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However, recent studies with deletion mutants have reported contrasting results concerning its actual importance for wall integrity. Here, we present functional characterization of the two members of the Pir family (Pir1 and Pir32) as well as protein structure modeling and mutagenesis studies to elucidate how Pir1, the most important family member, is incorporated into the cell wall. Our data show that Pir1 indeed is involved in \u03b2-1,3-glucan binding but its gene deletion did not affect cellular fitness. 3D structure modeling predicts that Pir1 has a core predominantly comprised of antiparallel \u03b2-sheets, surrounded by a large loop containing a variable number of canonical Pir repeat units. Mutagenesis studies indicate that two repeat units are required and sufficient for Pir1 surface localization, wall incorporation, and Pir1-mediated glucan binding. Altogether, our work provides novel mechanistic insights into Pir1 wall incorporation and functioning, and supports its proposed role as cell wall glucan crosslinker. At the same time, C. albicans also may have acquired alternative means to ascertain cell wall robustness.<\/jats:p>","DOI":"10.1093\/femsyr\/foaf042","type":"journal-article","created":{"date-parts":[[2025,8,6]],"date-time":"2025-08-06T11:38:57Z","timestamp":1754480337000},"source":"Crossref","is-referenced-by-count":2,"title":["Wall incorporation of the \u03b2-1,3-glucan cross-linking protein Pir1 in the human pathogen <i>Candida albicans<\/i> is facilitated by the presence of two or more Pir repeat units"],"prefix":"10.1093","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6879-1609","authenticated-orcid":false,"given":"Mar\u00eda","family":"Alvarado","sequence":"first","affiliation":[{"name":"Institute for Biomedicine, ETSIAMB, University of Castilla-La Mancha , 02008 Albacete ,","place":["Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana E","family":"Moreno-Mart\u00ednez","sequence":"additional","affiliation":[{"name":"Institute for 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,","place":["Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6669-0161","authenticated-orcid":false,"given":"Ver\u00f3nica","family":"Mix\u00e3o","sequence":"additional","affiliation":[{"name":"Barcelona Supercomputing Centre (BSC-CNS) , 08034 Barcelona ,","place":["Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0019-1735","authenticated-orcid":false,"given":"Toni","family":"Gabald\u00f3n","sequence":"additional","affiliation":[{"name":"Barcelona Supercomputing Centre (BSC-CNS) , 08034 Barcelona ,","place":["Spain"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Estibaliz","family":"Mateo","sequence":"additional","affiliation":[{"name":"University of the Basque Country (EHU) Department of Immunology, Microbiology and Parasitology, Faculty of Medicine and Nursing, , 48940 Bilbao 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