Characterization of the X-TfeXAC3266/X-TfiXAC3267 nuclease/inhibitor complex associated with the Xanthomonas citri type IV secretion system

• Grant number: 25/03876-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: June 01, 2025
• End date: May 31, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Shaker Chuck Farah
• Grantee: Giovanna Freire Braz
• 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

The type IV secretion system found in the phytopathogen Xanthomonas citri (X-T4SS) functions as a powerful nanomachine that plays a crucial role in bacterial competition, primarily mediating the secretion of toxins into other cells. The nuclease X-TfeXAC3266, predicted to act in the cytosol of the target cell, has a theoretical molecular mass of 92.5 kDa and contains AHH and XVIPCD protein domains, which grant the protein the ability to degrade DNA and be transported by X-T4SS, respectively. However, X-TfeXAC3266 can have its toxic activity inhibited by the cognate immunity protein X-TfiXAC3267, kept in X. citri's cytoplasm as a defense mechanism to prevent autointoxication. The initial hypothesis is that X-TfiXAC3267 could bind to the toxin's AHH catalytic domain. Thus, this project aims to characterize the yet unknown three-dimensional structure of the toxin-antitoxin complex formed by proteins X-TfeXAC3266 and X-TfiXAC3267, using methods such as heterologous expression in E. coli, purification by chromatography, and subsequent analysis by cryo-electron microscopy (cryo-EM) and/or crystallography. Understanding its structure will therefore clarify the molecular interactions established between the toxin and antitoxin of interest, elucidating the mechanism of nuclease inhibition and the potential protein domains involved; finally, the structural characterization of this complex will contribute to future studies on the functions of X-TfeXAC3266 em X. citri and its possible applications in biotechnology. (AU)