PDR Transporter ABC1 Is Involved in the Innate Azole Resistance of the Human Fungal Pathogen Fusarium keratoplasticum

Abstract

Fusarium keratoplasticum is arguably the most common Fusarium solani species complex (FSSC) species associated with human infections. Invasive fusariosis is a life-threatening fungal infection that is difficult to treat with conventional azole antifungals. Azole drug resistance is often caused by the increased expression of pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters of the ABCG sub-family. Most investigations of Fusarium ABC transporters associated with azole antifungal drug resistance are limited to plant pathogens. Through the manual curation of the entire ABCG protein family of four FSSC species including the fully annotated genome of the plant pathogen Nectria haematococca we identified PDR transporters ABC1 and ABC2 as the efflux pump candidates most likely to be associated with the innate azole resistance phenotype of Fusarium keratoplasticum. An initial investigation of the transcriptional response of logarithmic phase F. keratoplasticum cells to 16 mg/L voriconazole confirmed strong upregulation (372-fold) of ABC1 while ABC2 mRNA levels were unaffected by voriconazole exposure over a 4 h time-period. Overexpression of F. keratoplasticum ABC1 and ABC2 in the genetically modified Saccharomyces cerevisiae host ADΔΔ caused up to ∼1,024-fold increased resistance to a number of xenobiotics, including azole antifungals. Although ABC1 and ABC2 were only moderately (20% and 10%, respectively) expressed compared to the Candida albicans multidrug efflux pump CDR1, overexpression of F. keratoplasticum ABC1 caused even higher resistance levels to certain xenobiotics (e.g., rhodamine 6G and nigericin) than CDR1. Our investigations suggest an important role for ABC1 orthologues in the innate azole resistance phenotype of FSSC species.

Morphology, Phenotype, and Molecular Identification of Clinical and Environmental Fusarium solani Species Complex Isolates from Malaysia

“…It is quite likely that the rather distantly related isolate Fstr541 ( Figure 1 ) and possibly also the only voriconazole resistant F. falciforme isolate Ff0020, both of clinical origin, have different voriconazole resistance conferring mutations. One possibility may be gain of function mutations in a transcription factor that causes the overexpression of orthologs of the recently discovered multidrug efflux pump, F. keratoplasticum Abc1 [ 76 ].…”

Voriconazole Treatment Induces a Conserved Sterol/Pleiotropic Drug Resistance Regulatory Network, including an Alternative Ergosterol Biosynthesis Pathway, in the Clinically Important FSSC Species, Fusarium keratoplasticum

“…Although antifungal clinical breakpoints have not yet been established for fusaria, epidemiological cut-off values have been defined using the CLSI method [ 7 ]. We have previously shown that exposure to high but sub-growth inhibitory concentrations of VRC (16 mg/L) caused increased expression of cyp51A [ 25 ] and the PDR transporter abc1 [ 46 ]. F. keratoplasticum strain 2781 was initially grown on potato dextrose agar, PDA (Merck & Co., Kenilworth, NJ, USA) at 28 °C for a week.…”

“…Most intriguingly, however, this association held true across species boundaries with all VRC-resistant FSSC isolates (i.e., 3 F. keratoplasticum and 6 F. suttonianum isolates) possessing the 23 bp cyp51A promoter deletion [ 25 ]. We also recently reported a 372-fold induction of the mRNA levels of the pleiotropic drug resistance (PDR) transporter abc1 in response to VRC exposure [ 46 ]. When abc1 was expressed in S. cerevisiae it conferred up to 1024-fold increased resistance towards a range of xenobiotics including azoles [ 46 ].…”

“…We also recently reported a 372-fold induction of the mRNA levels of the pleiotropic drug resistance (PDR) transporter abc1 in response to VRC exposure [ 46 ]. When abc1 was expressed in S. cerevisiae it conferred up to 1024-fold increased resistance towards a range of xenobiotics including azoles [ 46 ].…”

Inhibitor-Resistant Mutants Give Important Insights into Candida albicans ABC Transporter Cdr1 Substrate Specificity and Help Elucidate Efflux Pump Inhibition

“…Over the past two decades, we have developed and refined a yeast-based efflux pump expression system ( 9 , 11 , 19 – 22 ) that allows us to study fungal efflux pumps in great detail. The overexpression of functional efflux pumps in the genetically modified host S. cerevisiae AD1-8u - has many advantages, of which one is the ability to isolate naturally selected inhibitor-resistant efflux pump mutants.…”