Functional studies of VirB2 homology from Staphylococcus aureus

• Grant number: 23/10432-7
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: November 01, 2023
• End date: December 31, 2024
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: Cristiane Rodrigues Guzzo Carvalho
• Grantee: Pedro Antônio França Henrique
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

In an era of increasing antimicrobial resistance and the emergence of multi-resistant bacteria, the transfer of plasmids through conjugation is of significant relevance. This process, long characterized as mediated by a specialized type IV secretion system (T4SS), has been relatively less explored in Gram-positive bacteria. Recently, bioinformatics analyses conducted by our research group revealed that, contrary to the existing literature, conjugative plasmids in these organisms may also contain homologs of the VirB2 protein, which in Gram-negatives assembles the pilus responsible for contacting target cells during transfer. To assess the importance of these VirB2 homologs, we chose to investigate the gene called trsB, present in the T4SS-encoding cluster of the plasmid pGO1 in Staphylococcus aureus. We intend to overexpress a copy fused with a 6×His-tag (already cloned into an inducible vector in S. aureus) to evaluate whether its secretion can be detected via Western Blot and if it is dependent on the presence of pGO1 in the same cell. In a complementary approach, we plan to utilize a gene silencing system for S. aureus based on CRISPR interference (CRISPRi) to inhibit a regulator of the T4SS and derepress the system, potentially facilitating the observation of the investigated phenotypes.