Amidase and Lysozyme Dual Functions in TseP Reveal a New Family of Chimeric Effectors in the Type VI Secretion System
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eLife Assessment
This valuable study describes how a single effector of the Type Six Secretion System (T6SS) has two distinct enzymatic functions that together may contribute to bacterial survival and dynamics in a community and provide potential for developing new antimicrobial compounds. The authors have deployed a range of methods in biochemistry, microbiology, and microscopy, generating solid data that support the main assertions. While the manuscript could benefit from additional clarifying experiments and a more detailed discussion of the methods, it will appeal to those studying T6SS, particularly those interested in effectors and bacterial enzymes.
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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.
Article activity feed
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eLife Assessment
This valuable study describes how a single effector of the Type Six Secretion System (T6SS) has two distinct enzymatic functions that together may contribute to bacterial survival and dynamics in a community and provide potential for developing new antimicrobial compounds. The authors have deployed a range of methods in biochemistry, microbiology, and microscopy, generating solid data that support the main assertions. While the manuscript could benefit from additional clarifying experiments and a more detailed discussion of the methods, it will appeal to those studying T6SS, particularly those interested in effectors and bacterial enzymes.
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Reviewer #1 (Public review):
Summary:
The manuscript performs a comprehensive biochemical, structural, and bioinformatic analysis of TseP, a type 6 secretion system effector from Aeromonas dhakensis that includes the identification of a domain required for secretion and residues conferring target organism specificity. Through targeted mutations, they have expanded the target range of a T6SS effector to include a gram-positive species, which is not typically susceptible to T6SS attack.
Strengths:
All of the experiments presented in the study are well-motivated and the conclusions are generally sound.
Weaknesses:
There are some issues with the clarity of figures. For example, the microscopy figures could have been more clearly presented as cell counts/quantification rather than representative images. Similarly, loading controls for the …
Reviewer #1 (Public review):
Summary:
The manuscript performs a comprehensive biochemical, structural, and bioinformatic analysis of TseP, a type 6 secretion system effector from Aeromonas dhakensis that includes the identification of a domain required for secretion and residues conferring target organism specificity. Through targeted mutations, they have expanded the target range of a T6SS effector to include a gram-positive species, which is not typically susceptible to T6SS attack.
Strengths:
All of the experiments presented in the study are well-motivated and the conclusions are generally sound.
Weaknesses:
There are some issues with the clarity of figures. For example, the microscopy figures could have been more clearly presented as cell counts/quantification rather than representative images. Similarly, loading controls for the secreted proteins for the westerns probably should be shown.
Also, some of the minor/secondary conclusions reached regarding the "independence" of the N and C term domains of the TseP are a bit overreaching.
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Reviewer #2 (Public review):
Summary:
Wang et al. investigate the role of TseP, a Type VI secretion system (T6SS) effector molecule, revealing its dual enzymatic activities as both an amidase and a lysozyme. This discovery significantly enhances the understanding of T6SS effectors, which are known for their roles in interbacterial competition and survival in polymicrobial environments. TseP's dual function is proposed to play a crucial role in bacterial survival strategies, particularly in hostile environments where competition between bacterial species is prevalent.
Strengths:
(1) The dual enzymatic function of TseP is a significant contribution, expanding the understanding of T6SS effectors.
(2) The study provides important insights into bacterial survival strategies, particularly in interbacterial competition.
(3) The findings have …
Reviewer #2 (Public review):
Summary:
Wang et al. investigate the role of TseP, a Type VI secretion system (T6SS) effector molecule, revealing its dual enzymatic activities as both an amidase and a lysozyme. This discovery significantly enhances the understanding of T6SS effectors, which are known for their roles in interbacterial competition and survival in polymicrobial environments. TseP's dual function is proposed to play a crucial role in bacterial survival strategies, particularly in hostile environments where competition between bacterial species is prevalent.
Strengths:
(1) The dual enzymatic function of TseP is a significant contribution, expanding the understanding of T6SS effectors.
(2) The study provides important insights into bacterial survival strategies, particularly in interbacterial competition.
(3) The findings have implications for antimicrobial research and understanding bacterial interactions in complex environments.
Weaknesses:
(1) The manuscript assumes familiarity with previous work, making it difficult to follow. Mutants and strains need clearer definitions and references.
(2) Figures lack proper controls, quantification, and clarity in some areas, notably in Figures 1A and 1C.
(3) The Materials and Methods section is poorly organized, hindering reproducibility. Biophysical validation of Zn²⁺ interaction and structural integrity of proteins need to be addressed.
(4) Discrepancies in protein degradation patterns and activities across different figures raise concerns about data reliability.
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Reviewer #3 (Public review):
Summary:
Type VI secretion systems (T6SS) are employed by bacteria to inject competitor cells with numerous effector proteins. These effectors can kill injected cells via an array of enzymatic activities. A common class of T6SS effector are peptidoglycan (PG) lysing enzymes. In this manuscript, the authors characterize a PG-lysing effector-TseP-from the pathogen Aeromonas dhakensis. While the C-terminal domain of TseP was known to have lysozyme activity, the N-terminal domain was uncharacterized. Here, the authors functionally characterize TsePN as a zinc-dependent amidase. This discovery is somewhat novel because it is rare for PG-lysing effectors to have amidase and lysozyme activity.
In the second half of the manuscript, the authors utilize a crystal structure of the lysozyme TsePC domain to inform the …
Reviewer #3 (Public review):
Summary:
Type VI secretion systems (T6SS) are employed by bacteria to inject competitor cells with numerous effector proteins. These effectors can kill injected cells via an array of enzymatic activities. A common class of T6SS effector are peptidoglycan (PG) lysing enzymes. In this manuscript, the authors characterize a PG-lysing effector-TseP-from the pathogen Aeromonas dhakensis. While the C-terminal domain of TseP was known to have lysozyme activity, the N-terminal domain was uncharacterized. Here, the authors functionally characterize TsePN as a zinc-dependent amidase. This discovery is somewhat novel because it is rare for PG-lysing effectors to have amidase and lysozyme activity.
In the second half of the manuscript, the authors utilize a crystal structure of the lysozyme TsePC domain to inform the engineering of this domain to lyse gram-positive peptidoglycan.
Strengths:
The two halves of the manuscript considered together provide a nice characterization of a unique T6SS effector and reveal potentially general principles for lysozyme engineering.
Weaknesses:
The advantage of fusing amidase and lysozyme domains in a single effector is not discussed but would appear to be a pertinent question. Labeling of the figures could be improved to help readers understand the data.
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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 …
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!
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