Aug 2026· mLife· Vol 5, pp. 417 - 431· 0 citations· 58 references
Medicine
TL;DR
Genetic analysis and structural modeling indicate that TseMt is delivered through a dedicated PAAR−VgrG−chaperone pathway, defining the structural basis, functional mechanism, and delivery pathway of the H4‐T6SS effector TseMt from a clinical P. aeruginosa isolate.
Abstract
Abstract Pseudomonas aeruginosa is a major nosocomial pathogen in which the type VI secretion system (T6SS) contributes to interbacterial competition and virulence. While most strains encode three T6SSs, additional T6SS clusters have been identified in clinical isolates through comparative genomics, but their functions and effector biology remain undefined. Here, we identify TseMt as a major antibacterial effector associated with an H4‐T6SS in a clinical P. aeruginosa isolate LYSZa7. TseMt is a periplasmically active toxin whose activity is neutralized by a cognate immunity protein, TsiMt. Biochemical assays show that TseMt binds membranes and forms ion‐conducting pores, establishing it as a pore‐forming effector. A 3.0 Å cryo‐electron microscopy structure reveals a distinct three‐domain architecture comprising an N‐terminal MIX‐like domain, a central α‐helical scaffold, and a C‐terminal toxin domain. Genetic analysis and structural modeling indicate that TseMt is delivered through a dedicated PAAR−VgrG−chaperone pathway. Together, these findings define the structural basis, functional mechanism, and delivery pathway of the H4‐T6SS effector TseMt from a clinical P. aeruginosa isolate and reveal its role in mediating bacterial competition.
TpeX is identified as a bactericidal, colicin-like pore-forming T6SS effector whose membrane activity is controlled by a cognate immunity protein, thereby expanding the repertoire of membrane-targeting weapons used by P. aeruginosa in interbacterial competition.
Chantal Soscia, Sébastien Reig, D. Lefebvre et al.· bioRxiv· 0 citations
The type VI secretion system (T6SS) is a contractile nanomachine used by Gram-negative bacteria to deliver effector proteins into target cells, contributing to both interbacterial competition and pathogenesis. Although T6SS gene clusters are present in recently isolated commensal and pathogenic Escherichia coli strains, they are absent from classical laboratory strains that have been propagated for decades in pure cultures, suggesting that T6SS can be lost in the absence of competition. Here, we combined experimental evolution with whole-genome sequencing to track the fate of the enteroaggregative Escherichia coli (EAEC) Sci1 T6SS during competition with either T6SS-susceptible or T6SS-immune bacteria. After ∼640 generations, T6SS activity was largely maintained during competition with T6SS-susceptible bacteria, whereas ∼90% of clones evolved with T6SS-immune bacteria lost or attenuated T6SS activity through diverse mutations within the sci1 promoter, essential T6SS structural genes, or the rfaH transcriptional antiterminator. We identified two RfaH-binding ops elements within the sci1 cluster, revealing antitermination as a regulatory element of EAEC T6SS transcription, which is conserved among Enterobacteriaceae. Our findings highlight how experimental evolution can reveal the selective forces shaping T6SS maintenance and identify new regulatory components controlling its activity.
Boris Taillefer, Jonas B. Desjardins, Eric Cascales· Current Biology· 0 citations
Key determinants of secretion specificity and endopilus stability are identified, revealing how minor sequence variations in conserved nanomachines drive functional adaptation to diverse environments.
A structural model of the EatA N-terminal domain in complex with TapA1 and TapA2 is investigated using site-directed mutagenesis and bacterial 2-hybrid assays, and results are consistent with the three proteins forming a stacked bundle of α-helices.
Eunice K. E. Lee, Kieran Bowran, Eleanor R. Boardman et al.· bioRxiv· 0 citations
Pseudomonas aeruginosa is a nosocomial pathogenic bacterium that causes a wide range of human infectious diseases. The type III secretion system (T3SS) serves as a key virulence determinant underlying the pathogenesis of this bacterium in acute infections. As the master transcriptional activator of T3SS, ExsA binds to target promoter regions and modulates the expression of all currently identified T3SS genes. In this study, we identified tyrosine phosphoprotein A (TypA) as a repressor that restricts expression of the T3SS in P. aeruginosa. TypA interacts with ExsA to block its binding to target promoters, thereby inhibiting T3SS expression. We show that the typA expression is induced in response to low calcium, low temperature, a biofilm lifestyle, and direct contact with host cells. Additionally, the absence of TypA caused a growth defect in P. aeruginosa at low temperatures. Collectively, these data confirm the significant role of TypA and reveal a novel molecular mechanism by which P. aeruginosa regulates T3SS.
Li-Wen Yin, Yi-Ming Li, Xue-Tao Gong et al.· Microorganisms· 0 citations
Sortase A (SrtA) enzymes covalently anchor surface proteins to Gram-positive bacterial cell walls, promoting colonization and virulence. In Streptococcus pneumoniae, previous studies identified both a domain-swapped dimer and an active refolded monomer, but the active enzyme has not been characterized at the structural and residue-specific level. Here, we performed quantitative proteomic comparisons of wild-type and SrtA knockout strains that confirmed the loss of multiple LPxTG-containing virulence factors, including ZmpB, NanA, and IgA1 protease, consistent with an essential role for SrtA in surface protein anchoring. To enable mechanistic studies, we established a biochemical framework to produce monomeric Streptococcus pneumoniae SrtA by refolding and developed a gel-based assay using recombinant substrates to monitor catalytic activity. The refolded monomer, but not the swapped dimer, catalyzed cleavage and transpeptidation of a canonical LPxTG substrate in a metal-independent manner under the conditions examined. We further report high-resolution NMR backbone assignments for the active monomer and identify substrate-induced chemical shift perturbations that localize to the active site. Together, these findings provide an integrated proteomic, biochemical, and NMR characterization of monomeric, catalytically active Streptococcus pneumoniae SrtA and reveal residue-specific interactions with a canonical LPNTG recognition peptide.
Eunjeong Lee, Blaine H. Gordon, J. Redzic et al.· Biomolecules· 0 citations
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