Enhanced efflux pump expression in Candida mutants results in decreased manogepix susceptibility.

Manogepix is a broad-spectrum antifungal agent that inhibits glycosylphosphatidylinositol (GPI) anchor biosynthesis. Using whole-genome sequencing, we characterized two efflux-mediated mechanisms in the fungal pathogens Candida albicans and Candida parapsilosis that resulted in decreased manogepix susceptibility. In C. albicans, a gain-of-function mutation in the transcription factor gene ZCF29 activated expression of ATP-binding cassette transporter genes CDR11 and SNQ2.

I nvasive fungal infections cause significant mortality and morbidity in humans, killing Ͼ1.5 million people annually (1,2). A rapidly growing immunocompromised population is at particular risk, including those undergoing chemotherapy or solid organ transplantation or those infected with HIV (3)(4)(5). Current antifungal treatments are limited to three major classes of drugs: azoles, polyenes, and echinocandins (6). Issues of safety, tolerability, and the evolution of fungal drug resistance necessitate the development of antifungals with new mechanisms of action.
To further explore the therapeutic potential of fosmanogepix, it is important to understand the potential for evolution of drug resistance. Spontaneous and serial passage experiments revealed that the GWT1 missense mutations V162A (heterozygous) and V163A in C. albicans and Candida glabrata, respectively, demonstrated 16and 32-fold increases in MGX MIC values (20). These mutations are hypothesized to impede drug binding to the target and differ from essential catalytic residues in Gwt1 (10,21). Off-target mutations driving increases in MGX MIC values are largely unknown, although mutations in EMP24 suppress toxicity of the Gwt1 inhibitor gepinacin (14). Emp24 facilitates quality control of GPI assembly by directing mature GPI-anchored proteins from the ER to the Golgi complex (22). Emp24 loss of function is predicted to release immature GPI-anchored proteins that accumulate during Gwt1 inhibition (23).
Spontaneous mutants of C. albicans (strain 5-3) and C. parapsilosis (strain 5-2) were identified that demonstrated decreased susceptibility to MGX and fluconazole (FLC) (20). The C. albicans mutant demonstrated 4-fold and 2-fold increases in the MICs of MGX and FLC, respectively, versus MICS of the isogenic wild-type strain, while the C. parapsilosis mutant demonstrated 8-fold and 4-fold increased MICs of MGX and FLC, respectively, versus MICs of the isogenic wild-type strain (20) ( Table 1). In vitro susceptibility assays were performed as described in CLSI M27-A3, except that the dilution scheme consisted of 2-fold serial dilutions from 5 M to 0.0049 M (1.792 g/ml to 0.00175 g/ml). MIC values were determined at 50% growth inhibition relative to that of drug-free controls at 48 h (24).
These two strains were not mutated in the GWT1 gene; thus, we hypothesized that elevated MIC values for MGX and FLC could result from enhanced expression of multidrug efflux pumps, affecting the susceptibility to the structurally and mechanistically distinct antifungal agents. Although drug efflux has been frequently associated with antifungal resistance (25), it has not been described for MGX. To assess efflux in the strains with elevated MGX MIC values, we incubated cultures with Nile red, which accumulates in lipid membranes and is actively extruded from cells by efflux (26). Nile red is a substrate for the ATP-binding cassette (ABC) transporters Cdr1 and Cdr2, and the major facilitator superfamily transporter Mdr1 (26), which are the efflux pumps most frequently resulting in azole resistance in Candida (25). For these experiments, unit equivalents of an optical density at 600 nm (OD 600 ) of 1 of log-phase cultures were washed 2 times in 1 ml buffer A (20 mM Na-HEPES, 150 mM NaCl, pH 7.5), resuspended in 1 ml buffer A, and incubated at 30°C for 2 h. Nile red was added to 7 M and incubated for 1 h. Stained cells were washed 2 times in buffer A, and then efflux was initiated by addition of glucose to 1% (wt/vol). Nile red fluorescence was determined by flow cytometry after 30 min (Beckman CytoFLEX, phycoerythrin (PE) filter A01-1-0052; analysis with CytExpert 2.3) and visualized by fluorescence microscopy on a Zeiss AxioObserver.Z1 (Chroma Tech ET Cy5 filter). Both C. albicans and C. parapsilosis MGX mutants with elevated MIC values accumulated ϳ60% less Nile red than their parental strains ( Fig. 1A and B), implicating drug efflux in the decreased susceptibility to MGX.
To identify mutations responsible for activation of drug efflux, we sequenced the genomes of the MGX mutants using the Illumina MiSeq platform (Genewiz). Adaptor sequences and low-quality reads were removed using Trimmomatic v0.39 (27). Paired reads were assembled to Candida Genome Database (28) C_albicans_SC5314_A21 (29) and C_parapsilosis_CDC317 (30) by using Bowtie2 v2.3.5.1 (31) ( Table 2). Missense  In C. albicans MGX r 5-3, a single heterozygous SNV resulted in a W986L substitution in the Zn(II) 2 Cys 6 transcription factor Zcf29. This gain-of-function mutation was previously identified in beauvericin-resistant C. albicans (36). Consistent with this connection, C. albicans MGX r 5-3 demonstrated a 32-fold increase in the MIC value versus that in the wild-type (WT) strain to beauvericin, and an engineered ZCF29/ZCF29 W986L C. albicans mutant demonstrated an 8-fold increase in the MGX MIC value versus that in the WT strain (Table 1). Transcript levels for efflux pump genes were assessed using reverse transcriptase quantitative real-time PCR (RT-qPCR). RNA was extracted from 10 ml log-phase yeast extract-peptone-dextrose (YPD) cultures grown at 30°C by using a Qiagen RNeasy minikit. RNA was treated with Qiagen RNase-free DNase and reverse transcribed using Bio-Rad iScript cDNA synthesis kit. qPCR was performed using Thermo Fisher Scientific SYBR green master mix and oligonucleotide primers described in Table S2 in the supplemental material. Data were analyzed using Bio-Rad CFX Manager 3.1. Transcript levels of efflux genes CDR11, SNQ2, and MDR1 were increased in MGX r 5-3 by 6.8, 13.3, and 2.2-fold, respectively (Fig. 1C), consistent with transcriptional profiling of C. albicans ZCF29/ZCF29 W986L (36). Transcript levels of CDR1 and FLU1 were unchanged (Fig. 1C).
No missense SNVs were detected in C. parapsilosis MGX r 5-2; however, a deletion was detected in the mitochondrial chromosome from bases 15430 to 17322. The boundaries of this deletion were defined using MitoDel v3.0 (37), which identified 2,087 reads across this junction. This deletion interrupts CAPAFMP06 (COX1), which encodes cytochrome c oxidase, and CAPAFMP06.3/CAPAFMP06. 4, intronic open reading frames (ORFs) to COX1 that encode enzymes with predicted roles in mRNA splicing (38). Consistent with a respiratory defect, C. parapsilosis MGX r 5-2 formed petite colonies on YP glucose-containing medium and did not grow on YP medium containing the nonfermentable carbon source glycerol ( Fig. 2A). Furthermore, when subcultured in glycerol-supplemented synthetic complete medium for 2 h and then stained with 10 nM mitochondrial membrane potential-dependent stain MitoTracker Red CMXRos (Invitrogen) for 30 min, C. parapsilosis MGX r 5-2 showed ϳ60% reduced staining relative to that of the parental strain. MitoTracker Red fluorescence was quantified by flow cytometry (Fig. 2B) (Beckman CytoFLEX, DsRed filter A01-1-0053) and visualized by fluorescence microscopy (Fig. 2C) on a Zeiss AxioObserver.Z1 (Chroma Tech ET HQ DsRed filter).
Respiratory competence is linked to drug susceptibility in diverse fungal pathogens and is often driven by efflux pump overexpression (39). Indeed, transcript levels of MDR1 were 8.5-fold upregulated in C. parapsilosis MGX r 5-2 (Fig. 1C), consistent with a previously described petite mutant of C. albicans with decreased susceptibility to FLC (40,41). In some Saccharomyces cerevisiae and C. glabrata petite mutants, the Pdr1/Pdr3 transcription factors induce expression of ABC transporter genes PDR5 (ortholog of CDR1), SNQ2, and YOR1 (42)(43)(44). This is not the case in C. parapsilosis MGX r 5-2, as transcript levels of CDR1 and SNQ2 were unchanged (Fig. 1C).
In conclusion, we have identified two efflux-mediated mechanisms conferring reduced susceptibility to MGX in two Candida species. In C. albicans, a gain-of-function

ACKNOWLEDGMENTS
Funding for this work was provided by Amplyx. S.D.L. was supported by an NSERC postdoctoral fellowship (516840-2018).
L.E.C. and L.W. are cofounders and shareholders in Bright Angel Therapeutics, a platform company for development of novel antifungal therapeutics. L.E.C. is a consultant for Boragen, a small-molecule development company focused on leveraging the unique chemical properties of boron chemistry for crop protection and animal health. M.K. is an employee of Amplyx. K.J.S. was an employee of Amplyx and is now an independent consultant at Hearts Consulting Group. S.D.L. has no conflicts.