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Biochemical and structural insights of a new endolysin encoded by the Xanthomonas citri lytic phage CP2

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Author(s):
Caccalano, Mario Nicolas ; Otero, Igor Vinicius Ramos ; Zamuner, Caio Felipe Cavicchia ; Linhares, Leonardo Alves ; Enright, Mark ; Ramos, Carlos Henrique Inacio ; Ferreira, Henrique
Total Authors: 7
Document type: Journal article
Source: International Journal of Biological Macromolecules; v. 338, p. 11-pg., 2026-01-01.
Abstract

The Gram-negative bacterium Xanthomonas citri subsp. citri (X. citri) is a phytopathogen causal agent of citrus canker, a severe disease affecting citrus crops worldwide. Disease control relies primarily on copper biocides, which pose toxicity risks to humans and the environment. Endolysins are enzymes that catalyze bacterial cell wall degradation and could serve as environmentally sustainable alternatives for the control of X. citri. Here, we conducted structural in silico studies with the endolysin CP2-07 encoded by the X. citri lytic phage CP2, followed by its heterologous expression and purification for biochemical characterization. Structural analysis revealed that CP2-07 is a globular endolysin that putatively lacks the typical beta-hairpin motif found in other lysozymes, while retaining conserved residues in its catalytic cleft. This new muramidase exhibited activity against purified peptidoglycan extracted from X. citri, demonstrating good thermostability (Tm of 52 degrees C), activity across a broad pH range (7-10), and optimal performance in the presence of 60 mM NaCl. Furthermore, CP2-07 displayed bactericidal action against X. citri at 100 mu g/mL and effectively promoted bacterial cell lysis during the mid-log phase, but only in the presence of EDTA (a membrane permeabilizer). This study lays the groundwork for exploring and developing novel biocatalysis-based strategies for controlling economically significant bacterial phytopathogens. (AU)

FAPESP's process: 21/10577-0 - Biology of Bacteria and Bacteriophages Research Center
Grantee:Shaker Chuck Farah
Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
FAPESP's process: 23/08535-2 - Construction of "Artilysins" for the control of Xanthomonas citri subsp. citri
Grantee:Igor Vinicius Ramos Otero
Support Opportunities: Scholarships in Brazil - Post-Doctoral
FAPESP's process: 18/21164-5 - Xanthomonas citri subsp. citri citrus canker in São Paulo. molecular epidemiology and biocontrol potential of bacteriophages and their depolymerases
Grantee:Henrique Ferreira
Support Opportunities: Regular Research Grants
FAPESP's process: 23/12177-4 - Multi-user equipment approved in grant 2021/10577-0: circular dichroism spectrophotometers JASCO, model J-1500
Grantee:Henrique Ferreira
Support Opportunities: Multi-user Equipment Program
FAPESP's process: 22/01814-0 - Evaluation of the lytic potential of endolysins encoded by Xanthomonas citri phages
Grantee:Mario Nicolas Caccalano
Support Opportunities: Scholarships in Brazil - Master