Exploring biotechnological applications of bacteria-killing type IV secretion systems.

• Grant number: 24/06039-0
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: June 01, 2024
• End date: May 31, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Shaker Chuck Farah
• Grantee: Wenny Camilla dos Santos Adan
• 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

Understanding the molecular details of how secretion systems recognize substrates for translocation opens up interesting possibilities for their control as well as the design of new biotechnological tools for the delivery of engineered effectors against targeted pathogens. Many Xanthomonadales, Burkholderales, and Neisseriales species carry a type IV secretion system (X-T4SS) specialized in translocating effector proteins (X-Tfes) into other gram-negative species, during bacterial interspecies warfare (Souza et al, 2015; Sgro et al, 2019). These effectors, known as X-Tfes, carry a carboxy terminal domain of <120 residues, termed XVIPCD, characterized by several conserved motifs and a glutamine-rich tail. We have shown that the XVIPCD is required for interaction with the T4SS coupling protein VirD4 and for X-T4SS-dependent translocation, have determined the solution NMR structure of the XVIPCD from one X-Tfe and have mapped the interaction between the central all-alpha domain of VirD4 (VirD4AAD) and the XVIPCD (Oka et al, 2022). The knowledge gained from these studies puts us in a position to create bacterial species better equipped to kill other bacterial species. For example, new X-Tfes could be created by fusing XVIPCDs to novel toxin domains and enteric bacteria carrying a functional X-T4SS and a full repertoire of X-Tfe/X-Tfi (toxin/antitoxin) pairs could be used to modify intestinal bacterial flora. The general objective of this post-doctoral project is to explore some biotechnological applications of bactericidal X-T4SSs. Specific objectives: i) Insert X-Tfe/X-Tfi pairs from other Xanthomonadales species into X. citri to augment its bactericidal activity ii) Create new X-Tfes by fusing XVIPCDs to novel toxin domainsiii) Increase the competitiveness of a common laboratory E. coli strain by transformation with sequences coding for a bactericidal X-T4SS and its X-Tfe/X-Tfi pairsiv) Convert a conjugative T4SS into a bactericidal X-T4SS by exchanging specific T4SS components or domainsv) Generate models of complete X-T4SSs from X. citri, S. maltophilia and other bacterial species