STRUCTURAL CHARACTERIZATION OF THE XANTHOMONAS CITRI’s T4SS INNER MEMBRANE COMPLEX

Type IV secretion systems (T4SSs) are versatile translocators employed by Gram-negative bacteria to transfer DNA or to deliver effector proteins into host cells. Minimized Type IV secretion machineries are formed by a multiprotein complex (VirB1-10/VirD4) that spans both the inner and the outer membrane of bacterial cells​. The architecture of the R388 conjugative plasmid T4SS (T4SSR388) was revealed by Cryo-EM. It is formed by two sub-complexes: a core complex (CC) that crosses the periplasm and makes a pore in the outer membrane (VirB7, VirB9 and VirB10), and an inner membrane complex (IMC) inserted in the inner membrane (VirB3, VirB4, VirB6, VirB8 and VirB10). The IMC and the CC are connected to each other by a flexible stalk (VirB6 and VirB10). The Xanthomonas citri T4SS outer membrane CC was structurally characterized (PDB: 6gyb), however, the periplasmic and the inner membrane complexes (IMCXac) remain uncharacterized. Therefore, the aim of this study is to characterize the T4SSXac IMC subunits, focusing on VirB6 and its possible partners. To this end we isolated the recombinant full length VirB6XAC. However, the purified protein did not yield suitable images under the electron cryomicroscope. To increase the size of VirB6 particles, we co-expressed VirB6 with the full-length VirB5. Western blotting experiments showed that VirB6 and VirB5 interact with each other when solubilized with DDM (n-dodecyl-β-D-maltoside). In another attempt to increase the VirB6 particle size, we co-expressed VirB2 with the VirB5/VirB6 operon. Indeed, VirB6 is expected to interact with VirB2 or VirB5 at different stages of the T4SS assembly and functioning. Overall, these studies contribute to a deeper understanding of the Xanthomonas citri T4SS architecture at different functional stages. This work was supported by the São Paulo Research Foundation (FAPESP 2024/03217-5, 2021/10577-0 and 2017/17303-7).