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

• Grant number: 25/22355-2
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: November 01, 2025
• End date: August 31, 2026
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Roberto Kopke Salinas
• Grantee: Delaram Taghavi
• 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 from the cytoplasm to the extracellular milieu or to a host cell. The Xanthomonas citri T4SS (T4SS-XAC) belongs to the class of minimized systems, being highly similar to the T4SS from Agrobacterium tumefaciens. They all consist of eleven VirB subunits (VirB1 to VirB11) and the coupling protein VirD4. However, the T4SS-XAC is an effector translocator that secretes toxins that kill other gram-negative bacteria, while the A. tumefaciens T4SS is a conjugation system that translocates protein-DNA complexes. 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). All T4SSs exhibit an outer membrane associated core complex (OMCC), which is connected by a stalk or a periplasmic ring to the inner membrane complex (IMC). In minimized systems, the IMC is composed of the N-terminal domain of VirB10 and the membrane proteins VirB3, VirB4, VirB6, and VirB8. Atomic-level three-dimensional structures of the OMCC from expanded and minimized T4SSs were obtained by single-particle reconstruction cryoelectron microscopy (Cryo-EM). Visualization of conjugative T4SSs in native environment using cryoelectron tomography (CryoET) indicated that the IMC displays six-fold symmetry, while density observed at the cytoplasmic side was attributed to a hexamer of dimers of the VirB4-ATPase. More recently, models of the entire T4SS from the Escherichia coli conjugation plasmid R388 and from the Dot/Icm system from Legionella pneumophila were built based on Cryo-EM data. Considering that structural characterization of individual IMC subunits could anticipate features of the complete IMC, we aim, in this proposal, to develop protocols to express, purify and characterize the T4SSXAC IMC subunits VirB6 and VirB8.