{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:52:29Z","timestamp":1760147549497,"version":"build-2065373602"},"reference-count":24,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2023,2,13]],"date-time":"2023-02-13T00:00:00Z","timestamp":1676246400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003977","name":"Israel\u2013China Science Foundation","doi-asserted-by":"publisher","award":["2444\/18","2017293"],"award-info":[{"award-number":["2444\/18","2017293"]}],"id":[{"id":"10.13039\/501100003977","id-type":"DOI","asserted-by":"publisher"}]},{"name":"USA\u2013Israel Binational Science Foundation","award":["2444\/18","2017293"],"award-info":[{"award-number":["2444\/18","2017293"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The opportunistic fungus Aspergillus fumigatus is the primary invasive mold pathogen in humans, and is responsible for an estimated 200,000 yearly deaths worldwide. Most fatalities occur in immunocompromised patients who lack the cellular and humoral defenses necessary to halt the pathogen\u2019s advance, primarily in the lungs. One of the cellular responses used by macrophages to counteract fungal infection is the accumulation of high phagolysosomal Cu levels to destroy ingested pathogens. A. fumigatus responds by activating high expression levels of crpA, which encodes a Cu+ P-type ATPase that actively transports excess Cu from the cytoplasm to the extracellular environment. In this study, we used a bioinformatics approach to identify two fungal-unique regions in CrpA that we studied by deletion\/replacement, subcellular localization, Cu sensitivity in vitro, killing by mouse alveolar macrophages, and virulence in a mouse model of invasive pulmonary aspergillosis. Deletion of CrpA fungal-unique amino acids 1\u2013211 containing two N-terminal Cu-binding sites, moderately increased Cu-sensitivity but did not affect expression or localization to the endoplasmic reticulum (ER) and cell surface. Replacement of CrpA fungal-unique amino acids 542\u2013556 consisting of an intracellular loop between the second and third transmembrane helices resulted in ER retention of the protein and strongly increased Cu-sensitivity. Deleting CrpA N-terminal amino acids 1\u2013211 or replacing amino acids 542\u2013556 also increased sensitivity to killing by mouse alveolar macrophages. Surprisingly, the two mutations did not affect virulence in a mouse model of infection, suggesting that even weak Cu-efflux activity by mutated CrpA preserves fungal virulence.<\/jats:p>","DOI":"10.3390\/ijms24043705","type":"journal-article","created":{"date-parts":[[2023,2,13]],"date-time":"2023-02-13T03:28:46Z","timestamp":1676258926000},"page":"3705","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Identification and Analysis of Fungal-Specific Regions in the Aspergillus fumigatus Cu Exporter CrpA That Are Essential for Cu Resistance but Not for Virulence"],"prefix":"10.3390","volume":"24","author":[{"given":"Hila","family":"Werner","sequence":"first","affiliation":[{"name":"Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ammar","family":"Abou Kandil","sequence":"additional","affiliation":[{"name":"Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zohar","family":"Meir","sequence":"additional","affiliation":[{"name":"Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5170-7215","authenticated-orcid":false,"given":"Yehonathan","family":"Malis","sequence":"additional","affiliation":[{"name":"Department of Pathology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yona","family":"Shadkchan","sequence":"additional","affiliation":[{"name":"Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gal","family":"Masrati","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nir","family":"Ben-Tal","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Koret","family":"Hirschberg","sequence":"additional","affiliation":[{"name":"Department of Pathology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nir","family":"Osherov","sequence":"additional","affiliation":[{"name":"Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,2,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1146\/annurev-micro-030117-020444","article-title":"Copper Acquisition and Utilization in Fungi","volume":"71","author":"Smith","year":"2017","journal-title":"Annu. Rev. Microbiol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"340","DOI":"10.1016\/j.fct.2017.10.042","article-title":"Protective role of quercetin against copper(II)-induced oxidative stress: A spectroscopic, theoretical and DNA damage study","volume":"110","author":"Jomova","year":"2017","journal-title":"Food Chem. Toxicol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"929","DOI":"10.1074\/jbc.M607646200","article-title":"Micromolar Intracellular Hydrogen Peroxide Disrupts Metabolism by Damaging Iron-Sulfur Enzymes","volume":"282","author":"Jang","year":"2007","journal-title":"J. Biol. Chem."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"28121","DOI":"10.1074\/jbc.R114.588814","article-title":"The Mismetallation of Enzymes during Oxidative Stress","volume":"289","author":"Imlay","year":"2014","journal-title":"J. Biol. Chem."},{"doi-asserted-by":"crossref","unstructured":"Raffa, N., Osherov, N., and Keller, N.P. (2019). Copper Utilization, Regulation, and Acquisition by Aspergillus fumigatus. Int. J. Mol. Sci., 20.","key":"ref_5","DOI":"10.3390\/ijms20081980"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"18945","DOI":"10.1074\/jbc.R115.649129","article-title":"Copper at the Fungal Pathogen-Host Axis","volume":"290","author":"Thiele","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"13549","DOI":"10.1074\/jbc.R111.316406","article-title":"Copper Homeostasis at the Host-Pathogen Interface","volume":"287","author":"Hodgkinson","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"33949","DOI":"10.1074\/jbc.M109.070201","article-title":"A Role for the ATP7A Copper-transporting ATPase in Macrophage Bactericidal Activity","volume":"284","author":"White","year":"2009","journal-title":"J. Biol. Chem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2174","DOI":"10.1016\/j.celrep.2017.05.075","article-title":"Aspergillus fumigatus Copper Export Machinery and Reactive Oxygen Intermediate Defense Counter Host Copper-Mediated Oxidative Antimicrobial Offense","volume":"19","author":"Wiemann","year":"2017","journal-title":"Cell Rep."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e00351-17","DOI":"10.1128\/IAI.00351-17","article-title":"Host and Pathogen Copper-Transporting P-Type ATPases Function Antagonistically during Salmonella Infection","volume":"85","author":"Ladomersky","year":"2017","journal-title":"Infect. Immun."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e12864","DOI":"10.1111\/cmi.12864","article-title":"The Aspergillus fumigatus transcription factor AceA is involved not only in Cu but also in Zn detoxification through regulating transporters CrpA and ZrcA","volume":"20","author":"Cai","year":"2018","journal-title":"Cell. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"737","DOI":"10.1002\/iub.1437","article-title":"Copper-transporting ATPases: The evolutionarily conserved machineries for balancing copper in living systems","volume":"67","author":"Migocka","year":"2015","journal-title":"IUBMB Life"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2186","DOI":"10.1080\/21505594.2021.1958057","article-title":"Transcriptional response of Aspergillus fumigatus to copper and the role of the Cu chaperones","volume":"12","author":"Anabosi","year":"2021","journal-title":"Virulence"},{"doi-asserted-by":"crossref","unstructured":"Antsotegi-Uskola, M., Markina-I\u00f1arrairaegui, A., and Ugalde, U. (2020). Copper Homeostasis in Aspergillus nidulans Involves Coordinated Transporter Function, Expression and Cellular Dynamics. Front. Microbiol., 11.","key":"ref_14","DOI":"10.3389\/fmicb.2020.555306"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1108","DOI":"10.1080\/21505594.2020.1809324","article-title":"Cu transporter protein CrpF protects against Cu-induced toxicity in Fusarium oxysporum","volume":"11","author":"Guarro","year":"2020","journal-title":"Virulence"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"3520","DOI":"10.1073\/pnas.97.7.3520","article-title":"The high copper tolerance of Candida albicans is mediated by a P-type ATPase","volume":"97","author":"Weissman","year":"2000","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1128\/EC.5.1.207-211.2006","article-title":"The akuB(KU80) mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus","volume":"5","author":"Kress","year":"2006","journal-title":"Eukaryot Cell"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"W344","DOI":"10.1093\/nar\/gkw408","article-title":"ConSurf 2016: An improved methodology to estimate and visualize evolutionary conservation in macromolecules","volume":"44","author":"Ashkenazy","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"755","DOI":"10.1093\/bioinformatics\/14.9.755","article-title":"Profile hidden Markov models","volume":"14","author":"Eddy","year":"1998","journal-title":"Bioinformatics"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"14.1.1","DOI":"10.1002\/0471142735.im1401s83","article-title":"The isolation and characterization of murine macrophages","volume":"83","author":"Zhang","year":"2008","journal-title":"Curr. Protoc. Immunol."},{"key":"ref_21","first-page":"109","article-title":"Animal Models of Aspergillosis","volume":"68","author":"Desoubeaux","year":"2018","journal-title":"Comp. Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1357","DOI":"10.1016\/j.addr.2012.09.039","article-title":"Fusion protein linkers: Property, design and functionality","volume":"65","author":"Chen","year":"2012","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"21459","DOI":"10.1074\/jbc.M301652200","article-title":"Binding of peptides with basic and aromatic residues to bilayer membranes: Phenylalanine in the myristoylated alanine-rich C kinase substrate effector domain penetrates into the hydrophobic core of the bilayer","volume":"278","author":"Zhang","year":"2003","journal-title":"J. Biol. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1093\/jac\/48.1.75","article-title":"Resistance to itraconazole in Aspergillus nidulans and Aspergillus fumigatus is conferred by extra copies of the A. nidulans P-450 14alpha-demethylase gene, pdmA","volume":"48","author":"Osherov","year":"2001","journal-title":"J. Antimicrob. Chemother."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/4\/3705\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T18:32:49Z","timestamp":1760121169000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/4\/3705"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,2,13]]},"references-count":24,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2023,2]]}},"alternative-id":["ijms24043705"],"URL":"https:\/\/doi.org\/10.3390\/ijms24043705","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2023,2,13]]}}}