gms | German Medical Science

Introduction and research question: Virulence-associated type III secretion systems (T3SS) serve the injection of bacterial effector proteins into eukaryotic host cells. These effector proteins modulate host cell biology in order to promote colonization and infection. The core of T3SS is a cell envelope-spanning macromolecular machine called injectisome. It is 6 MDa complex consisting of more than 20 different proteins. Our general picture of the assembly of the injectisome reveals a conceptual problem at the early assembly of the needle filament. In this project, we want to clarify whether the secretin assembles onto the base before the onset of secretion of early substrates or whether needle adapter and filament secretion and assembly precedes assembly of the secretin complex. Also identify factors that regulate achievement of the correct path of assembly.

Methods: We utilize in vivo photocrosslinking, a technique that exploits the encoding of the artificial UV-inducible crosslinking amino acid p-benzoyl-phenylalanine to identify protein-protein interactions and to delineate assembly pathways. Also a highly sensitive luciferase-based analysis of type III secretion function.

Results: So far, the experiments that we have done, we got positive crosslinking from PrgJ-SpaP interaction. About PrgH-InvG interaction we didn’t get the results expected, we only obtained unspecific bands around 109 kDa for positive and negative UV samples. Concerning NanoLuc luciferase analysis we found that later induction of invG can complement pre-existing T3SS.

Conclusion and outlook: For InvG-PrgH interaction we need deeper experiments, e.g., membrane fractionation to increase the sensitivity of the analysis. About, crosslinking of PrgJ-SpaP we have to repeat the experiment with a ΔinvG strain to see if that impacts the PrgJ-SpaP interaction. From Secretion kinetics results we have to investigate if after adding the rhamnose, InvG assembles arround the needle filament or it’s a new T3SS.