Inhaltszusammenfassung:
Type III Secretion Systems (T3SS) are a key factor to the virulence of Salmonella Typhimurium. They are membrane-spanning syringe-like molecular machines that inject effector proteins through a hollow needle-like filament directly into the target cells. One prominent hallmark of T3SS is the secretion hierarchy, with distinct stages of assembly and secretion that follow a strict sequential and temporal order. InvE, the Salmonella pathogenicity island-1 (SPI-1) homolog of SctW, is thought to be a vital regulator for the switch between intermediate and late substrates. The so-called gatekeeper is thought to block substrate specificity switching until host cell contact is established. It is suggested that interaction of SctW with the major export apparatus protein InvA, the SPI-1 homolog of SctV, is vital for the regulation of secretion, and several positions in SctW and SctV have been proposed as part of the interaction interface. The aim of this study was to investigate this interaction and the role of InvE as a gatekeeper. The secretion phenotype of ∆invE was assessed with a NanoLuc (NL) luciferase-based T3SS secretion assay. The findings in this study agree with previous data and additionally, newly quantify the abolishment of translocators and oversecretion of effectors in a variety of contexts. Additionally, it was found that tagging of the N-terminus interfered with InvE function, whereas C-terminal tagging can be used for further studies. The results hint at the importance of the N-terminal secretion signal of InvE. Several mutants of specific amino acid residues in InvA have been assessed for their secretion phenotype by NL and TCA-based secretion assays, for protein expression by SDS-PAGE, and for assembly by BN PAGE. The positions were mapped on a predicted nonameric ring structure of InvA and identify regions that are essential for overall function of the T3SS. Interaction between InvA and InvE was investigated by in vivo photocrosslinking. Although some interactions of InvA with other proteins could be identified, no interaction between these two proteins could be identified. The study confirms that the gatekeeper undoubtedly plays an integral part in establishing the secretion hierarchy and in substrate specificity switching between intermediate and late secretion. Nevertheless, the data is incomplete and cannot confirm a possible gatekeeper mechanism, but rather suggests possible improvements for further experiments and options for further scientific exploration.
