from extracellular (secreted) to the bacterial surface (retained). We propose a model similar to MotAB, where the identified PG-binding domain of SiiA might function as a molecular plug to regulate H+-flux. Upon attractant binding, structural changes could modulate the molecular interface between CheM and the SiiAB channel, thus enabling PG-binding of SiiA and H+-flux. The energy harvested from the PMF would then be transferred to the SPI-4 T1SS by means of an energy-rich conformation resulting in retention of the SiiE molecule. In summary, we identified a unique link between a bacterial chemosensor and a virulence function resulting in precise spatio-temporal regulation of Salmonella adhesion in response to environmental signals. With that, our findings contribute to the general understanding of host cell sensing by bacterial pathogens.
Projektbezogene Publikationen (Auswahl)
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(2017). Scarless deletion of up to seven methyl-accepting chemotaxis genes with an optimized method highlights key function of CheM in Salmonella Typhimurium. PLoS One, 12(2):e0172630
Hoffmann S., Schmidt C., Walter S., Bender J.K. & Gerlach R.G.
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(2017). Structural and functional dissection reveals distinct roles of Ca2+-binding sites in the giant adhesin SiiE of Salmonella enterica. PLoS Pathogens 13: e1006418
Peters, B., Stein, J., Klingl, S., Sander, N., Sandmann, A., Taccardi, N., Sticht, H., Gerlach, R. G., Muller, Y. A., & Hensel, M.
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(2018). High-throughput quantification of bacterial-cell interactions using virtual colony counts. Frontiers in Cellular and Infection Microbiology, 8:43
Hoffmann S., Walter S., Blume A.K., Fuchs S., Schmidt C., Scholz A. & Gerlach R.G.
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(2019). Structural and functional characterisation of SiiA, an auxiliary protein from the SPI-4-encoded type 1 secretion system from Salmonella enterica. Molecular Microbiology 112: 1403-1422
Kirchweger, P., Weiler, S., Egerer-Sieber, C., Blasl, A.-T., Hoffmann, S., Schmidt, C., Sander, N., Merker, D., Gerlach, R. G., Hensel, M. & Muller, Y. A.
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(2020) Recombinant protein production and purification of SiiD, SiiE and SiiF - components of the SPI-4-encoded type I secretion system from Salmonella Typhimurium. In: bioRχiv
Klingl, S., Kordes, S., Schmid, B., Gerlach, R. G., Hensel, M. & Muller, Y. A.
