The Medical Triazole Voriconazole Can Select for Tandem Repeat Variations in Azole-Resistant Aspergillus Fumigatus Harboring TR34/L98H Via Asexual Reproduction

Journal of Fungi

Article The Medical Triazole Voriconazole Can Select for Tandem Repeat Variations in Azole-Resistant Aspergillus Fumigatus Harboring TR34/L98H via Asexual Reproduction

1, 2,3 2,3, 1 Jianhua Zhang * , Jan Zoll , Tobias Engel †, Joost van den Heuvel , Paul E. Verweij 2,3,4 and Alfons J. M. Debets 1

1 Laboratory for Genetics, Wageningen University and Research, 6708 PB Wageningen, The Netherlands; [email protected] (J.v.d.H.); [email protected] (A.J.M.D.) 2 Department of Medical Microbiology, Radboud University Medical Center, 6500 HB Nijmegen, The Netherlands; [email protected] (J.Z.); [email protected] (T.E.); [email protected] (P.E.V.) 3 Center of Expertise in Mycology Radboudumc/CWZ, 6500 HB Nijmegen, The Netherlands 4 Center for Infectious Diseases Research, Diagnostics and Laboratory Surveillance, National Institute for Public Health and the Environment (RIVM), 3720 BA Bilthoven, The Netherlands * Correspondence: [email protected] Currently affiliated to Laboratory for Medical Microbiology and Public Health, 7555 BB Hengelo, The Netherlands. † The work for this study was performed at the Radboud University Medical Center.

Received: 1 September 2020; Accepted: 6 November 2020; Published: 11 November 2020

Abstract: Azole-resistant Aspergillus fumigatus isolates recovered at high frequency from patients, harbor mutations that are associated with variation of promoter length in the cyp51A gene. Following the discovery of the TR34/L98H genotype, new variations in tandem repeat (TR) length and number of repeats were identified, as well as additional single nucleotide polymorphisms (SNPs) in the cyp51A gene, indicating that the diversity of resistance mutations in A. fumigatus is likely to continue to increase. Investigating the development routes of TR variants is critical to be able to design preventive interventions. In this study, we tested the potential effects of azole exposure on the selection of TR variations, while allowing haploid A. fumigatus to undergo asexual reproduction. Through experimental evolution involving voriconazole (VOR) exposure, an isolate harboring 3 TR34 /L98H evolved from a clinical TR34/L98H ancestor isolate, confirmed by whole genome 3 sequencing. TR34 /L98H was associated with increased cyp51A expression and high VOR and posaconazole MICs, although additional acquired SNPs could also have contributed to the highly 3 azole-resistant phenotype. Exposure to medical azoles was found to select for TR34 , thus supporting the possibility of in-host selection of TR34 variants.

Keywords: azole resistance; tandem repeats variants; asexual reproduction; Aspergillus fumigatus; antifungal agents

1. Introduction In recent decades, the emergence of azole resistance in Aspergillus fumigatus, a saprobic ﬁlamentous fungus, has caused challenges as this fungus is a main cause of opportunistic fungal diseases in humans and animals. In the past ﬁve years, the azole resistance frequency increased from 7.6% in 2013 to 15% in 2018 in the Netherlands [1]. Up to 90% of resistant A. fumigatus isolates cultured from patient samples exhibit a resistance mechanism characterized by tandem repeats (TR) in the promotor region of the cyp51A gene in combination with short nucleotide polymorphisms (SNPs) in the gene, typically

J. Fungi 2020, 6, 277; doi:10.3390/jof6040277 www.mdpi.com/journal/jof J. Fungi 2020, 6, 277 2 of 13

TR34/L98H and TR46/Y121F/T289A. Cyp51A encodes the target enzyme 14α-demethylase in the cell membrane and these mutations confer resistance to triazole antifungals including itraconazole (ITR), voriconazole (VOR), isavuconazole (ISA) and posaconazole (POS) [2–5]. In addition to clinical cultures, identical TR-associated resistance mutations were recovered from environmental samples, especially in decaying plant material that contained azole fungicide residues [6]. Numerous studies have confirmed a relation between environmental resistance selection and azole-resistant infection in humans, and the concept of an environmental route of resistance selection is now generally accepted [7,8]. TR-mediated resistance mutations could be distinguished from single resistance SNPs that were commonly found in resistant A. fumigatus isolates from patients that received chronic azole therapy (patient route). However, the association between TR-mediated resistance mutations and resistance selection in the environment was recently challenged when an azole-resistant A. fumigatus isolate harboring TR120 was recovered from a patient that had received chronic azole therapy [5]. Whole genome sequencing of a previously cultured wild type (WT) A. fumigatus isolate and the TR120 isolate proved that the 3 TR resistance mechanism had emerged through patient therapy. Recently a TR34 /L98H variant was recovered from a cystic fibrosis patient who also harbored a TR34/L98H isolate. Microsatellite typing 3 showed that the TR34 /L98H and TR34/L98H shared eight of nine markers, which supports possible in vivo acquisition of an additional TR [9]. Apparently not the resistance selection route, but other factors such as chemical characteristics of the azole, the reproduction mode the fungus goes through and azole stress duration might determine which resistance mutation is selected for [10]. It is important to understand how TR-mediated resistance mutations develop. TR-mediated resistance is highly dominant, especially TR34, accounting for up to 70% of resistance mutations in surveillance studies [11]. Furthermore, over time increased variation in TRs is observed with both increasing TR length, i.e., TR34, TR46, TR53, and TR120, as well as increasing numbers of TR-duplications, 3 4 3 i.e., TR46, TR46 and TR46 [5,12,13] and TR34 and TR34 [9].The repeated promoter sequence in the 3 4 variants TR46, TR53, TR120 TR46 , and TR46 all contain the repeated sequence of TR34. It has been shown that the repeat sequence in TR34 is bound by both the sterol regulatory element binding protein SrbA, and the CCAAT binding complex (CBC). Sterol biosynthesis and azole tolerance is governed by the opposing actions of SrbA and the CCAAT Binding. The mechanism underpinning TR34-driven overexpression of cyp51A is a result of the duplication of SrbA binding sites but not CBC [14]. Therefore, the extra two copies of 34 bp, largely increase the overexpression of cyp51A due to the extended SrbA binding sites. In a previous study we have shown that sexual reproduction can play a role in extending the promoter length under laboratory conditions by unequal crossing over between tandem repeats during meiosis. Through sexual crossing of two A. fumigatus isolates harboring TR46, an isolate with 3 TR46 was recovered in the progeny [4]. However, sexual reproduction in A. fumigatus has not yet been found in the environment and may be restricted to highly specific conditions. Furthermore, the sexual cycle requires compatible strains of opposite mating type and is highly unlikely to occur in the clinical setting. It is unclear whether TR diversification can occur in asexually reproducing haploid cultures. Our hypothesis is that also asexual reproduction can extend the promoter length, since asexual reproduction involves numerous mitotic divisions and promoter repeats may further expand by replication slippage or unequal sister chromatid recombination [15,16]. Additionally, as TR-mediated resistance was shown to emerge in an azole-treated patient we investigated if exposure to VOR could play a role in TR-variations through the asexual cycle. In this study, we performed experimental evolution which allows for asexual reproduction to occur using a clinical A. fumigatus TR34/L98H isolate and exposure to VOR.

2. Materials and Methods Culture media and antifungal agents: Malt extract agar (MEA), used for counting conidia and measuring the mycelial growth rate, was purchased from Sigma Company (Sigma, Aldrich, Germany). Minimal Medium (MM) was used for culturing the heterokaryon. MM consists of 6.0 g NaNO3, 1.5 g J. Fungi 2020, 6, 277 3 of 13

KH2PO4, 0.5 g MgSO4. 7H2O, 0.5 g KCl, 10 mg of FeSO4, ZnSO4, MnCl2, and CuSO4 and 15 g agar + 1000 mL H2O (pH 5.8) [17]. The antifungal agents ITR, VOR and POS were purchased from Sigma Company (Sigma Aldrich, Germany) [18]. The evolution of resistance through asexual reproduction under antifungal agents pressure: we started the evolutionary experiment with clinical isolate TR34/L98H (V30-40) as ancestral strain under exposure to 2 µg/mL of VOR, which allows the asexual sporulation of ancestor TR34/L98H (V30-40). In the evolutionary experiment, a new cycle was started every seven days. This evolutionary line was inoculated by placing a droplet (5 µL) of asexual conidia suspension from the ancestor colony into a 30 mL glass bottle (infusion bottle, VWR, Netherlands) with MEA supplemented with VOR and the bottle was incubated at 37 ◦C. The colonies grown in these bottles represented the first cycle. After seven days, A. fumigatus conidia were harvested with 5 mL saline (distilled water with NaCl 0.8 g/L) supplemented with Tween 80 (0.05%), by washing off the fungal growth with beads. Asexual conidia suspensions were stored and an inoculum of 1% (50 µL) of the conidia was used to initiate a next selection cycle [18]. This procedure was repeated every seven days until 15 cycles were completed. The analysis of mutations (promoter length and coding region) of the cyp51A gene: at the end of the evolutionary experiment, the whole population was diluted and plated on agar plates supplemented with 2 µg/mL of VOR. After two days of growth under 37 ◦C, 20 A. fumigatus colonies were randomly selected. Single resistant colonies were inoculated in 3 mL Malt Extract broth and grown overnight at 37 ◦C. Mycelial mats were recovered and subjected to a DNA extraction protocol [19]. The full coding sequence of the cyp51A gene and its promoter region were determined by amplification and subsequent sequencing as previously described [20,21]. The cyp51A sequence under accession number AF338659 in GenBank was used for comparison to detect mutations, cyp51A-PF(50-ATGGTGCCGATGCTATGG-30) and cyp51A-PR (50-CTGTCTCACTTGGATGTG-30). The presence of TRs in the promotor region of the gene was investigated by amplifying part of the promoter region of the cyp51A gene using appropriate primers (50-TGAGTTAGGGTGTATGGTATGCTGGA-30 and 50-AGCAAGGGAGAAGGAAAGAAGCACT-30). The cycling program consisted of a 3-min denaturation step at 94 ◦C, followed by 35 cycles of 30 s at 94 ◦C, 45 s at 55 ◦C, and 45 s at 72 ◦C and a final elongation step of 5 min at 72 ◦C[13]. The amplified DNA fragments were purified with a QIAquick PCR purification kit (Qiagen). DNA sequencing of the forward strand of each fragment was performed by Eurofins sequencing company (Ebersberg, Germany). The resulting sequences were aligned in CLUSTALW46 using the program BioEdit47. If genetics changes were detected, the early evolutionary cycles were traced back following the same protocol. Testing for isogeneity between ancestor and evolved isolates: In order to test whether the evolved A. fumigatus colony was isogenic with the ancestor strain, we applied heterokaryon compatibility testing, microsatellite locus genotyping, and whole genome sequencing (WGS). Heterokaryon compatibility test: as a result of high polymorphism for heterokaryon incompatibility het-genes involved in self/nonself recognition in fungal populations, heterokaryon compatibility is mostly restricted to clonally related isolates and, therefore two randomly picked isolates from nature are typically heterokaryon incompatible [22,23]. In this study heterokaryon compatibility of strains was tested following standard methods [24–26]. Recessive markers (nitrate non-utilizing mutations nia and 2 cnx) were introduced by ultra violet (UV 60 s; 20 erg/mm /s) radiation in ancestor isolate TR34/L98H and any evolved genotypes. Nia and cnx mutants were isolated on basis of chlorate resistance and characterized for their ability to utilize urea, hypoxanthine, nitrite and nitrate as sole N-source (a nia (chlorate resistant, nitrate non-utilising, hypoxanthine and nitrite utilising) and a cnx (chlorate resistant, nitrate and hypoxanthine non-utilising, nitrite utilising), see Supplemental Figure S1 and Table S1). The isolates with complementing nia and cnx markers were co-inoculated on the medium with nitrate as the sole N-source. Heterokaryons of compatible complementing strains show typical vigorous growth after 5 days, whereas inability to form heterokaryons results in spares growth of the individual homokaryons [27]. J. Fungi 2020, 6, 277 4 of 13

Microsatellite locus genotyping: genetic relationships between the ancestor and evolved genotype were established based on a categorical analysis of six microsatellite markers (STR3 A, B, C and STR4 A, B, C) [13]. Whole genome sequencing: for ancestral TR34/L98H and evolved genotypes we performed WGS. Environmental isolate TR34/L98H (V62-10) was used as control. Mapping and variant calling: Raw FASTQ files were trimmed and filtered by TRIMMOMATIC (v 0.27, LEADING:3, TRAILING:3, SLIDINGWINDOW:4:15, MINLENGTH:70) [28]. The resulting trimmed reads were aligned using bwa mem (v 0-7-15, default parameters) [29] using the A. fumigatus Af293 as reference (assembly ASM265v1). Reads with mapping quality lower than 20 were filtered using samtools (v 0.1.19) [30]. We then removed duplicates using picard tools (v.1.109, http://broadinstitute. github.io/picard/) and performed realignment around indels with GATK (v 3.7-0) [31]. The resulting BAM files were combined in mpileup format (samtools, mpileup, default parameters, v 0.1.19) [30], after which SNPs and indels were separately called using varscan (v 2.3.9, using mpileup2snps and mpileup2indel, respectively, -output-vcf 1, -min-var-freq 0.05) [32]. The resulting vcf files were inspected for genetic differences between any pairwise comparison, using vcfR (v 1.8.0) [33], for which we used a minimum allele frequency difference of 0.6 and a minimum coverage of 10 to filter SNPs and indels. Every resulting variant was then visually inspected in all samples using IGV (v 2.4.14) [34]. Susceptibility testing and cyp51A gene expression of ancestor and evolved isolates: In vitro susceptibility testing was performed using a broth microdilution method according to EUCAST protocol E.DEF 60 9.2 [13,18]. The cyp51A expression was analyzed from duplicate cultures of the ancestor TR34/L98H isolate and evolved genotypes and a WT control isolate (V256-07). Strains were cultured for 16 h in 50 mL of Vogel’s MM at 37 ◦C at 200 rpm. Harvested mycelia were snap-frozen in liquid nitrogen and homogenized with a MagNALyser. RNA was isolated using RNAzolB (Sigma, Aldrich, Germany) according the manufacturers protocol. cDNA was synthesized using random hexamer primers and Transcriptor reverse transcriptase (Roche, Meylan, France). Real-time PCR was performed for the cyp51A and actin genes using the PCR Master kit (Roche, Meylan, France). Cyp51A and actin amplification was performed using specific primer/probe sets for each gene (cyp51A: forward primer 50- GTGCTCCTTGCTTCACCTG-30, reverse primer 50- TCCTGCTCCTTAGTAGCCTGGTT-30, probe 50-6Fam-AGTGACAGCCCTCAGCGACGAA- BBQ-30; Actin: forward primer 50-ATTGCTCCTCCTGAGCGTAAATAC-30, reverse primer 50-GAAGGACCGCTCTCGTCGTAC-30, probe 50-6Fam-TCTGGCCTCTCTGTCCACCTTCCA-BBQ-30). Expression levels were calculated using the delta–delta–Ct method and normalized for WT expression levels [35]. The mycelium growth rate and conidia production of ancestor and evolved genotype: the mycelial growth rate (MGR) and conidia production of ancestor TR34/L98H and evolved genotypes were assayed both in the presence (2 µg/mL) and absence of VOR. We inoculated 5 µL of conidia suspension in a 30 mL glass bottle of MEA medium with and without 2 µg/mL of VOR. After four days of incubation at 37 ◦C, conidia were harvested with 5 mL saline (distilled water with NaCl 0.8 g/L) supplemented with Tween 80 (0.05%), by washing off the fungal conidia with beads. The total conidia production was measured in three replicates by a Casy® TT cell counter (OLS OMNI Life Science, Germany). For MGR determination, we inoculated 2 µL of the conidia suspension of all cultures onto Petri dishes with solid MEA medium with and without 2 µg/mL of VOR. After four days of incubation at 37 ◦C, the MGR was determined by averaging the colony diameters (in mm) as measured in two randomly chosen perpendicular directions. Competition between the ancestor and evolved isolate: In order to investigate the competition between the ancestor TR34/L98H and evolved genotypes, we visually selected a spontaneous white color mutant from the ancestral TR34/L98H by microscopic inspection of a three-day-old culture grown on an MEA plate covered with a high-density of conidial sporeheads. In order to check whether the color has effect on VOR susceptibility and competition with the other isolate, TR34/L98H green conidia and white conidia were mixed (ratio 1:1) and co-inoculated in the center of an agar-plate in the presence and absence of VOR. After two and half days of growth, the distribution of white and green conidia was recorded via camera. After the confirmation of the color effect, a mixed inoculum of conidia J. Fungi 2020, 6, x FOR PEER REVIEW 5 of 13

J. Fungiplate 2020in the, 6, 277presence and absence of VOR. After two and half days of growth, the distribution5 of of 13 whiteJ. Fungi and 2020 green, 6, x FORconidia PEER was REVIEW recorded via camera. After the confirmation of the color effect, a5 ofmixed 13 inoculum of conidia from the white ancestral TR34/L98H strain and the green evolved genotypes (ratio fromplate the white in the ancestralpresence TRand absence/L98H strainof VOR. and After the tw greeno and evolved half days genotypes of growth, (ratio the distribution 1:1) was plated of on 1:1) was plated on MEA-plates34 in the presence and absence of 2 µg/mL VOR. After two days of MEA-plateswhite and in green the presence conidia was and recorded absence via of camera. 2 µg/mL Af VOR.ter the After confirmation two days of the ofgrowth, color effect, the a distribution mixed growth, the distribution of white and green conidia was recorded in the same way. of whiteinoculum and green of conidia conidia from was the white recorded ancestral in the TR same34/L98H way. strain and the green evolved genotypes (ratio 1:1) was plated on MEA-plates in the presence and absence of 2 µg/mL VOR. After two days of 3. Results 3. Resultsgrowth, the distribution of white and green conidia was recorded in the same way. 3.1. Evolution of Azole Resistance 3.1. Evolution3. Results of Azole Resistance At the end of the 15-week evolutionary experiment, 20 single conidia cultures of this line were At the end of the 15-week evolutionary experiment, 20 single conidia cultures of this line were investigated3.1. Evolution for ofchanges Azole Resistance in the promoter region regarding length and coding region of cyp51A gene investigated for changes in the promoter region regarding length and coding region of cyp51A gene by by PCR.At The the sequence end of the results 15-week showed evolutionary that 100% experiment, (20/20 colonies) 20 single had conidia genetic cultures changes of this in linethe werecyp51A PCR. The sequence results showed that 100% (20/20 colonies) had genetic changes in the cyp51A gene. gene.investigated All isolates for contained changes in three the promoter copies of region 34 bp regardingin the promoter length andregion coding and regiona point of mutation cyp51A gene L98H Allon isolates theby PCR.coding contained The region. sequence threeThus, results copiescompared showed of 34 with bpthat inthe 100% the ance promoter(20/20stral colonies)isolate region an had additional and genetic a point changes copy mutation of in34 the bp L98H cyp51Ahad been on the codinggained.gene. region. The All previousisolatesThus, contained compared evolutionary three with cyclescopies the ancestral wereof 34 bpthen in isolate analyzed,the promoter an additional which region showed copyand a of thatpoint 34 the bpmutation genetic had been L98H change gained. Thefirst previouson emerged the coding evolutionary in region. the fifth Thus, cycles cycle compared were with then awith percentage analyzed, the ance whichstral of isolate5%. showed By an the additional that eight the geneticcycle, copy ofthe change 34 bpproportion had first been emerged of gained. The previous evolutionary cycles were then analyzed, which showed that the genetic3 change inTR the343 fifth/L98H cycle harboring with a isolates percentage had ofincreased 5%. By to the 50%, eight while cycle, 100% the of proportion the colonies of harbored TR34 /L98H TR34 harboring3/L98H first emerged in the fifth cycle with a percentage of 5%. By the eight3 cycle, the proportion of isolatesat the 12th had increasedcycle (Figure to 50%, 1). while 100% of the colonies harbored TR34 /L98H at the 12th cycle (Figure1). TR343/L98H harboring isolates had increased to 50%, while 100% of the colonies harbored TR343/L98H at the 12th cycle (Figure 1).

Figure 1. The percentage of isolates harboring TR343/L98H3 over the evolutionary cycle. Figure 1. The percentage of isolates harboring TR34 /L98H over the evolutionary cycle. Figure 1. The percentage of isolates harboring TR343/L98H over the evolutionary cycle. 3 3.2.3.2. The The Ancestor AncestorTR TR3434//L98HL98H and Evolved TR TR34343/L98H/L98H Are Are Isogenic Isogenic 3.2. The Ancestor TR34/L98H and Evolved TR343/L98H Are Isogenic ToTo exclude exclude contamination, contamination, wewe usedused the self/nonse self/nonselflf compatibility compatibility test test and and microsatellite microsatellite locus locus To exclude contamination, we used the self/nonself compatibility test and microsatellite locus genotypinggenotyping methodsmethods to to assess assess whether whether the the evolved evolved isolate isolate and ancestral and ancestral isolate isolateare isogenic. are isogenic. Both genotyping methods to assess whether the evolved isolate and ancestral isolate are isogenic. Both Bothgenotypes genotypes were were found found to be to compatible be compatible (Figure (Figure 2) and2) isogenic and isogenic (Table (Table 1). 1). genotypes were found to be compatible (Figure 2) and isogenic (Table 1).

Figure 2. Heterokaryon compatibility test on agar with nitrate as the sole N-source. Left: ancestor Figure 2. Figure 2.Heterokaryon Heterokaryon compatibilitycompatibility test on3 agar with with nitrate nitrate as as the the sole sole N-source. N-source. Left: Left: ancestor ancestor TR34/L98H nia mutant, right: evolved TR343/L98H cnx mutant. Middle, the mixture of the TR34/L98H TRTR3434/L98H/L98H nia nia mutant, mutant, right:right: evolvedevolved TR343/L98H/L98H cnx cnx mutant. mutant. Middle, Middle, the the mixture mixture ofof the the TR TR34/L98H34/L98H 3 nia and TR34 /L98H cnx mutants from left and right. The ﬁgure shows that the strains were heterokaryon compatible: the strains are not able to grow individually on nitrate as a N-source but can grow together through complementation in a heterokaryon. From top to bottom, 4 replicates are shown. J. Fungi 2020, 6, x FOR PEER REVIEW 6 of 13

nia and TR343/L98H cnx mutants from left and right. The figure shows that the strains were heterokaryon compatible: the strains are not able to grow individually on nitrate as a N-source but can grow together through complementation in a heterokaryon. From top to bottom, 4 replicates are J. Fungishown.2020, 6 , 277 6 of 13

Table 1. Microsatellite locus genotyping. Table 1. Microsatellite locus genotyping. STR 3 * STR 4 * STR 3 * STR 4 * Type Number 3A 3B 3C 4A 4B 4C Type Number 3A 3B 3C 4A 4B 4C Ancestor TR34/L98H 31 9 8 6 8 19 Ancestor TR34/L98H3 31 9 8 6 8 19 Evolved TR334 /L98H 31 9 8 6 8 19 Evolved TR34 /L98H 31 9 8 6 8 19 * STR3 A, B, C and STR4 A, B, C: six microsatellite markers. * STR3 A, B, C and STR4 A, B, C: six microsatellite markers.

WGS was performed on the ancestor TR34/L98H, the TR343/L98H, and an environmental WGS was performed on the ancestor TR /L98H, the TR 3/L98H, and an environmental TR /L98H TR34/L98H control isolate (raw data are available34 via NCBI,34 BioProjectID: PRJNA666755). TR3434/L98H control isolate (raw data are available via NCBI, BioProjectID: PRJNA666755). TR /L98H and and TR343/L98H were confirmed to be isogenic, sharing 99.9% of their genomes. Only three34 SNPs were TR 3/L98H were confirmed to be isogenic, sharing 99.9% of their genomes. Only three SNPs were found34 to differ between ancestral TR34/L98H and TR343/L98H, while 14,500 SNPs were found between found to differ between ancestral TR /L98H and TR 3/L98H, while 14,500 SNPs were found between TR343/L98H and the environmental 34TR34/L98H control34 isolate (Supplemental Table S2). These three 3 TRSNPs34 / L98Hincluded and one the in environmental gene AFUA_1G06920 TR34/L98H Serine control/threonine isolate protein (Supplemental kinase which Table may S2). Thesebe involved three SNPsin included one in genegrowth AFUA_1G06920 Serine/threonine(http://www.as protein kinasepergillusgenome.org/cgi- which may be involved inbin/locus.pl?locus=AFUA_1G06920&org growth (http://www.aspergillusgenome.organism=A_fumigatus_Af293);/cgi-bin/locus.pl?locus= AFUA_1G06920&organismand an= A_fumigatus_Af293AFUA_5G06450/Vacuolar); and anprotein AFUA_5G06450 sorting protein/Vacuolar DigA protein which sorting may protein be involved DigA which in vacuole may be involvedorganization in vacuole organization (http://www.aspergillusgenome.org(http://www.as/cgi-binpergillusgenome.org/cgi-/locus.pl?locus=AFUA_ 5G06450&organismbin/locus.pl?locus=AFUA_5G06450&organism=A_fumigatus_Af293);=A_fumigatus_Af293); The third gene involved AtrR, The athird transcription gene involved factor AtrR, that hasa transcription been reported factor to play that ahas role been in regulating reported tocyp51A play aexpression role in regulating [36]. cyp51A expression [36]. 3.3. Susceptibility Testing and Cyp51a Gene Expression of Ancestor and Evolved Isolates 3.3. Susceptibility Testing and Cyp51a Gene Expression of Ancestor and Evolved Isolates 3 In vitro susceptibility testing showed increased MICs of VOR and POS in the TR34 /L98H In vitro susceptibility testing showed increased MICs of VOR and POS in the TR343/L98H isolate isolate (Table2). Expression levels assayed from Cyp51A /actin mRNA ratios showed that cyp51A (Table 2). Expression levels assayed3 from Cyp51A/actin mRNA ratios showed that cyp51A gene gene expression levels in the TR34 /L98H were significantly higher compared to WT and ancestral expression levels in the TR343/L98H were significantly higher compared to WT and ancestral TR34/L98H (ANOVA: p < 0.05) (Figure3). TR34/L98H (ANOVA: p < 0.05) (Figure 3). 3 Table 2. In vitro azole susceptibility profiles of TR34/L98H and TR34 /L98H. Table 2. In vitro azole susceptibility profiles of TR34/L98H and TR343/L98H. MIC (µg/mL) MIC (µg/mL) Isolate Code Genotype ITR VOR POS Isolate Code Genotype ITR VOR POS V30–40 TR34/L98H >16 4 0, 5 V30–40 TR34/L98H3 >16 4 0, 5 v284–37 TR34 /L98H >16 >16 >8 v284–37 TR343/L98H >16 >16 >8

33 FigureFigure 3.3. ExpressionExpression ofof cyp51Acyp51Ain in thethe TR TR3434 /L98H/L98H isolate, comparedcompared withwith thethe TRTR3434//L98HL98H ancestorancestor andand aa wildwild typetype (WT)(WT) controlcontrol isolate.isolate.

3.4. The Role of SNPs in the Observed Azole-Resistant Phenotypes As the AtrR gene is involved in regulation of cyp51A expression, mutations in this gene might be associated with an azole-resistant phenotype. We therefore analyzed an isolate from the eighth cycle, J. Fungi 2020, 6, x FOR PEER REVIEW 7 of 13

3.4. The Role of SNPs in the Observed Azole-Resistant Phenotypes J. Fungi 2020, 6, 277 7 of 13 As the AtrR gene is involved in regulation of cyp51A expression, mutations in this gene might be associated with an azole-resistant phenotype. We therefore analyzed an isolate from the eighth whichcycle, harboredwhich harbored the TR34 the/L98H TR34 mutation,/L98H mutation, but showed but showed a change a inchange VOR phenotypein VOR phenotype (MIC >16 (MICµg/mL) >16 andµg/mL) POS (MICand POS> 8 µ(MICg/mL) > compared8 µg/mL) withcompared the ancestor with the TR 34ancestor/L98H isolate. TR34/L98H This isolate,isolate. indicatedThis isolate, as Transientindicated TR as34 Transient/L98H, contained TR34/L98H, a mutation contained in a AtrR, mutation which in supports AtrR, which a possible supports role a of possible AtrR in role azole of resistanceAtrR in azole (Supplemental resistance Table (Supplemental S3). This isolate Table also S3). harbored This isolate an additional also harbored non-resistant an additional related SNPnon- 3 inresistant protein related PtaB, which SNP wasin protein not found PtaB, in which the ﬁnally was evolvednot found TR in34 the/L98H finally isolate, evolved indicating TR343/L98H that this isolate, line 3 didindicating not survive that inthis competition line did not with survive the TR in34 competition/L98H line with (Supplemental the TR343/L98H Table line S4). (Supplemental Cyp51A expression Table levelS4). ofCyp51A the Transient expression TR34 level/L98H of isolate the Transient was higher TR34 than/L98H that isolate of the was ancestor higher TR 34than/L98H, that butof the lower ancestor than 3 theTR expression34/L98H, but level lower of TR than34 / L98Hthe expression (Supplemental level Figure of TR S2).343/L98H These (Supplemental observations indicate Figure thatS2). inThese the 3 evolvedobservations TR34 /indicateL98H, the that overexpression in the evolved of TR cyp51A343/L98H, is the the combined overexpression eﬀect of of AtrR cyp51A and is three the combined copies of 3 34effect bp (see of AtrR Supplemental and three Figure copies S2). of Furthermore,34 bp (see Suppl theemental fact that inFigure our evolution S2). Furthermore, experiment the TR fact34 /thatL98H in 3 replacedour evolution Transient experiment TR34/L98H TR indicates343/L98H areplaced selective Transient advantage TR of34 TR/L98H34 /L98H indicates over a Transient selective TR advantage34/L98H underof TR34 VOR3/L98H pressure. over Transient TR34/L98H under VOR pressure.

3.5. The Mycelium Growth Rate and Conidia Production of Ancestor and Evolved TR Genotypes 3.5. The Mycelium Growth Rate and Conidia Production of Ancestor and Evolved TR3434 Genotypes 3 Under VOR (2 µg mL) exposure, TR 3L98H showed 60% increased mycelium growth rate and Under VOR (2 µg/mL)/ exposure, TR3434//L98H showed 60% increased mycelium growth rate and p 2.62.6 times times the the conidia conidia production production compared compared with with the the ancestral ancestral TR 34TR/L98H34/L98H (ANOVA: (ANOVA:< p0.05, < 0.05, Figure Figure4). 3 However,4). However, in the in absencethe absence of VOR of VOR the the TR 34TR/34L98H3/L98H isolate isolate showed showed a ﬁtnessa fitness cost cost with with lower lower amounts amounts ofof conidiaconidia productionproduction (30%(30% less)less) andand decreased decreased myceliummycelium growthgrowth raterate comparedcompared toto the the ancestral ancestral TRTR3434//L98HL98H(Figure (Figure4 ).4).

33 FigureFigure 4. 4.The The growth growth characteristics characteristics of of TR TR3434/L98H/L98H and and TR TR3434//L98H,L98H, includingincludingthe the phenotype, phenotype, growth growth raterate and and conidia conidia production production when when cultured cultured in the in presence the presence and absence and absence of voriconazole of voriconazole (VOR) (2 (VOR)µg/mL). (2 µg/mL). 3 3.6. Competition between TR34/L98H and TR34 /L98H

3.6. Competition between TR34/L98H and TR343/L98H 3 Fitness differences between TR34/L98H and TR34 /L98H in presence or absence of VOR can be visualizedFitness when differences conidia arebetween co-inoculated TR34/L98H in the and center TR343 of/L98H an agar-plate. in presence As aor control, absence TR of34 VOR/L98H can with be greenvisualized conidia when and conidia a derived are mutantco-inoculated with white in the conidia center of were an agar-plate. mixed. They As showeda control, the TR same34/L98H fitness with withgreen equal conidia appearance and a derived of white mutant and green with conidia white atconidia the rim were of the mixed. colony They in the showed presence the andsame absence fitness ofwith VOR equal after appearance 2.5 days of of growth white and at 37 green◦C (Figure conidia5B,D). at the This rim indicatesof the colony that in there the presence is no color and e ff absenceect on 3 fitnessof VOR of after the white-color 2.5 days of mutation.growth at Under37 °C (Figure VOR-free 5B,D). conditions, This indicates TR34 / L98Hthat there with is green no color conidia effect was on clearly outcompeted by the faster growing TR34/L98H with white conidia as the rim has exclusively 3 white conidia (Figure5A). However, in the presence of VOR (2 µg/mL), TR34 /L98H outcompeted the J. Fungi 2020, 6, x FOR PEER REVIEW 8 of 13

J. Fungi 2020, 6, 277 8 of 13 fitness of the white-color mutation. Under VOR-free conditions, TR343/L98H with green conidia was clearly outcompeted by the faster growing TR34/L98H with white conidia as the rim has exclusively white conidia (Figure 5A). However, in the presence of VOR (2 µg/mL), TR343/L98H outcompeted the ancestor TR34/L98H isolate, showing only green conidia at the rim but no white conidia (Figure5C) ancestorafter two TR days.34/L98H isolate, showing only green conidia at the rim but no white conidia (Figure 5C) after two days.

Figure 5. Competition between TR34/L98H (white conidia) and TR343/L98H3 (green conidia) in the Figure 5. Competition between TR34/L98H (white conidia) and TR34 /L98H (green conidia) in the absence (panel A) and presence (panel C) of VOR. Control between TR343/L98H3 (green conidia) and absence (panel A) and presence (panel C) of VOR. Control between TR34 /L98H (green conidia) and TR343/L98H3 (white conidia) in the absence (panel B) and presence (panel D) of VOR after two and half TR34 /L98H (white conidia) in the absence (panel B) and presence (panel D) of VOR after two and half days of growth at 37 °C. days of growth at 37 ◦C.

4.4. Discussion Discussion OurOur results results indicate indicate that that cyp51Acyp51A promoterpromoter elongation elongation in in A.A. fumigatus fumigatus isis possible possible via via asexual asexual 3 reproductionreproduction when when exposed exposed to to VOR. VOR. After After five five evolutionary evolutionary cycles cycles the the TR TR34343/L98H/L98H genotype genotype first first emerged,emerged, and and subsequently subsequently became became the the dominant dominant genotype genotype with with higher higher VOR VOR and and POS POS MICs MICs than than thethe ancestor. ancestor. The The extra extra 34 34 bp bp promoter promoter repeat repeat resulted resulted in in a a1.5 1.5 times times higher higher expression expression of of cyp51Acyp51A 3 comparedcompared with with the the TR TR3434/L98H/L98H ancestor ancestor isolate. isolate. TR TR34343/L98H/L98H manifested manifested better better fitness fitness compared compared with with thethe ancestor ancestor TR TR3434/L98H/L98H when when grown grown in in the the presence presence of of VOR, VOR, in in terms terms of of mycelium mycelium growth growth rate rate and and conidiaconidia production. production. These These results results indicate indicate that that under under strong strong azole azole selection selection the the TR TR copy copy number number may may increaseincrease through through asexual asexual reproduction reproduction in in A.A. fumigatus fumigatus. .Thus Thus A.A. fumigatus fumigatus maymay employ employ both both the the sexual sexual andand asexual asexual cycle cycle to to expand expand promotor promotor repeat repeatss and and adapt adapt to to the the azole azole environment environment [4]. [4]. MechanisticallyMechanistically this this can can be be explained explained by by replication replication slippage slippage or or unequal unequal sister sister chromatid chromatid recombinationrecombination during during mitosis mitosis [37]. [37 ].Such Such processes processes may may be rare be rareper persingle single mitotic mitotic division division but are but relevantare relevant during during growth growth and andsporulation sporulation of A. of A.fumigatus fumigatus culturescultures that that involve involve numerous numerous mitotic mitotic divisions.divisions. Typically, Typically, after after one one week week of of growth, growth, a a colony colony of of A.A. fumigatus fumigatus startedstarted from from a asingle single conidium conidium maymay contain contain more more than than 10 109 9conidia.conidia. Apart Apart from from nuclear nuclear divisions divisions leading leading to to multinucleate multinucleate mycelium, mycelium, duringduring sporulation sporulation each each conidium conidium results results from from a amitotic mitotic division division in in a aphialide phialide cell cell of of a aconidiophore. conidiophore. DuringDuring each each of of these these numerous numerous mitotic mitotic divisions, divisions, there there is is a apossibility possibility of of replication replication slippage slippage and and unequalunequal sister sister chromatid chromatid recombination recombination that that may may lead lead to to an an increase increase in in the the TR TR number number (Figure (Figure 66).). Replication slippage may occur during DNA replication in the mitotic cell cycle. Particularly during the S stage of interphase (from N-2N, Figure6A), when DNA is replicated. Local misalignment of the template and nascent strands during a brief detachment of the DNA polymerase may lead to slippage and the TRs obtained are expanded [38,39] (Figure6A). After the replication stage, two sister chromatids are generated and at S/G2 phase unequal cross over may occur between the TRs of two sister chromatids, resulting in a daughter cell with three copies of 34 bp after chromatid segregation [40] (Figure6B).

J. Fungi 2020, 6, x FOR PEER REVIEW 8 of 12

244 4. Discussion 245 Our results indicate that CYP51A promoter elongation in A. fumigatus is possible via asexual 246 reproduction when exposed to VOR. After 5 evolutionary cycles the TR343/L98H genotype first 247 emerged, which subsequently became the dominant genotype. The extra 34 bp repeat resulted in a 248 1.5 times higher expression of CYP51A compared with the TR34/L98H ancestor isolate, and higher 249 VOR and POS MICs. From an evolutionary perspective, TR343/L98H manifested a better fitness 250 compared with the ancestor TR34/L98H when grown in the presence of VOR, in terms of mycelium 251 growth rate and spore production. These results indicate that the TR copy number may increase 252 through asexual reproduction in A. fumigatus, resulting in a more resistant azole phenotype. Thus A. 253 fumigatus may employ both the sexual and asexual cycle to expand promotor repeats and adapt to 254 the azole environment [4]. 255 Mechanistically this can be explained by replication slippage or unequal sister chromatid 256 recombination during mitosis [35]. Such processes may be rare per single mitotic division but are 257 relevant during growth and sporulation of A. fumigatus cultures that involve numerous mitotic 258 divisions. Typically, after one week of growth, a colony of A. fumigatus started from a single spore 259 may contain more than 109 spores. Apart from nuclear divisions leading to multinucleate mycelium, 260 during sporulation each spore results from a mitotic division in a phialide cell of a conidiophore. 261 During each of these numerous mitotic divisions, there is a possibility of replication slippage and 262 unequal sister chromatid recombination that may lead to an increase in the TR number (Figure 6). J. Fungi 2020, 6, 277 9 of 13 263 264

265 266 Figure 6. Possible mechanisms of extension from two to three 34 bp tandem repeats (TRs) in the 267 promoter region of the cyp51A gene during the mitotic cell cycle. (A) Replication slippage: when strand 268 slippageFigure occurs 6. during Possible DNA mechanisms replication of in extension the S-phase from of two the to mitotic three 34bp cell cycle, TRs in a DNAthe promoter strandwith region of the 269 the repeatcyp51A may loop gene out during resulting the mitotic in addition cell cycle. of the A. repeat Replication number slippage: [15]. (B when) Unequal strand sister slippage chromatid occurs during 270 recombination:DNA replication during the in S /theG2 S-phase phase, unequal of the mitotic crossing cell over cycle, may a DNA occur strand between with the the repeats repeat of may the loop out 271 sister chromatidsresulting resultingin addition in anof extrathe repeat copy ofnumber the repeat [15]. on B. oneUnequal chromatid. sister chromatid recombination: during 272 the S/G2 phase unequal crossing over may occur between the repeats of the sister chromatids resulting Our study has shown the acquisition of an extra TR in a clinical isolate, which is remarkably 273 in an extra copy of the repeat on one chromatid.3 different from a previous study, in which TR34 was recovered through exposure of recombinant KU80 KU80 274isolate akuB Replicationto tebuconazole slippage may under occur laboratory during conditionsDNA replication [7]. Recombinant in the mitotic isolate cell cycle.akuB Particularly 275had an increasedduring the mutation S stage frequency of interphase due to a(from defect N-2N, in the DNAFigure repair 6A), whichwhen mayDNA have is facilitatedreplicated. Local 3 276TR triplication.misalignment Our of results the template indicate and that nascent in-host strands selection during of azole-resistanta brief detachment TR34 of theA. DNA fumigatus polymerase 277isolates may is possible lead to and slippage may furtherand the limit TRs treatmentobtained are options. expanded Our [36,37] laboratory (Figure observations 6A). After support the replication 3 the recent report of recovery of genetically closely related TR34 /L98H and TR34/L98H isolates from a cystic fibrosis patient. The patient had been treated with VOR and POS (MC Arendrup, personal 3 communication), which could have selected TR34 /L98H harboring A. fumigatus strains in the lung [9]. 3 However, the TR34/L98H patient isolate was recovered after the isolates harboring TR34 /L98H and no 3 ancestral isolate was available [9], therefore it is unclear whether the TR34 -variant evolved in the host 3 under VOR and POS selection from a TR34 or vice versa. Our study shows that the TR34 -variant can 3 evolve from a TR34 ancestor through asexual reproduction. The TR34 /L98H patient isolates showed no phenotypic change regarding VOR and POS MICs compared to the TR34/L98H patient isolate [9]. 3 However, the TR34 /L98H isolate we obtained through exposure to VOR showed higher VOR and POS 3 MICs compared with the ancestor. The TR34 /L98H isolate harbored three SNPs compared with the TR34/L98H ancestor, one of which (AtrR) was found to be a transcription factor reported to play a role in regulating cyp51A expression. This mutation has been confirmed to contribute to the high VOR and POS resistance phenotype as analysis of an isolate obtained from the 8th cycle showed high resistance (VOR MIC > 16 µg/mL and POS MIC >8 µg/mL) in the TR34/L98H background. The cyp51A expression level of this isolate was higher compared with the TR34/L98H ancestor (Supplemental Figure S2), 3 3 but lower than final fixed TR34 /L98H, which might have contributed to a fitness benefit of TR34 /L98H compared to the Transient TR34/L98H under VOR selection pressure. Understanding the role of AtrR in the VOR resistance phenotype was beyond the scope of our research, and further experiments would be required. As antifungal drug and other stressor exposures in the lung are uncontrolled [41], whole genome sequencing would be required to gain more insights into the genetic background of J. Fungi 2020, 6, 277 10 of 13 the patient isolates in relation to the observed triazole phenotypes. Our observations are in line with observations of in-host selection of triazole resistance mutations, including TR120 from an ancestor isolate without a TR [5]. Nevertheless, despite TR34/L98H being the most prevalent azole-resistance 3 mechanism, an isolate with TR34 has been reported only once indicating that specific conditions 3 4 might be required for its selection. Isolates with three copies (TR46 ) and four copies (TR46 ) have been found and acquisition of an extra TR was demonstrated in two environment isolates carrying TR46/Y121F/T289A through unequal crossover during sexual reproduction [4]. 3 There are various possible reasons for the low frequency of TR34 isolates. There may be a fitness 3 cost of TR34 isolates in the azole-free environment, which was also observed in the isolate that we analyzed. The consequence of a fitness cost is that the genotype will disappear in competition with WT isolates unless it acquires compensatory mutations that restore its fitness. In our experiment, 3 TR34 /L98H is likely not to have undergone compensatory evolution to overcome its fitness cost in the azole-free environment. Alternatively, other factors related to azole selection pressure (type of azole, 3 concentration, duration) may be important. Furthermore, TR34 may not be stable in the long term. Although TR-mediated azole-resistance mutations are generally associated with environmental resistance selection and single resistance mutations with in-host resistance selection, there is increasing awareness that the correlation between resistance-mutation characteristics and route of selection is artificial [10]. Indeed, A. fumigatus harboring single resistance mutations has been recovered from the environment and from azole-naïve patients, which is consistent with environmental resistance rather than in-host selection [10]. Alternatively, TR120 was shown to develop through in-host 3 selection [5]. Our observation of TR34 selection through exposure to a medical triazole underscores that characteristics of the fungus and the azole environment are the critical factors that determine which genotype will eventually evolve. In this study, we provide evidence for selection of TR-variants in TR34 isolates through asexual reproduction during VOR exposure, adding to the strategies A. fumigatus can employ to adapt to the azole environment. Future work should further characterize factors that facilitate selection of TR-variants including genetic background of the isolate, concentration and chemical properties of the azole in order to enable development of preventive interventions.

Supplementary Materials: The Supplementary data will be available online at http://www.mdpi.com/2309-608X/ 6/4/277/s1. Figure S1: Isolation and classification of nitrate non-utilising mutants based on resistance to chlorate. 3 Figure S2: Expression of cyp51A in the TR34 /L98H isolate, compared with ancestry isolates TR34/L98H and Transient TR34/L98H, and a WT control isolate. Table S1: The characteristics of chlorate resistant nia and cnx mutants for their ability to utilise urea, hypoxanthine, nitrite and nitrate as sole N-source. Table S2: The SNPs 3 calling between the ancestor TR34/L98H (V30-40) and evolved isolate TR34 /L98H. Table S3: In vitro azole 3 susceptibility profiles of TR34/L98H, Transient TR34/L98H and TR34 /L98H Table S4: The gene mutations in 3 TR34 /L98H and Transient TR34/L98H isolate compared with the TR34/L98H(V30-40) ancestor. Author Contributions: Conceptualization, Methodology, Validation, Formal Analysis, Investigation, Data Curation J.Z. (Jianhua Zhang), J.Z. (Jan Zoll), T.E., J.v.d.H.; Resources, P.E.V.; Draft Preparation, J.Z. (Jianhua Zhang), P.E.V., A.J.M.D., J.v.d.H., J.Z. (Jan Zoll); Writing—Review and Editing, J.Z. (Jianhua Zhang), P.E.V., A.J.M.D., J.v.d.H., J.Z. (Jan Zoll). All authors have read and agreed to the published version of the manuscript. Funding: We gratefully acknowledge funding from the China Scholarship Council to J.Z. (Jan Zoll). Acknowledgments: We thank the technician MarlouTehupeiory-Kooreman from the Department of Medical Microbiology, Radboud University Medical Centre for MIC test and Marc F.P.M. Maas for the illustration of Figure6 . Conflicts of Interest: The authors declare no conflict of interest. J. Fungi 2020, 6, 277 11 of 13

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