Expression and activation control of the T6SS4 machinery in Yersinia pseudotuberculosis

• Applicant: Dr. Anne-Sophie Herbrüggen
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 494589699

The type 6 secretion system (T6SS) is a versatile nanomachine that is used by bacteria to compete with other pro- and eukaryotes in densely occupied niches to obtain a growth advantage over competitors for space and nutrients. It consists of 13 core components which are assembled into a baseplate where tube, sheath and spike proteins are mounted into a phage tail-like structure. Upon contraction of the sheath, the tube and spike, which is decorated with effector proteins with deleterious functions for the opponent, are propelled out of the bacterial cell into the opponent resulting in growth inhibition or cell death. Yersinia pseudotuberculosis (Y. pstb) encodes four different T6SSs (T6SS1-4). However, all four clusters are mostly repressed under standard laboratory conditions impeding functional analysis of T6SS use in Y. pstb. Recently, we could show that a unique hexameric regulator - named RovC - binds to DNA upstream of T6SS4 gene cluster and promotes its expression, elevating functional research on this T6SS. The preliminary analysis revealed that both, expression and functional assembly of the T6SS4s, seem to occur by molecular mechanisms that are significantly different to the well-studied T6SSs of V. cholerae and P. aeruginosa. Moreover, regulation and firing of T6SS4 is subjected to a multi-layered control circuit implicating also the formation of heterogeneous subpopulations (phenotypic heterogeneity) and multiple stress responses. To elucidate this novel complex control circuit, we will: • Identify and characterize regulators that control expression of rovC using reporter fusion to test transposon- and gene bank libraries. • Investigate the molecular mechanism for T6SS4 phenotypic heterogeneity and its benefits for Y. pstb by sorting and subsequent analysis of the T6SSON and T6SSOFF populations (e.g., microscopy or RNA-sequencing). • Determine the post-translational regulatory cascade that controls assembly and firing of T6SS4 in Y. pstb using phos-tag® analysis to identify post-translationally modified T6SS4 components and pull-down assays and subsequent mass spectrometry of T6SS4 binding partners to characterize post-translational regulators. Furthermore, we will induce cell wall modifications to explore the trigger mechanism of T6SS firing. Taken together, we will identify crucial mechanisms that control expression, functional assembly and firing of T6SS4 in Y. pstb. Our data will reveal important information about how and when Y. pstb T6SS4 is employed, which will shed light on the purpose of T6SSs in Y. pstb.

• DFG Programme: Research Grants