Functional analysis and characterization of the type I secretion system and its substrate, the giant adhesin SiiE, of Salmonella enterica

Salmonella enterica is a facultative intracellular pathogen, able to invade various hosts and successfully replicate within them. Invasion of polarized cells by S. enterica serovar Typhimurium (STM) occurs in dependence of the type 1 secretion system (T1SS), encoded on Salmonella Pathogenicity Island 4 (SPI4). The 595 kDa non-fimbrial adhesin SiiE is the substrate of the SPI4-T1SS and mediates the first close contact to the host cells apical side. This allows for the type 3 secretion system (T3SS) of the SPI1 to translocate its effector proteins into the host cells cytosol, leading to actin remodeling, membrane ruffle formation and finally uptake of the pathogen. The SPI4-T1SS belongs to the family of ATP-binding cassette (ABC) transporters and is characteristically composed of the ATPase SiiF in the inner membrane (IM), the periplasmic adaptor protein (PAP) SiiD and the secretin SiiC. Further there are two non-canonical proteins encoded, namely SiiA and SiiB, which are known to form a proton channel in the IM. Every single subunit was found to be essential for invasion of polarized cells. The substrate SiiE is transiently retained on the cell surface during secretion process and protrudes the lipopolysaccharide (LPS) layer, a step essential for adhesion. Following translocation of the SPI1-T3SS effector proteins, SiiE is released into the extracellular space. Utilizing a variety of techniques, I was able to show that the transient retention of SiiE only occurs in the outer membrane (OM) protein SiiC and not in the whole two membrane-spanning T1SS. My analyses showed that the proton channel SiiAB is involved in initial steps of secretion and not necessary for release of SiiE, further narrowing down possible modes of action. I found a potential proteolytic cleavage site in the N-terminal part of SiiE, essential for release of the adhesin and discovered a potential retention domain in its N-terminus, too bulky to pass through the secretin. Additionally, I gained first hints that the large cytosolic domain of SiiB is not only involved in SiiE retention mechanism, but also in flagellar-dependent movement under swarming conditions. Using dual-color 3D direct stochastic optical reconstruction microscopy (dSTORM), I was able to localize SiiAB in the IM and SiiB not only at the SPI4-T1SS, but during SiiE retention maximum primarily at the flagellum. Intriguingly, the synthetic expression of siiAB as well as synthetic expression of the flagellar stator unit motAB both showed an increase of velocity. Furthermore, I successfully established murine and human intestinal organoid cell culture for microscopic and quantitative analyses of STM and S. Paratyphi A (SPA) invasion processes. Thus, with this work I was able to reveal new insights of the SPI4-T1SS, its substrate SiiE and the non-canonical subunits SiiAB that pave the way for further SPI4-T1SS investigations and also other secretion systems and their associated subunits