Characterization of the Efflux Capability and Substrate Specificity of Aspergillus fumigatus PDR5-like ABC Transporters Expressed in Saccharomyces cerevisiae

Abstract

This research analyzed six Aspergillus fumigatus genes encoding putative efflux proteins for their roles as transporters. The A. fumigatus genes abcA, abcC, abcF, abcG, abcH, and abcI were cloned into plasmids and overexpressed in a Saccharomyces cerevisiae strain in which the highly active endogenous ABC transporter gene PDR5 was deleted. The activity of each transporter was measured by efflux of rhodamine 6G and accumulation of alanine β-naphthylamide. The transporters AbcA, AbcC, and AbcF had the strongest efflux activities of these compounds. All of the strains with plasmid-expressed transporters had more efflux activity than did the PDR5-deleted background strain. We performed broth microdilution drug susceptibility testing and agar spot assays using an array of compounds and antifungal drugs to determine the transporter specificity and drug susceptibility of the strains. The transporters AbcC and AbcF showed the broadest range of substrate specificity, while AbcG and AbcH had the narrowest range of substrates. Strains expressing the AbcA, AbcC, AbcF, or AbcI transporter were more resistant to fluconazole than was the PDR5-deleted background strain. Strains expressing AbcC and AbcF were additionally more resistant to clotrimazole, itraconazole, ketoconazole, and posaconazole than was the background strain. Finally, we analyzed the expression levels of the genes by reverse transcription-quantitative PCR (RT-qPCR) in triazole-susceptible and -resistant A. fumigatus clinical isolates. All of these transporters are expressed at a measurable level, and transporter expression varied significantly between strains, demonstrating the high degree of phenotypic variation, plasticity, and divergence of which this species is capable. IMPORTANCE One mechanism behind drug resistance is altered export out of the cell. This work is a multifaceted analysis of membrane efflux transporters in the human fungal pathogen A. fumigatus. Bioinformatics evidence infers that there is a relatively large number of genes in A. fumigatus that encode ABC efflux transporters. However, very few of these transporters have been directly characterized and analyzed for their potential role in drug resistance. Our objective was to determine if these undercharacterized proteins function as efflux transporters and then to better define whether their efflux substrates include antifungal drugs used to treat fungal infections. We chose six A. fumigatus potential plasma membrane ABC transporter genes for analysis and found that all six genes produced functional transporter proteins. We used two fungal systems to look for correlations between transporter function and drug resistance. These transporters have the potential to produce drug-resistant phenotypes in A. fumigatus. Continued characterization of these and other transporters may assist in the development of efflux inhibitor drugs.

Aspergillus fumigatusand aspergillosis: From basics to clinics

“…2017 ). Furthermore, these efflux pumps have different substrate specificity, i.e ., a narrow or broad-spectrum and functionality, with CDR1B showing the broadest substrate specificity ( Esquivel et al. 2020 ).…”

Functional Genomic and Biochemical Analysis Reveals Pleiotropic Effect of Congo Red on Aspergillus fumigatus

“…An A. fumigatus strain lacking b-1,3-glucan, although poorly growing, does not show different susceptibility to CR (11)(12)(13)(14). Membrane transport and cell wall permeability may also play a part in CR activity in this fungus as there is a role for the hydrophobin RodA and two ABC transporters, AbcF and AbcH, in CR susceptibility (15,16). A functional genomic study of Congo red activity in S. cerevisiae has allowed the identification of CRH (Congo red-hypersensitive) genes controlling susceptibility to Congo red (2).…”

Itraconazole resistance of Trichophyton rubrum mediated by the ABC transporter TruMDR2

“…atrD was shown to be involved in protection against cytotoxic compounds, such as cycloheximide, nigericin, valinomycin or the cyclosporine derivative PSC 833, but its activity towards ITC was not demonstrated. 24 Recently, Esquivel et al 25 studied the efflux capability and substrate specificity of 6 A. fumigatus ABC-type transporters (abcA, abcC, abcF, abcG, abcH and abcI) in S cerevisiae. These six transporters are more related to TruMDR3 than to TruMDR2.…”