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

Overexpression of ATP-binding cassette (ABC) transporters is a major cause of drug resistance in fungal pathogens. Milbemycins, enniatin B, beauvericin, and FK506 are promising leads for broad-spectrum fungal multidrug efflux pump inhibitors. The characterization of naturally generated inhibitor-resistant mutants is a powerful tool to elucidate structure-activity relationships in ABC transporters. We isolated 20 Saccharomyces cerevisiae mutants overexpressing Candida albicans ABC pump Cdr1 variants resistant to fluconazole efflux inhibition by milbemycin α25 (8 mutants), enniatin B (8), or beauvericin (4). The 20 mutations were in just 9 residues at the centers of transmembrane segment 1 (TMS1) (6 mutations), TMS4 (4), TMS5 (4), TMS8 (1), and TMS11 (2) and in A713P (3), a previously reported FK506-resistant "hot spot 1" mutation in extracellular loop 3. Six Cdr1-G521S/C/V/R (TMS1) variants were resistant to all four inhibitors, four Cdr1-M639I (TMS4) variants were resistant to milbemycin α25 and enniatin B, and two Cdr1-V668I/D (TMS5) variants were resistant to enniatin B and beauvericin. The eight milbemycin α25-resistant mutants were altered in four amino acids as follows: G521R, M639I, A713P, and T1355N (TMS11). These four Cdr1 variants responded differently to various types of inhibitors, and each exhibited altered substrate specificity and kinetic properties. The data infer an entry gate function for Cdr1-G521 and a role for Cdr1-A713 in the constitutively high Cdr1 ATPase activity. Cdr1-M639I and -T1355N possibly cause inhibitor resistance by altering TMS contacts near the substrate/inhibitor-binding pocket. Models for the interactions of substrates and different types of inhibitors with Cdr1 at various stages of the transport cycle are presented.

Identification and functional characterization of a PDR transporter in Tripterygium wilfordii Hook.f. that mediates the efflux of triptolide.

KEY MESSAGE: TwPDR1, a PDR transporter from Tripterygium wilfordii Hook.f., was proved to efflux triptolide and its stability could be enhanced by A1033T mutation. Triptolide, an abietane-type diterpene in Tripterygium wilfordii Hook.f., possesses many pharmacological activities. However, triptolide is in short supply and very expensive because it is present at low amounts in natural plants and lack alternative production methods. Transporter engineering, which increases the extracellular secretion of secondary metabolites in in vitro culture systems, is an effective strategy in metabolic engineering but is rarely reported. In this study, TwPDR1, a pleiotropic drug resistance-type ATP binding cassette transporter, was identified as the best efflux pump candidate for diterpenoids through bioinformatics analysis. TwPDR1 was located in the plasma membrane, highly expressed in adventitious roots, and induced by methyl jasmonate. The triptolide efflux function of TwPDR1 was confirmed by transient expression in tobacco BY-2 cells and by downregulation via RNA interference in the native host. However, the overexpression of TwPDR1 had a limited effect on the secretion of triptolide. As shown by previous studies, a single amino acid mutation might increase the abundance of TwPDR1 by increasing protein stability. We identified the A1033 residue in TwPDR1 by sequence alignment and confirmed that A1033T mutation could increase the expression of TwPDR1 and result in the higher release ratio of triptolide (78.8%) of the mutants than that of control (60.1%). The identification and functional characterization of TwPDR1 will not only provide candidate gene material for the metabolic engineering of triptolide but also guide other transporter engineering researches in the future.

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

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.

AaABCG40 Enhances Artemisinin Content and Modulates Drought Tolerance in Artemisia annua.

The phytohormone Abscisic acid (ABA) regulates plant growth, development, and responses to abiotic stresses, including senescence, seed germination, cold stress and drought. Several kinds of researches indicate that exogenous ABA can enhance artemisinin content in A. annua. Some transcription factors related to ABA signaling are identified to increase artemisinin accumulation through activating the artemisinin synthase genes. However, no prior study on ABA transporter has been performed in A. annua. Here, we identified a pleiotropic drug resistance (PDR) transporter gene AaPDR4/AaABCG40 from A. annua. AaABCG40 was expressed mainly in roots, leaves, buds, and trichomes. GUS activity is primarily observed in roots and the vascular tissues of young leaves in proAaABCG40: GUS transgenic A. annua plants. When AaABCG40 was transferred into yeast AD12345678, yeasts expressing AaABCG40 accumulated more ABA than the control. The AaABCG40 overexpressing plants showed higher artemisinin content and stronger drought tolerance. Besides, the expression of CYP71AV1 in OE-AaABCG40 plants showed more sensitivity to exogenous ABA than that in both wild-type and iAaABCG40 plants. According to these results, they strongly suggest that AaABCG40 is involved in ABA transport in A. annua.

AaPDR3, a PDR Transporter 3, Is Involved in Sesquiterpene ß-Caryophyllene Transport in Artemisia annua.

Artemisinin, a sesquiterpenoid endoperoxide, isolated from the plant Artemisia annua L., is widely used in the treatment of malaria. Another sesquiterpenoid, ß-caryophyllene having antibiotic, antioxidant, anticarcinogenic and local anesthetic activities, is also presented in A. annua. The role played by sesquiterpene transporters in trichomes and accumulation of these metabolites is poorly understood in A. annua and in trichomes of other plant species. We identified AaPDR3, encoding a pleiotropic drug resistance (PDR) transporter located to the plasma membrane from A. annua. Expression of AaPDR3 is tissue-specifically and developmentally regulated in A. annua. GUS activity is primarily restricted to T-shaped trichomes of old leaves and roots of transgenic A. annua plants expressing proAaPDR3: GUS. The level of ß-caryophyllene was decreased in transgenic A. annua plants expressing AaPDR3-RNAi while transgenic A. annua plants expressing increased levels of AaPDR3 accumulated higher levels of ß-caryophyllene. When AaPDR3 was expressed in transformed yeast, yeasts expressing AaPDR3 accumulated more ß-caryophyllene, rather than germacrene D and ß-farnesene, compared to the non-expressing control.

A pleiotropic drug resistance transporter is involved in reduced sensitivity to multiple fungicide classes in Sclerotinia homoeocarpa (F.T. Bennett).

Dollar spot, caused by Sclerotinia homoeocarpa, is a prevalent turfgrass disease, and the fungus exhibits widespread fungicide resistance in North America. In a previous study, an ABC-G transporter, ShatrD, was associated with practical field resistance to demethylation inhibitor (DMI) fungicides. Mining of ABC-G transporters, also known as pleiotropic drug resistance (PDR) transporters, from RNA-Seq data gave an assortment of transcripts, several with high sequence similarity to functionally characterized transporters from Botrytis cinerea, and others with closest blastx hits from Aspergillus and Monilinia. In addition to ShatrD, another PDR transporter showed significant over-expression in replicated RNA-Seq data, and in a collection of field-resistant isolates, as measured by quantitative polymerase chain reaction. These isolates also showed reduced sensitivity to unrelated fungicide classes. Using a yeast complementation system, we sought to test the hypothesis that this PDR transporter effluxes DMI as well as chemically unrelated fungicides. The transporter (ShPDR1) was cloned into the Gal1 expression vector and transformed into a yeast PDR transporter deletion mutant, AD12345678. Complementation assays indicated that ShPDR1 complemented the mutant in the presence of propiconazole (DMI), iprodione (dicarboximide) and boscalid (SDHI, succinate dehydrogenase inhibitor). Our results indicate that the over-expression of ShPDR1 is correlated with practical field resistance to DMI fungicides and reduced sensitivity to dicarboximide and SDHI fungicides. These findings highlight the potential for the eventual development of a multidrug resistance phenotype in this pathogen. In addition, this study presents a pipeline for the discovery and validation of fungicide resistance genes using de novo next-generation sequencing and molecular biology techniques in an unsequenced plant pathogenic fungus.