Biology subjects

Pei, T.-T.

Publications and source records attributed to Pei, T.-T..

5 recordsLinked to original sources

bioRxiv2024

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

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-domains 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 TsePC4+ 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 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.

microbiology↗

bioRxiv2023

LLPS condensates of Fha initiate the inside-out assembly of the type VI secretion system

The type VI secretion system (T6SS) is one of the most powerful nanomachines employed by Gram-negative pathogens for penetrating diverse cell envelopes, including bacteria and fungi, to deliver potent effectors into target cells. While the membrane-anchored contractile tubular structure of the T6SS is well characterized, the assembly process remains poorly understood. The prevailing model suggests that the assembly of T6SS initiates from its outer-membrane component. Here, we report a distinct model that the cytoplasmic protein Fha initiates T6SS assembly in Acidovorax citrulli, an important plant pathogen. Fha dictates the formation of the inner-membrane complex and the baseplate, and directly interacts with these key components. Importantly, imaging and biochemical assays reveal that Fha undergoes liquid-liquid phase separation (LLPS), forming condensates that selectively recruit essential T6SS proteins, which are otherwise dispersed in cells. Fha also exhibited conserved functions in human pathogens Vibrio cholerae and Pseudomonas aeruginosa. These findings unveil an inside-first LLPS-driven model for T6SS assembly and suggest LLPS might be broadly involved in mediating the assembly of bacterial macromolecular complexes and facilitating interspecies interactions and pathogenesis. Significance statementThe T6SS plays a pivotal role in interspecies competition and host-microbe interactions by delivering toxins to various prokaryotes and eukaryotes. Its crucial function relies on a membrane-anchored macromolecular structure comprising at least 13 conserved components. However, the mechanisms governing the efficient assembly of its diverse cytosolic and membrane-bound components remain elusive. Here, we identify Fha, a conserved cytosolic protein, as a key initiator of T6SS assembly. Fha recruits multiple structural and effector components, forming LLPS condensates. Fha homologs of plant and human pathogens exhibit conserved functions. Our findings not only unveil an inside-first assembly model for the T6SS, initiating from inner-membrane and baseplate components, but also suggest LLPS may have a broader impact on bacterial physiology beyond intracellular activities.

microbiology↗

bioRxiv2022

Systematic mutagenesis reveals critical effector functions in the assembly and dueling of the H1-T6SS in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important human pathogen that can cause severe wound and lung infections. It employs the type VI secretion system (H1-T6SS) as a molecular weapon to carry out a unique dueling response to deliver toxic effectors to neighboring sister cells or other microbes after sensing an external attack. However, the underlying mechanism for such dueling is not fully understood. Here, we examined the role of all H1-T6SS effectors and VgrG proteins in assembly and signal sensing by ectopic expression, combinatorial deletion and point mutations, and imaging analyses. Expression of effectors targeting the cell wall and membrane resulted in increased H1-T6SS assembly. Deletion of individual effector and vgrG genes had minor- to-moderate effects on H1-T6SS assembly and dueling activities. The dueling response was detectable in the P. aeruginosa mutant lacking all H1-T6SS effector activities. In addition, double deletions of vgrG1a with either vgrG1b or vgrG1c and double deletions of effector genes tse5 and tse6 severely reduced T6SS assembly and dueling activities, suggesting their critical role in T6SS assembly. Collectively, these data highlight the diverse roles of effectors in not only dictating antibacterial functions but also their differential contributions to the assembly of the complex H1-T6SS apparatus.

microbiology↗

bioRxiv2022

A dueling-competent signal-sensing module guides precise delivery of cargo proteins into target cells by engineered Pseudomonas aeruginosa

To recognize and manipulate a specific microbe of a crowded community is a highly challenging task in synthetic biology. Here, we introduce a highly-selective protein delivery platform, termed DUEC, which responds to direct contact of attacking cells by engineering the tit-for-tat/dueling response of H1-T6SS (type VI secretion system) in Pseudomonas aeruginosa. Using a Cre-recombinase-dependent reporter, we screened H1-T6SS secreted substrates and developed Tse6N as the most effective secretion tag for Cre delivery. DUEC cells can discriminately deliver the Tse6N-Cre cargo into the cytosol of T6SS+ but not T6SS- Vibrio cholerae cells in a mixed population. These data demonstrate that the DUEC cell is not only a prototypical physical-contact sensor and delivery platform but also may be coupled with recombination-based circuits with the potential for complex tasks in mixed microbial communities.

microbiology↗

bioRxiv2021

Breaching the cell-envelope barriers of gram-positive and fungal microbes by a type VI secretion system in Acidovorax citrulli

The type VI secretion system (T6SS) is a double-tubular toxin-injection nanomachine widely found in gram-negative human and plant pathogens. The current model depicts that the T6SS spear-like Hcp tube is powered by the contraction of an outer sheath to drill through the envelope of a neighboring cell, achieving cytosol to cytosol delivery. However, gram-positive bacteria seem to be impenetrable to such T6SS action. Here we report that a plant pathogen Acidovorax citrulli (AC) deploys a highly potent T6SS to kill a range of bacteria including Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Mycobacterium smegmatis as well as fungal species including Candida albicans and Pichia pastoris. Using bioinformatic and biochemical assays, we identified a group of T6SS effectors and characterized one effector RhsB that is critical for interspecies interaction. We report that RhsB contains a conserved YD-repeat domain and a C-terminal nuclease domain. Toxicity of RhsB was neutralized by its downstream immunity proteins through direct interaction. RhsB was cleaved at the C-terminal end and a catalytic mutation within the internal aspartic protease abolished such cleavage. Collectively, the T6SS of AC displays potent activities to penetrate the cell envelope barriers of gram-positive and fungal species, highlighting the greatly expanded capabilities of T6SS in modulating microbiome compositions in complex environments.

microbiology↗