Abstract Description

The manifestation of fungal co-infection in patients with COVID-19 has been a matter of concern due to the potential for increased disease severity. While immune dysregulation following SARS-CoV-2 infection creates conditions that facilitate fungal infections, the possibility of fungal infections contributing to viral invasion remains unclear. This study sought to investigate the role of fungal proteases, specifically yeast kexin protease (Kex2p) from Candida albicans, in activating the latent SARS-CoV-2 spike glycoprotein. A candidal recombinant Kex2p was isolated from E. coli BL21 (DE3) crude lysate. The protein production was confirmed by SDS-PAGE and immunodetection with an anti-His-tag antibody, revealing the expected 85 kDa band. Functional assays were then performed to assess the ability of Kex2p to 1) cleave a fluorogenic 20-mer peptide mimicking the SARS-CoV-2 spike glycoprotein, 2) transduce pseudovirions into RAW R264.7 macrophages, and 3) characterise the immune response associated with pseudovirion entry. Recombinant furin, the protein typically involved in spike activation in humans, served as a reference serine protease control. Analysis of the fluorogenic peptide cleavage assay data using the Kex2p revealed that this protease could cleave the mimetic SARS-CoV-2 spike glycoprotein (p > 0.05) as well as activate a full-length spike glycoprotein associated with pseudovirions to transduce a macrophage cell line (p > 0.05) with the same biochemical efficiency as recombinant furin. The Kex2p-mediated pseudovirion entry was characterised by the production of pro-inflammatory IL-6 and IFNG. The detected levels were also comparable (p > 0.05) to those observed when furin-mediated pseudovirion entry. Together, these findings provide enzymatic evidence that C. albicans Kex2p can prime the SARS-CoV-2 spike glycoprotein, suggesting a plausible mechanism by which fungal co-infection could facilitate viral entry. This study highlights a previously unrecognised dimension of viral-associated mycoses interplay, underscoring the importance of considering fungal proteases as important microbial factors that can potentially aggravate SARS-CoV-2 pathogenesis.