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

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

The type VII secretion system (T7SS) is a membrane-embedded protein export pathway found in mycobacteria and Gram-positive bacteria. Recently it was shown that Mycobacterium abscessus uses its ESX-4 variant of the T7SS to secrete a toxin, EatA, which targets arabinogalactan present in the mycobacterial cell envelope. Prior to its export, EatA forms a complex with a pair of small proteins from the WXG100 family, TapA1 and TapA2. Here we investigated a structural model of the EatA N-terminal domain in complex with TapA1 and TapA2 using site-directed mutagenesis and bacterial 2-hybrid assays. Our results are consistent with the three proteins forming a stacked bundle of α-helices. Structural modelling also predicted an interaction of the EatA-TapA1-TapA2 complex with EsxT-EsxU, a second pair of WXG100-family proteins that are likely required for the mechanistic operation of ESX-4. Whilst we could demonstrate a potential interaction between TapA2 and EsxT by bacterial 2-hybrid analysis, we were not able to purify a complex of all five proteins.

A cryptic aromatic binding pocket in the highly thermostable GpgS from Mycobacterium hassiacum hints at MGLP pathway crosstalk

Mycobacteria have long posed a threat to human health, being responsible for a range of hard‐to‐treat infections such as tuberculosis, leprosy, and other chronic diseases caused by nontuberculous mycobacteria. Their intrinsic resistance to antibiotics and resilience when exposed to environmental stress complicate treatment and underscore the need for discovering novel drug targets and developing new therapeutic strategies. Mycobacterium hassiacum, a thermophilic mycobacterium, is a promising source of stable proteins suitable for detailed structural analysis. In this study, we investigated its glucosyl‐3‐phosphoglycerate synthase (GpgS), an enzyme that plays a key role in the biosynthesis of methylglucose lipopolysaccharides in Mycobacterium tuberculosis and is implicated in responses to nitrogen starvation and thermal stress in different mycobacterial species. We confirmed the potential of M. hassiacum as a source of enzymes amenable to structural studies, as its GpgS ortholog yielded not only the first mycobacterial GpgS crystallographic structure at near‐atomic resolution (1.10 Å) but also multiple high‐resolution structures under different pH conditions and in complex with various substrates. These structural insights revealed a previously unrecognized aromatic binding pocket that is not only cryptic in nature but also capable of accommodating the substrates p‐aminobenzoic acid and p‐hydroxybenzaldehyde, which have been found to be relevant in its genetic context, thereby establishing a direct link between GpgS activity and broader metabolic pathways in mycobacteria. Our findings establish M. hassiacum as a valuable model for structural enzymology in mycobacteria and highlight GpgS as a potential drug target. The detailed structural data provided here offer a foundation for rational drug design, opening new avenues for the development of inhibitors targeting this key, potentially pleiotropic enzyme.

Conserved copper(I)-binding auxiliary domains link virulence-associated LPMOs to copper homeostasis in the intestinal environment

Structural and Functional Interrogation of Active Streptococcus pneumoniae Sortase A

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.

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.

Genome-Wide Identification of the Collectin Family in Silver Pomfret (Pampus argenteus) and Innate Immune Functions of a Novel Truncated Member PaCL-11b against Vibrio parahaemolyticus.

The systematic identification of the Collectin family in silver pomfret is presented and the multifunctional immune roles of PaCL-11b in defense against V. parahaemolyticus are revealed, providing a theoretical basis for the prevention and control of bacterial diseases in silver pomfret aquaculture.