Construction of "Artilysins" for the control of Xanthomonas citri subsp. citri

• Grant number: 23/08535-2
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Effective date (Start): August 01, 2024
• Effective date (End): July 31, 2026
• Field of knowledge: Agronomical Sciences - Agronomy - Plant Health
• Principal Investigator: Henrique Ferreira
• Grantee: Igor Vinicius Ramos Otero
• Host Institution: Instituto de Biociências (IB). Universidade Estadual Paulista (UNESP). Campus de Rio Claro. Rio Claro , SP, Brazil
• Associated research grant: 21/10577-0 - Biology of Bacteria and Bacteriophages Research Center, AP.CEPID

Abstract

The Gram-negative bacteria Xanthomonas citri subsp. citri (X. citri) is the etiological agent of Asian Citrus Canker (ACC), the primary bacterial disease affecting citrus cultivation in Brazil and worldwide. The current strategy of applying copper compounds in field to prevent the spread of X. citri has raised concerns regarding environmental impact and the possible induction of copper resistance in these bacteria. Among the alternatives to copper use, the utilization of bacteriophages (phages) and/or their enzymes capable of controlling the growth of X. citri stands out. Endolysins are hydrolases derived from phages, efficiently lysing the peptidoglycan of Gram-positive bacteria. However, these enzymes have low efficiency against Gram-negative bacteria. Artilysins are modified endolysins, expressed with peptides capable of destabilizing or destroying the outer membrane of Gram-negative bacteria. The destruction or destabilization of the outer membrane of Gram-negative bacteria allows the modified enzyme to reach the peptidoglycan and consequently lyse the cell wall of these bacteria. Thus, the present project aims to obtain an artilysin capable of destabilizing or destroying the outer membrane of X. citri, degrading the bacterial cell wall of this organism, and acting as a protective antimicrobial agent against citrus canker. For this purpose, the association of five endolysins with three polycationic peptides will be analyzed. The antimicrobial action of artilysins will be evaluated in in vitro and in vivo assays. Initially, four endolysins will be selected from phages infecting X. citri, from the phage library of the Bacteria Genetics Laboratory (LGB - IB/RC). A fifth endolysin will be selected from previously published works and used as a control. Three polycationic peptides will be selected and/or modified from previously published works. A modular expression system will be created based on the Golden Gate assembly technology to obtain different combinations of endolysins and selected polycationic peptides. The present project foresees the creation of 14 new artilysins and the obtaining of an enzyme with proven activity against Gram-negative bacteria. All 15 artilysins will be produced by heterologous expression in Escherichia coli BL21 (DE3) or Pichia pastoris KM71 and purified by affinity chromatography. All enzymes will be characterized for muralytic activity in X. citri. Enzymes with proven muralytic activity will be characterized for optimal temperature and pH. After this stage, the logarithmic growth reduction will be evaluated against three isolates of X. citri, aiming to select the artilysin with the best antimicrobial activity. From this selection, the inhibitory concentration for the artilysin with the best activity will be defined, as well as its stability in relation to pH and temperature. Activity against X. citri in biofilm will also be determined. Finally, in vivo activity will be assessed by protective potential of the artilysin against X. citri infection in Citrus sinensis (sweet orange) leaves. The results of this project will contribute to the development of new ways to control X. citri and potentially control ACC. As part of CEPID B3, the created artilysins may be further used in the control of other relevant bacteria. Finally, the proposed modular expression system may also be used in future stages of optimization and/or the creation of new artilysins.