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

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Abstract

Peptidoglycan (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, from Aeromonas dhakensis , represents a family of effectors with dual amidase-lysozyme activities. In vitro PG-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 in Aeromonas hydrophila and Pseudomonas syringae exhibited 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 TseP C4+ effector that successfully lyses otherwise resistant Bacillus subtilis cells, enabling the T6SS to inhibit B. subtilis in 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 Statement

Antimicrobial 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.

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  1. Thank you very much for this amazing! I love learning about novel T6S effectors. This one is especially cool for so many reasons, like the fact that it contains both amidase and glycosidase activities, which as you say is rare among these types of proteins. I was curious about the amidase domain that you describe in this current study. You mention that it "lacks any recognizable conserved motif". Your study very clearly demonstrates that it is an amidase that cleaves bacterial peptidoglycan. I was wondering if you have conducted any analysis regarding how is this motif/domain related to other amidase domains, such as the CHAP domain. Is the N-terminal sequence of TseP similar at all to any known CHAP domains? What about the structure of the N-terminus, does that show any significant similarity to CHAP domains? This is so cool! Thank you so much for your time and for publishing these really interesting findings!