Heterologous expression of PilP to study the interaction with secretin of type IV pilus

• Grant number: 22/12975-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: January 01, 2023
• End date: June 30, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Cristiane Rodrigues Guzzo Carvalho
• Grantee: Guilherme Camargo Pompeu e Silva
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Type IV Pili (T4P) are retractable extracellular filaments of bacteria that allow twitching motility over surfaces. In the phytopathogen Xanthomonas citri pv. citri, T4P were also associated with biofilm formation and adhesion processes. In this organism, the secretin complex that forms the outer membrane channel for the passage of these filaments is being studied by electron cryomicroscopy (cryoEM) of particles purified directly from their cells. The structure of the secretin formed by 14 copies of PilQ surrounded by 7 copies of the peptidoglycan binding protein TsaP was thus determined (unpublished data). To investigate how this channel couples to the base of the T4P assembly machinery in the inner membrane, we decided to investigate its interaction with PilP, the lipoprotein responsible for anchoring one complex to the other in the periplasm. We intend to clone the HR C-terminal domain of the protein into a vector for heterologous expression and purification by affinity chromatography. The purified protein can then be added to the secretin preparations to try to observe the in vitro interaction by negative stain in a transmission electron microscope and by complementary techniques. It is expected to reveal the position and relative orientation of PilP in contact with the N0 domain of PilQ in the complex, which can be investigated in higher resolution by cryoEM.