P3-07 Structural study of type IV secretion system protein VirJ

Abstract

Brucella spp. survival and replication inside host cells requires a type IV secretion system (T4SS). The T4SS translocates effectors proteins into the host cell to modulate the intracellular fate of the bacterium in order to establish a secure niche were it actively replicates. Although many VirB substrates have been identified in Brucella, we still know very little about the secretion mechanism that mediate their translocation across the two membranes and the periplasmic space. In Gram negative bacteria, T4SSs are composed of 12 proteins, VirB1 through VirB11 and VirD4 according to the nomenclature used for the prototypal Agrobacterium tumefaciens VirB/D T4SS. VirD4 serves as cytoplasmic receptor for substrate binding to facilitate entry into the translocation channel. The T4SS of Brucella is homologous to the VirB/D system, but lacks the virD4 gene raising questions about effector translocation in this system. VirJ has been identified as a periplasmic protein essential for the translocation of two Brucella effectors BPE123 and SepA. These two effectors are initially secreted in the periplasm before being translocated to the host cell in a VirJ-dependent manner by the T4SS. Indeed, VirJ also interacts with T4SS components VirB5 and VirB8 and thus could facilitate delivery of selected effectors to the T4SS machinery. In this study we have investigated the structural properties of VirJby biochemistry and modelling. We first purified the VirJ protein to investigate its biochemical properties. Multi-angle light scattering (MALS) experiments show that VirJ is monomeric in solution domain. We then predicted the structure of monomeric VirJ using alfafold 2. The structure reveals that VirJ contain 2 domains separated by a short linker. Domain 2 adopts a hydrolase fold with eight b-sheet surrounded by six α helices that appears to be conserved in A. tumefaciens VirJ. Analysis of structure-sequence conservation points towards a potential active site composed of one serine, one histidine and one aspartate. We then present the results of VirJ-effectors interactions and discuss potential mechanism for VirJ-guided translocation.