Identification and Analysis of Fungal-Specific Regions in the Aspergillus fumigatus Cu Exporter CrpA That Are Essential for Cu Resistance but Not for Virulence

Hila Werner, Ammar Abou Kandil, Zohar Meir, Yehonathan Malis, Yona Shadkchan, Gal Masrati, Nir Ben-Tal, Koret Hirschberg, Nir Osherov*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

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’s 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 1–211 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 542–556 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 1–211 or replacing amino acids 542–556 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.

Original languageEnglish
Article number3705
JournalInternational Journal of Molecular Sciences
Volume24
Issue number4
DOIs
StatePublished - Feb 2023

Funding

FundersFunder number
Israel–China Science Foundation2017293, 2444/18
United States - Israel Binational Science Foundation
Tel Aviv University

    Keywords

    • Aspergillus fumigatus
    • Cu P-type ATPase
    • Cu export
    • Cu resistance
    • protein trafficking
    • virulence

    Fingerprint

    Dive into the research topics of 'Identification and Analysis of Fungal-Specific Regions in the Aspergillus fumigatus Cu Exporter CrpA That Are Essential for Cu Resistance but Not for Virulence'. Together they form a unique fingerprint.

    Cite this