Characterizing the interaction of a type VII-secreted antimycobacterial toxin with its small helical partner proteins

Helicolysins emerge as a new family of metzincin metallopeptidases in host-pathogen interactions.

The Hom family and canilysin are defined as helicolysins, a previously uncharacterized metzincin subfamily distinguished by a conserved Thr-turn and an accessory ND, and implicates these proteins in host-pathogen interactions, adhesion, and immunomodulation.

Functional characterization of a pore‐forming effector TseMt from the H4 type VI secretion system of Pseudomonas aeruginosa

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.

Discovery of a regulatory node that coordinates cell envelope assembly in mycobacteria

Mycobacteria possess a complex double-membrane cell envelope critical for survival and pathogenesis. Proper assembly of this architecture requires the biosynthesis and transport of major components, including arabinogalactan (AG) polysaccharides and mycolic acids (MAs), but how these processes are effectively coordinated is unknown. Here, we discover an essential membrane complex that serves as a regulatory node in mycobacterial envelope biogenesis. The acyltransferase TmaT and the arabinofuranosyltransferase AftD physically interact; cryo-EM structures reveal a 1:1 stoichiometry, and present a novel fold for TmaT, featuring a central channel that binds co-factor for acetylation in the periplasm. We establish that the TmaT-AftD interaction, and the catalytic activities of both enzymes, are required for MA transport across the cell envelope, as well as AG ligation to the cell wall, the final stage of AG biosynthesis. The TmaT-AftD complex coordinates the two major envelope assembly pathways, presenting a structural vulnerability for future anti-mycobacterial drug development.

Mutation-induced heterogeneity of the β7-β8 loop of the Staphylococcus aureus class A sortase leading to enhanced catalytic efficiency characterized by NMR and enzyme kinetics

Bacterial sortase enzymes are cysteine transpeptidases at the surface of Gram-positive bacteria that ligate substrates to the cell wall. In addition, these enzymes are powerful tools in protein engineering applications via sortase-mediated ligation (SML) due to their covalent attachment of two substrates, with one containing a pentapeptide recognition motif with sequence LPXTG, where X=any amino acid, and the second, an N-terminal glycine. The class A sortase from Staphylococcus aureus (saSrtA) was the first to be identified, and over 25 years later, the most widely used SML variants continue to be derivatives of a directed-evolution-identified pentamutant of saSrtA, or saSrtA5M. We previously characterized P94, a position mutated in saSrtA5M that interacts directly with a structurally conserved loop (the β7-β8 loop) near the active site of wild-type saSrtA only in the inactive conformation. This work revealed that the single P94X mutation dramatically affects relative saSrtA activity, as well as specificity for the P2 (or X) position in the LPXTG recognition motif. This is largely driven by Km effects. Here, we further interrogated P94 by probing structural changes in the active, apo state of saSrtA in the presence of the P94D mutation, as well as via mutations in Y187, the β7-β8 loop residue hypothesized to interact directly with P94. The saSrtA enzyme is allosterically activated by calcium; therefore, we were interested if P94D would induce structural changes in the calcium-bound apo enzyme. We used 1H-15N NMR experiments to compare spectra between enzymatically inactive variants of saSrtA with and without the P94D mutation. We also used NMR to calculate relative binding affinities for a pentapeptide substrate to these variants, as well as enzymatically inactive saSrtA5M. Our NMR data, in combination with enzymatic assays using active variants confirmed differences in the active, apo states of these enzymes. Overall, this work provides additional atomic detail regarding the importance of the P94 residue in saSrtA substrate recognition.

The Involvement of the Cryptococcal Yeast Kexin Protease in the Proteolytic Activation of the Uncleaved Influenza Virus Hemagglutinin Glycoprotein

Findings provide the first biochemical and complementary computational evidence that C. neoformans Kex2p may recognize influenza hemagglutinin, supporting its potential role in hemagglutinin activation during cryptococcal‐influenza co‐infection.

The Type VI secretion effector TpeX expands the pore-forming virulence arsenal of Pseudomonas aeruginosa