The Neosartorya (Aspergillus) fischeri antifungal protein NFAP2 has low potential to trigger resistance development in Candida albicans in vitro

Due to the increase in the number of drug-resistant Candida albicans strains, new antifungal compounds with limited potential for development of resistance are urgently needed. NFAP2, an antifungal protein (AFP) secreted by Neosartorya ( Aspergillus ) fischeri , is a promising candidate. We investigated the ability of C. albicans to develop resistance to NFAP2 in a microevolution experiment compared with generic fluconazole (FLC). C. albicans adapted to only 1 × minimum inhibitory concentration (MIC) of NFAP2 compared with 32 × MIC of FLC. Genome analysis revealed non-silent mutations in only two genes in NFAP2-resistant strains and in several genes in FLC-resistant strains. Resistance development to NFAP2 did not influence cell morphology. The susceptibility of NFAP2-resistant strains did not change to FLC, amphotericin B, micafungin, terbinafine. These strains did not show altered susceptibility to AFPs from Penicillium chrysogenum , except one which had less susceptibility to P. chrysogenum antifungal protein B. FLC-resistant strains had decreased susceptibility to terbinafine and NFAP2, but not to other drugs and AFPs from P. chrysogenum . NFAP2- and FLC-resistant strains showed decreased and increased NFAP2 binding and uptake, respectively. The development of resistance to NFAP2 decreased tolerance to cell wall, heat, and UV stresses. The development of FLC resistance increased tolerance to cell wall stress and decreased tolerance to heat and UV stresses. Resistance to NFAP2 did not have significant metabolic fitness cost and could not increase virulence, compared with resistance to FLC. Importance Due to the increasing number of (multi)drug-resistant strains, only a few effective antifungal drugs are available to treat infections caused by opportunistic Candida species. Therefore, the incidence of hard-to-treat candidiasis has increased dramatically in the past decade, and the demand to identify antifungal compounds with minimal potential to trigger resistance is substantial. The features of NFAP2 make it a promising candidate for the topical treatment of Candida infection. Data on the development of resistance to AFPs in C. albicans are lacking. In this study, we provide evidence that NFAP2 has low potential to trigger resistance in C. albicans in vitro and the developed resistance mechanisms to NFAP2 are not associated with severe phenotypic changes compared with development of resistance to generic FLC. These results suggest the slow emergence of NFAP2-resistant Candida strains and that NFAP2 can reliably be used long-term in the clinic. ### Competing Interest Statement The authors have declared no competing interest.