{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T08:21:04Z","timestamp":1787732464611,"version":"build-2784847793"},"reference-count":40,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T00:00:00Z","timestamp":1787529600000},"content-version":"vor","delay-in-days":23,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,8,1]],"date-time":"2026-08-01T00:00:00Z","timestamp":1785542400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["MicrobiologyOpen"],"published-print":{"date-parts":[[2026,8]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    Infectious diseases typically involve a single pathogen harming its host. However, in clinical settings, co\u2010infections also occur. This study investigated the potential interaction between\n                    <jats:italic>Cryptococcus<\/jats:italic>\n                    (\n                    <jats:italic>C<\/jats:italic>\n                    .)\n                    <jats:italic>neoformans<\/jats:italic>\n                    and influenza virus by assessing whether the cryptococcal yeast kexin protease (Kex2p) could activate the uncleaved hemagglutinin glycoprotein. Protein\u2010protein docking was performed using HADDOCK and independently validated using AlphaFold3. 6x His\u2010tagged truncated\n                    <jats:italic>KEX2<\/jats:italic>\n                    gene was expressed in\n                    <jats:italic>E. coli<\/jats:italic>\n                    BL 21 (DE3), and the recombinant protein was purified using nickel ion affinity chromatography. Successful protein production was confirmed by sodium dodecyl sulfate (SDS)\u2010polyacrylamide gel electrophoresis, anti\u2010His immunoblotting, and mass spectrometry. Recombinant Kex2p was evaluated in a biochemical peptide cleavage assay using a fluorogenic 12\u2010mer 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\u2009=\u2009\u2212119.2\u2009\u00b1\u20098.7; RMSD\u2009=\u20091.1\u2009\u00b1\u20090.2\u2009\u00c5), comparable to the furin\u2010hemagglutinin complex (HADDOCK score\u2009=\u2009\u2212115.9\u2009\u00b1\u20095.7; RMSD\u2009=\u20092.1\u2009\u00b1\u20091.3\u2009\u00c5). AlphaFold 3 independently reproduced the HADDOCK\u2010predicted binding orientations, yielding moderate\u2010confidence complexes for hemagglutinin\u2010furin (ipTM\u2009=\u20090.50, pTM\u2009=\u20090.56; RMSD\u2009=\u20093.634\u2009\u00c5) and hemagglutinin\u2010Kex2p (ipTM\u2009=\u20090.41, pTM\u2009=\u20090.58; RMSD\u2009=\u20093.243\u2009\u00c5). In enzymatic assays, recombinant Kex2p exhibited a significantly higher Vmax than furin (\n                    <jats:italic>p<\/jats:italic>\n                    \u2009=\u20090.0411), indicating greater catalytic activity toward the hemagglutinin cleavage peptide. These findings provide the first biochemical and complementary computational evidence that\n                    <jats:italic>C. neoformans<\/jats:italic>\n                    Kex2p may recognize influenza hemagglutinin, supporting its potential role in hemagglutinin activation during cryptococcal\u2010influenza co\u2010infection.\n                  <\/jats:p>","DOI":"10.1002\/mbo3.70384","type":"journal-article","created":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T06:22:17Z","timestamp":1787638937000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["The Involvement of the Cryptococcal Yeast Kexin Protease in the Proteolytic Activation of the Uncleaved Influenza Virus Hemagglutinin Glycoprotein"],"prefix":"10.1002","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1422-5206","authenticated-orcid":false,"given":"Nolwazi F.","family":"Ntshangase","sequence":"first","affiliation":[{"name":"University of the Free State Bloemfontein South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Khwezi","family":"Mdana","sequence":"additional","affiliation":[{"name":"University of the Free State Bloemfontein South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2915-4543","authenticated-orcid":false,"given":"Nozethu","family":"Mjokane","sequence":"additional","affiliation":[{"name":"University of the Free State Bloemfontein South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Winschau F. van","family":"Zyl","sequence":"additional","affiliation":[{"name":"University of the Free State Bloemfontein South 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