Understanding the structural and functional aspects of proteins involved in the transport and assimilation of anions in bacteria - role in virulence, pathogenesis and biotechnological potential

• Grant number: 23/15874-8
• Support Opportunities: Regular Research Grants
• Start date: February 01, 2024
• End date: January 31, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Andrea Balan Fernandes
• Grantee: Andrea Balan Fernandes
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Proteins involved in the capture and assimilation of ions, specifically anions, are of great importance in cells as they make such elements available for biological functions, including participation in the regulation of metabolism and activation/repression of essential pathways. Sulfate and phosphate perform essential functions in cells and the lack of these anions has implications for cell growth, virulence, and pathogenesis. In Xanthomonas citri, the system responsible for phosphate import during anion deficiency is an ABC transporter (PstSCAB system), whose activation is related to the activity of the two-component PhoR/PhoB system. In Pseudomonas aeruginosa, the interaction between the two systems is mediated by the PhoU protein, but there is no information for X. citri. Similarly, sulfate import in this bacterium is mediated by at least three ABC transporters, one of which, the SsuABC system, imports sulfate from sulfonated compounds and requires the activity of two enzymes for desulfonization. The enzymes are SsuD and SsuE, an oxide reductase and a monooxygenase, respectively, which work in association to transform sulfate into sulfide. In this proposal, we intend to continue the research developed in the group and characterize the structures and function of the complex formed by the phosphate transport system PstSCAB and the two-component sensor system PhoR/PhoB and the oxidoreductase/mono-oxygenase complex SsuD/SsuE. Structural studies of these processes are highlighted by their academic and biotechnological relevance, especially considering the lack of resolved structures for many components of the PstSCAB transporter and the PhoR-PhoB two-component system in complex, such as the SsuD/E complex. Furthermore, these proteins have several potential applications, such as biosensors for quantifying anions in environmental samples, medical diagnosis, and detection of food contamination. The proteins will be characterized by bioinformatics methods, biophysical and structural tests (SAXS, crystallization, cryo-electron microscopy). Significant advances have already been achieved, including the expression and purification of all target proteins, identification of promising conditions for thermal stabilization and crystallization, and the characterization of protein-protein interactions. The expected results have great relevance in understanding the mechanisms of virulence and pathogenesis of pathogenic microorganisms contributing significantly to the advancement of knowledge in this area and to the development of therapeutic strategies. (AU)