Structural studies of the inner membrane subunits of the Xanthomonas citri Type IV Secretion System using NMR spectroscopy and cryoelectron microscopy

• Grant number: 24/03217-5
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: April 01, 2024
• End date: May 31, 2025
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Roberto Kopke Salinas
• Grantee: José Edwin Neciosup Quesñay
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 21/10577-0 - Biology of Bacteria and Bacteriophages Research Center, AP.CEPID

Abstract

Bacterial Type IV Secretion Systems (T4SSs) are nanomachines that transport effector proteins or protein-DNA complexes to the extracellular milieu or to a target cell. The Xanthomonas citri T4SS (XAC-T4SS) belongs to the class of minimized T4SSs. Minimized T4SSs consist of eleven VirB subunits (VirB1 to VirB11) and the coupling protein VirD4. Expanded T4SSs have acquired additional subunits, which are presumably required for specialized functions. Examples of expanded T4SSs are the DotD/Icm from L. pneumophila, the Cag system from H. pylori, and the Tra conjugation system encoded by the IncF plasmid (T4SSF). The XAC-T4SS is an effector translocator system that secretes toxins to kill other gram-negative bacteria. All T4SSs exhibit an outer membrane associated core complex (OMCC), which is connected by a stalk (or a channel) to the inner membrane complex (IMC). Recent cryoelectron microscopy (Cryo-EM) based modelling of the complete T4SS (without VirD4 and VirB11) showed that the conjugative T4SS IMC is composed by the N-terminal transmembrane helices of VirB10 and VirB8, and by the polytopic membrane proteins VirB3 and VirB6. Furthermore, an hexamer of the VirB4-ATPase was seen at the cytoplasmic side of the IMC. While the atomic-level three-dimensional structure of the XAC-T4SS OMCC was obtained using single-particle Cryo-EM, high-resolution structural information about the XAC-T4SS IMC or the complete XAC-T4SS have not been obtained to date. Considering that structural characterization the IMC is an extremely challenging task, the study of isolated subunits could anticipate features of the complete assembly revealing precious information. Therefore, in this proposal we aim to develop protocols to express and purify the XAC-T4SS VirB6 and VirB8 and to use biophysical methods (NMR and ITC) to test their interactions with VirB10, VirB2 and VirB5; the last two Vir subunits should form the system's pilus. In addition, we will test the expression of larger constructs corresponding to VirB3/VirB4/VirB5/VirB6 and VirB10. Purification of this T4SS sub-assembly could provide suitable samples for structural characterization by negative staining transmission electron microscopy and cryo-EM.