Aug 2026· MicrobiologyOpen· Vol 15· 0 citations· 40 references
Medicine
TL;DR
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.
Abstract
ABSTRACT Infectious diseases typically involve a single pathogen harming its host. However, in clinical settings, co‐infections also occur. This study investigated the potential interaction between Cryptococcus (C.) neoformans and influenza virus by assessing whether the cryptococcal yeast kexin protease (Kex2p) could activate the uncleaved hemagglutinin glycoprotein. Protein‐protein docking was performed using HADDOCK and independently validated using AlphaFold3. 6x His‐tagged truncated KEX2 gene was expressed in E. coli BL 21 (DE3), and the recombinant protein was purified using nickel ion affinity chromatography. Successful protein production was confirmed by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis, anti‐His immunoblotting, and mass spectrometry. Recombinant Kex2p was evaluated in a biochemical peptide cleavage assay using a fluorogenic 12‐mer peptide mimicking the hemagglutinin HA1/HA2 site as substrate, with furin serving as the reference protease. HADDOCK predicted favorable binding of Kex2p to hemagglutinin (HADDOCK score = −119.2 ± 8.7; RMSD = 1.1 ± 0.2 Å), comparable to the furin‐hemagglutinin complex (HADDOCK score = −115.9 ± 5.7; RMSD = 2.1 ± 1.3 Å). AlphaFold 3 independently reproduced the HADDOCK‐predicted binding orientations, yielding moderate‐confidence complexes for hemagglutinin‐furin (ipTM = 0.50, pTM = 0.56; RMSD = 3.634 Å) and hemagglutinin‐Kex2p (ipTM = 0.41, pTM = 0.58; RMSD = 3.243 Å). In enzymatic assays, recombinant Kex2p exhibited a significantly higher Vmax than furin (p = 0.0411), indicating greater catalytic activity toward the hemagglutinin cleavage peptide. These 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 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.
A. Rodríguez-Banqueri, T. Goulas, Marina Girbal-González et al.· Journal of Molecular Biology· 0 citations
Bacillus thuringiensis (Bt) is well-known for its bio-larvicidal properties. Cytolytic (Cyt) protein is one of Bt larvicidal proteins. This protein requires proteolytic activation to remove partial N- and C-terminal regions. In this study, to improve proteolytic cleavage specificity, proteinase K cleavage sites at both end termini of Cyt2Aa2 protein were substituted by a trypsin cleavage sites L33R, S37K, S228R, and F237K. Afterward, the engineered Cyt2Aa2 proteins were heterologous expressed in Escherichia coli. Most of the mutants were produced as inclusion proteins similar to the wild type but their capability of solubilization and trypsin activation was significantly reduced, particularly for L33R, S37K, and F237K. The S228R mutant could be partially solubilized and activated. Moreover, the new trypsin cleavage site at N-terminus of L33R resulted in an aberrant toxic Cyt2Aa2 protein against E. coli. The L33R mutant limited E. coli growth during protein synthesis. Remarkably, although the capability of solubilization of the mutant proteins was reduced, their mosquito larvicidal activity (except L33R and L33R/F237K) was comparable to the wild type. These findings demonstrate that although the amino acid residues at N- and C-terminal regions of Cyt2Aa2 are eliminated from active protein, they are necessary for protein production and for preventing toxicity against E. coli during heterologous expression.
Chontida Tangsongcharoen, B. Promdonkoy, C. Krittanai et al.· Current Applied Science and...· 0 citations
This study depicted and characterized the phage-derived endolysin LysPEF52H, providing a new alternative antimicrobial strategy for controlling bacterial contamination and ensuring food safety.
The molecular weight of endolysin LysPEF52H was predicted using an online platform, and its three-dimensional (3D) structure was modeled and validated through a Ramachandran plot. Subsequently, LysPEF52H was expressed via a prokaryotic expression system, followed by purification and concentration determination. Its secondary structure and protein conformation were analyzed using circular dichroism spectroscopy and fluorescence spectroscopy. Moreover, molecular docking was used to preliminarily investigate the interactions between this endolysin and small molecules/metal ions. Finally, the physicochemical properties and antibacterial activity of LysPEF52H were examined, and its application for bacterial inactivation on chicken samples was evaluated.
The predicted molecular weight of endolysin LysPEF52H was 46808.1 Da, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirmed an apparent molecular weight of approximately 45 kDa. The target protein was successfully eluted using elution buffer containing 200 mmol/L imidazole, and the original concentration was quantified as 0.107 mg/mL. The secondary structure of LysPEF52H predominantly consisted of β-sheets (46%) and random coils (48%), with a minor contribution from α-helixes (6%). Conformational changes in LysPEF52H occurred between 25 °C and 45 °C, accompanied by an increase in fluorescence intensity. Above 45 ℃, the protein gradually denatured, leading to a decrease in fluorescence intensity. LysPEF52H exhibited metal ion affinity. Additionally, ʟ-alanine exhibited the strongest docking affinity to LysPEF52H. Antimicrobial spectrum assays and scanning electron microscopy demonstrated that LysPEF52H exhibited antimicrobial activity against Escherichia coli 105H and Staphylococcus aureus 8M. LysPEF52H also exhibited wide pH tolerance and good antibacterial activity after heat treatment. Its antimicrobial activity was influenced by salt concentration, metal ions, and surfactants. In addition, measurable antibacterial effects were observed on chicken artificially contaminated with E. coli 105H and S. aureus 8M treated with LysPEF52H at 4, 25, and 37 ℃.
The phage-derived endolysin LysPEF52H shows potential as an antimicrobial agent for future applications.
Jinling Wang, Yangyang Yang, Haijun Xu et al.· Food Quality and Safety· 0 citations
Deletion of fabR reduced host cell adherence, increased serum sensitivity, enhanced IgG deposition, and decreased vitronectin binding compared with the wild-type strain, and ΔfabR mutants from multiple NTHi strains showed increased metabolic activity, indicating a conserved role for FabR in metabolic regulation.
Martina Janoušková, Yu-Ching Su, Sandra Jonsson et al.· Journal of Infectious Diseas...· 0 citations
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.
Eunice K. E. Lee, Kieran Bowran, Eleanor R. Boardman et al.· bioRxiv· 0 citations
Newcastle disease virus (NDV), a significant avian paramyxovirus, depends on the acquisition of host-derived membranes for viral envelope assembly during budding. However, the relationship between NDV budding efficiency and host metabolic reprogramming remains incompletely understood. Our previous research demonstrated that the highly virulent strain Herts/33 and the non-virulent strain LaSota differ significantly in budding efficiency, which is attributed to the difference in ubiquitination levels at the K247 site of the M protein. In this study, using recombinant viruses generated in our previous study, we investigated the effects of the K247 ubiquitination site on host cell metabolism. Through RNA sequencing and LC–MS/MS, systematically profiled the transcriptional and metabolic alterations induced by these viruses in HeLa cells. We also compared organ pathology in three-week-old SPF chicks infected with Herts/33 versus LaSota. The results showed that NDV infection broadly reprograms host sphingolipid metabolism. Compared with rLaSota-WT, rLaSota-R247K exhibited higher budding efficiency, linked to accelerated ceramide depletion and a stronger innate immune response. Specifically, rLaSota-R247K infection upregulated interferon- and interleukin-related components such as ISG15, CXCL8, TNF-α, CXCL10, NOD2, CD274, OAS, and IFNB1. Furthermore, we confirmed that blocking the ceramide synthesis pathway significantly suppresses NDV-M protein-mediated budding of VLPs and virions. Pathologically, Herts/33 induced more severe tissue damage than LaSota. Together, these findings indicate that the K247 residue of the NDV M protein enhances viral budding and promotes rapid utilization of host sphingolipids. This work provides mechanistic insight into NDV budding and highlights virus–host metabolic interactions that may inform future antiviral strategies.
Jun Dai, Fan-Xin Liu, Yiyi Feng et al.· Poultry Science· 0 citations