Amidase and Lysozyme Dual Functions in TseP Reveal a New Family of Chimeric Effectors in the Type VI Secretion System

Abstract

AbstractPeptidoglycan (PG) serves as an essential target for antimicrobial development. An overlooked reservoir of antimicrobials lies in the form of PG-hydrolyzing enzymes naturally produced for polymicrobial competition, particularly those associated with the type VI secretion system (T6SS). Here we report that a T6SS effector TseP, fromAeromonas dhakensis, represents a family of effectors with dual amidase-lysozyme activities.In vitroPG-digestion coupled with LC-MS analysis revealed the N-domain’s amidase activity, which is neutralized by either catalytic mutations or the presence of the immunity protein TsiP. The N-domain, but not the C-domain, of TseP is sufficient to restore T6SS secretion in T6SS-defective mutants, underscoring its critical structural role. Using pull-down and secretion assays, we showed that these two domains interact directly with a carrier protein VgrG2 and can be secreted separately. Homologs inAeromonas hydrophilaandPseudomonas syringaeexhibited analogous dual functions. Additionally, N- and C-domains display distinctive GC contents, suggesting an evolutionary fusion event. By altering the surface charge through structural-guided design, we engineered the TsePC4+effector that successfully lyses otherwise resistantBacillus subtiliscells, enabling the T6SS to inhibitB. subtilisin a contact-independent manner. This research uncovers TseP as a new family of bifunctional chimeric effectors targeting PG, offering a potential strategy to harness these proteins in the fight against antimicrobial resistance.Significance StatementAntimicrobial resistance urgently demands global interventions, and the bacteria cell wall remains a promising target. Our research introduces a novel family of bifunctional, cell-wall-damaging T6SS effectors. More importantly, we demonstrate an effective strategy to enable an otherwise ineffective enzyme to target both Gram-negative and Gram-positive bacteria. Our findings highlight a promising path forward using cell-wall-damaging effectors, a largely untapped resource, in the fight against antimicrobial resistance.