Medical Mycology, 2017, 0, 1–14doi: 10.1093/mmy/myx081

Advance Access Publication Date: 0 2017Original Article

Original Article

Transcriptional profile of the human skin

pathogenic fungus Mucor irregularis in response

to low oxygen

Wenqi Xu1, Jingwen Peng1, Dongmei Li2, Clement K. M. Tsui3,

Zhimin Long4, Qiong Wang1, Huan Mei1 and Weida Liu1,∗

1Department of Mycology, Institute of Dermatology, Chinese Academy of Medical Sciences and PekingUnion Medical College, Nanjing 210042, Jiangsu, People’s Republic of China, 2Department of Microbi-ology and Immunology, Georgetown University Medical Center, Washington, DC 20057, USA, 3Divisionof Infectious Diseases, University of British Columbia, Vancouver, BC V6H 3Z6, Canada and 4Demo Lab,Shanghai AB Sciex Analytical Instrument Trading Co., Ltd, IBP, Shanghai, 200335, People’s Republic ofChina∗To whom correspondence should be addressed. Dr. Weida Liu, No. 12 Jiangwangmiao Street, Nangjing 210042, Jiangsu,People’s Republic of China. Tel: +86 25 85470580; Fax: +86 25 85414477;E-mail: liumyco@hotmail.com

Received 18 January 2017; Revised 28 April 2017; Accepted 25 August 2017; Editorial Decision 10 May 2017

Abstract

Mucormycosis is one of the most invasive mycosis and has caused global concern in pub-lic health. Cutaneous mucormycosis caused by Mucor irregularis (formerly Rhizomucorvariabilis) is an emerging disease in China. To survive in the human body, M. irregularismust overcome the hypoxic (low oxygen) host microenvironment. However, the ex-act molecular mechanism of its pathogenicity and adaptation to low oxygen stressenvironment is relatively unexplored. In this study, we used Illumina HiSeq technol-ogy (RNA-Seq) to determine and compare the transcriptome profile of M. irregularisCBS103.93 under normal growth condition and hypoxic stress. Our analyses demon-strated a series of genes involved in TCA, glyoxylate cycle, pentose phosphate pathway,and GABA shunt were down-regulated under hypoxic condition, while certain genes inthe lipid/fatty acid metabolism and endocytosis were up-regulated, indicating that lipidmetabolism was more active under hypoxia. Comparing the data with other importanthuman pathogenic fungi such as Aspergillus spp., we found that the gene expressionpattern and metabolism in responses to hypoxia in M. irregularis were unique and differ-ent. We proposed that these metabolic changes can represent a species-specific hypoxicadaptation in M. irregularis, and we hypothesized that M. irregularis could use the intra-lipid pool and lipid secreted in the infection region, as an extracellular nutrient source tosupport its hypoxic growth. Characterizing the significant differential gene expression inthis species could be beneficial to uncover their role in hypoxia adaptation and fungal

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pathogenesis and further facilitate the development of novel targets in disease diagnosisand treatment against mucormycosis.

Key words: Mucor irregularis, cutaneous mucormycosis, hypoxic response, lipid/fatty acid metabolism, endocytosis.

Introduction

Mucormycosis, a fungal disease typically occurs in sinuses,lungs and epidermal tissues, is spreading rapidly and ex-hibiting high rates of morbidity and mortality. The incidentrate of mucormycosis has increased by 7.4% per year (from0.7 to 1.2 cases/million persons) in the last decade.1 Mu-cormycosis is most commonly caused by members of Mu-corales. This fungal infection can be transmitted by sporesin the air, ingestion, or direct contact with injured skin.2,3

Based on clinical presentations, mucormycosis can be di-vided into two types—invasive and cutaneous mucormyco-sis. The former is a severe life-threating fungal infection,commonly prevalent in individuals with impaired immu-nity, while the latter has mostly a milder condition whichcan manifest at even immunocompetent patients.

Mucor irregularis (renamed from Rhizomucor vari-abilis4) was first isolated from a skin lesion, which had beenpresented as a primary cutaneous infection in the hand ofa Chinese patient in 1991.5 Since then, approximately 30cases of primary cutaneous infection caused by M. irreg-ularis have been documented,6–8 of which 23 cases werefrom China. In contrast to the angioinvasive mucormycosis(commonly caused by Rhizopus oryzae), infection causedby M. irregularis presents as a chronic disease, tending tobe limited to dermal and subcutaneous tissues without vas-cular invasion. Most patients with M. irregularis infectionswere immunocompetent or at least had no apparent im-munodeficiency, but some patients were badly disfigureddue to misdiagnoses in early stage.9–13

M. irregularis is a unique pathogen in its temperaturetolerance among the Mucorales. In general, Mucorales spp.are highly thermotolerant or even thermophilic, with max-imum growth temperatures up to 37–45◦C. However, M.irregularis cannot grow above 37◦C, which may be one ofthe major reasons for its inability to cause deep-tissue infec-tions.14 Therefore, this pathogen may have special genetictraits for environmental adaptation that differ from otherMucorales species for high temperature tolerance. To date,most studies on mucormycosis have focused on species thatcause invasive infections,12,13,15 but the knowledge for dis-ease management remains limited, especially the pathogen-esis/pathophysiology of M. irregularis is poorly understood.

To survive in a human host, the pathogenic fungi need totolerate and overcome in vivo micro-environmental stressconditions. One of the stresses is hypoxia (low oxygen)condition. It is well established that the oxygen levels inmost human tissues are considerably below atmospheric

level (20.9%); the oxygen concentration varies across dif-ferent tissues in the body, and ranging from 2.5% inthe kidney to 9% in the lung. Furthermore, oxygen par-tial pressure of the skin is only 41 mmHg (∼6% oxygenconcentration).16,17 Since oxygen is a critical componentto many essential biochemical processes, the ability to sur-vive under hypoxic conditions has been hypothesized tobe a necessary virulence attribute of human pathogenicfungi.18–21 To understand how human pathogenic fungiadapt and survive in low oxygen conditions, several inves-tigations have examined the global fungal transcriptomeresponses to hypoxia in Aspergillus nidulans, A. fumiga-tus, Candida albicans, and Cryptococcus neoformans.22–25

The results demonstrated that, pathogenic fungi possessdifferent mechanisms to maintain energy in order to sur-vive and grow in oxygen-limited environments. However,some transcriptome changes and gene regulation patternsare common and can be found across multiple species,such as the up-regulation of genes involved in glycolysis,steroid, and secondary metabolite metabolism, as well asthe down-regulation of genes responsible for ribosomal andpurine/pyrimidine biosynthesis.22–25

In contrast to these invasive pathogens, far less is knownabout hypoxia responses of M. irregularis, which is con-fined to causing skin lesions. For better understanding of itsadaptation in reduced oxygen level condition and its conse-quential significance in the pathogenicity of mucormycosisduring skin infection, we performed transcriptome sequenc-ing (RNA-seq) to examine the transcriptional responses ofM. irregularis to hypoxia. The RNA-Seq data of M. ir-regularis (CBS103.93) under atmospheric environment wascompared to the one under 6% O2 concentration. The lat-ter was used to mimic the skin oxygen concentration.16,17

The main goal of the study is to provide an overview ofgenes that are up- or down- regulated by low level of oxy-gen stress, particularly on genes involved in pathogenesisand hypoxia pathway that may distinguish it from otherclinical important pathogenic fungi.

Methods

Growth comparison under normoxic and hypoxicatmospheres

Mucor irregularis standard strain CBS103.93 was main-tained at the National Fungi Strain Reserve Center (Nan-jing, China). Since the fungus causes skin infection, it was

Xu et al. 3

cultured on solid medium instead of liquid broth, which canbe used for blood or blood vessel infection fungus. The fun-gus was initially incubated on Malt Extract Agar (OXOID,Basingstoke, UK) at 27◦C for 5 days in normoxic condition(20.9% O2), then the mycelia was homogenized and filteredthrough filter paper with average pore size of 40 µm. Theconcentration of spores (conidia) was determined with ahemocytometer under a microscope, and 5 µl conidial sus-pensions adjusted to 1 × 103 conidia/µl were spotted ontothe center of the plates. Plates were pre-incubated at 27◦Cunder normoxic condition for 12 hours, then either keptnormoxic (20.9% O2) or shifted to a hypoxia chamber(HuaXi Elctronics Technetronic, Changsha, China) with6% O2 and incubated until the mycelia covered the entirecultivation dish. The diameter of the colony was measuredand averaged from two separate experiments.

To compare the growth of M. irregularis on mediumwith or without triglycerides in normoxic and hypoxic con-ditions, 5 µl conidial suspensions were spotted onto thecenter of the plates containing 70% MEA or 70% MEAsupplemented with 7.5% (v/v) triglycerides, respectively,followed by incubation at 27◦C in a hypoxic atmosphere(6% O2) for 3 days.

RNA extraction and RNA-Seq library sequencing

Six plates of M. irregularis were pre-incubated for 3 days at27◦C under normoxic condition. Of these, three plates werethen incubated in hypoxic condition (6% O2), while the re-maining three plates were still kept under normoxic condi-tion. After 6 hours of incubation, all the repeat plates werecollected, and the mycelium was ground to fine powder fortotal RNA isolation using Qiagen RNeasy Plant Mini kit(Qiagen, Hilden, Germany). Total RNA concentration wasquantified with an Ultrospec 2100 Pro (Amersham Phar-macia, Little Chalfont, England).

Messenger RNA (mRNA) was purified by polyA se-lection method using oligo(dT) beads and was separatedin fragmentation buffer to elute 100-bp to 400-bp frag-ments. Total mRNAs were then reverse-transcribed intocomplementary DNAs (cDNAs) for library construction.RNA-seq transcriptome library was prepared accordingto a TruSeqTM RNA sample preparation kit from Illu-mina Technology (San Diego, California) using 5 µg ofRNA. Afterward, double-stranded cDNA was synthesizedusing a SuperScript double-stranded cDNA synthesis kit(Invitrogen, Carlsbad, California) with random hexamerprimers (Illumina, San Diego, California). Then the syn-thesized cDNA was subjected to end-repair, phosphoryla-tion and ‘A’ base addition according to Illumina’s libraryconstruction protocol. Libraries were size selected for tar-

geting fragments of 200–300 bp on 2% Low Range Ul-tra Agarose followed by 15 cycles of polymerase chainreaction (PCR) amplification using Phusion DNA poly-merase (NEB, Ipswich, Massachusetts). After quantificationby TBS-380 mini-fluorometer (Promega, Madison, Wiscon-sin), the paired-end RNA-seq sequencing library was se-quenced by Illumina HiSeq platform (2 × 150 bp readlength) at Biozeron Biotech Company (Shanghai, China).The sequencing of M. irregularis under normoxia and hy-poxia were performed, respectively.

Transcriptome data processing and assembly

After Illumina sequencing, the raw reads were in FASTQformat. The adapter sequences and reads of poor qualitywere trimmed (ambiguous bases and quality value ≤5).Reads obtained for M. irregularis under normoxia and hy-poxia conditions were assembled de novo, respectively byTrinity (http://trinityrnaseq.sourceforge.net/) using the de-fault parameters (–min_contig length 200 –min kmer cov1 –max reads per graph 20000 –group pairs distance 500–path reinforcement distance 75), which had been reportedin other publications.26,27 The contigs and unigenes of lessthan 200 bp were discarded due to low annotation rate.27,28

The raw data of M. irregularis under normoxia and hypoxiawere deposited in NCBI as BioProject PRJNA347489, inwhich SRA483033, SRA483036, and SRA483037 were de-rived from normoxia, and SRA483176, SRA483180, andSRA483179 from hypoxic condition. The assembled se-quencing data were deposited in the NCBI-TSR database(TSR: GFBC00000000).

Functional annotation

Functional annotations were performed by sequencecomparison to public databases including the NCBInonredundant protein database (NR database,http://www.ncbi.nlm.nih.gov), Swiss-Prot database(http://www.expasy.ch/sprot), clusters of orthologousgroups for eukaryotic complete genomes database (KOG)(ftp://ftp.ncbi.nih.gov/pub/COG/KOG/kyva), and Kyotoencyclopedia of genes and genomes (KEGG) pathwaydatabase (http://www.genome.jp/kegg/) using BLASTXalignment with an E-value of 1.0E−5, respectively. Inaddition, Blast2GO program and BlastX29 were also usedto perform GO annotation of unigenes, and then WEGO30

software was used to perform GO classification, assigningbiological processes, molecular functions, and cellularcomponents.

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Identification of differentially expressed genes

To better interpret the expression levels of each uni-gene, FPKM (Fragments Per kb per Million reads)31 wasused to eliminate the influence of differences of genelengths and sequencing depth. Then, the adjusted expres-sion level can be used for direct comparison of differ-ences between samples. The false discovery rate (FDR)method was also introduced to determine the thresholdP-value in multiple tests using Cufflink software pack-age. An FDR ≤ 0.05 was used as the threshold to de-termine the significance of gene expression differences be-tween the two tested temperatures.32 GO functional en-richment and KEGG pathway analysis were carried outby Goatools (https://github.com/tanghaibao/Goatools) andKOBAS (http://kobas.cbi.pku.edu.cn/home.do).33

Confirmation of gene transcripts usingquantitative RT-PCR

The total RNA used for RNA-Seq was also employed toverify the expression levels of selected target genes. First-strand cDNA was synthesized from 2 µg DNase-treatedtotal RNA using the HiScript R© Q RT SuperMix for qPCR(+gDNA wiper) (Vazyme, Nanjing, China). Quantitativereal time polymerase chain reaction (qPCR) amplificationsin 20 µl volumes, containing 10 µl SYBR Green MasterMix (Vazyme, Nanjing, China), 20 ng template, and 4pmol of each primer, were performed using the StratageneMx3000p Real-Time PCR instrument (Agilent Technolo-gies, Hansen, Connecticut). The temperature profile was95◦C for 5 min, 40 cycles of 95◦C for 15 s, 55◦C for25 s, and 72◦C for 25 s. The reaction was conducted intriplicate. The threshold values for each target gene werenormalized using the glyceraldehyde-3-phosphate dehydro-genase gene (GAPDH). The relative expression was esti-mated by employing the 2−��CT method.34 Seven predictedgenes involved in glycolysis/gluconeogenesis, GABA shun,glycerolipid metabolism, fatty acid metabolism pathwayswere selected according to the RNA-seq data. The RT-qPCR primers were designed with MacVector 11 (Accelrys,San Diego, California) (Table S1).

Results

Comparison of growth status under hypxiaand normoxia

The effect of hypoxia on the growth of pathogenic fungusM. irregularis was tested under 6% O2 conditions in anoxygen-controlled chamber, which was mimicking the levelof oxygen in human skin. After incubation for 3 days, the

Figure 1. Growth of Mucor irregularis CBS103.93 in normoxic and hy-poxic culture. A total of 5 × 103 conidia was spot inoculated onto MEAplates initially incubated in air at 27◦C. After 12 h, plates were eitherkept normoxic (20.9% O2) or shifted to a hypoxic condition (6% O2) andincubated for a further 3 days. B. The diameters of the colonies weremeasured over 78 h and are expressed in cm. Values represent the meanof three biological replicates. Bar = 2 cm.

sizes of M. irregularis colonies on MEA plates were alwayssmaller than those kept in the normoxic incubator (Fig. 1A).The average of colony diameters for M. irregularis myceliaunder hypoxia condition was 5.3 ± 0.2 cm after 72 hours,while the colonies were 7.6 ± 0.3 cm in diameter undernormoxic growth condition (Fig. 1B).

Transcriptomes of M. irregularis under normoxiaand hypoxia

To better understand the transcriptomic profile of M. ir-regularis in low oxygen condition, total RNAs were ex-tracted from two conditions in triplicates. A total of 126and 205 million reads were generated from the hypoxicand normoxic samples, respectively (Table S2), from whichvariable sequences with Q30 bases > 92 % and G+C%about 44 % were assigned to 30,761 consensus unigenes(Table S2). The mean length of these unigenes was 1247bp, with N50 of 1761bp (Table S3). The variance amongthe three biological replicates from normoxic and hy-poxic conditions was evaluated using the scatter plots ofgene expression (Fig. S1). The correlation among the three

Xu et al. 5

Figure 2. Sequence similarity and species distribution of the top BLASTxhits against the NR database for each unigene.

replicates in each tested condition was significantly higherthan the results between the two conditions for any partic-ular given replicate, comfirming the data were reliable fordownstream analysis.

Functional annotation of the unigenes

A total of 26,693 (86.76%) genes had positive hits at leastonce in the NR, the Swiss-Prot protein, KEGG or KOGdatabase, in which 85.4% of the sequences were homol-ogous to the gene sequences listed in the NR databasewhen the e-value frequency distribution has fixed signif-icant hits as 1.0E−60 (Fig. 2A). However, the blast hitsagainst SwissProt, KEGG and KOG databases only re-sulted in 58.30% (17,933 transcripts), 38.73% (11,915transcripts) and 8.44% (2597 transcripts) of positive hitsfrom genomic sequences, respectively (Table S3). The clos-est related species to M. irregularis in the NR database wasRhizopus delemar with a 36.98% identity between the twospecies, and only 6802 (22.11%) unigenes could be anno-tated, by searching against the NR database (Fig. 2B), whilethe remaining unigenes were either hypothetical genes orfunctionally uncharacterized.

Based on the NR annotation and gene ontologyclassification, 21085 unigenes were assigned with GOterms. The GO-annotated unigenes were distributed in 45

Figure 3. GO annotations of nonredundant consensus sequences. Best hits were aligned to the GO database, and 21085 unigenes were assigned to atleast one GO term. Most consensus sequences were grouped into three major functional categories, namely biological process, cellular component,and molecular function.

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Figure 4. Histogram of clusters of orthologous groups (KOG) classification. All unigenes were aligned to KOG database for prediction and classificationbased on possible functions.

categories in terms of biological processes, cellular compo-nents, and molecular functions clusters (Fig. 3, Table S4).For the biological processes category, the genes related tometabolic processes (7585, accounting for 35.97%) weredominant, followed by those related to cellular processes(6617, 31.38%), then single-organism processes (3744,17.76%) and biological regulation (2218, 10.52%) (Fig. 3).Among the cellular components category, cell part andcell (both 4594, 21.79%) were the dominant groups, fol-lowed by organelles (2555, 12.12%) and membrane (2335,11.07%) (Fig. 3). In terms of molecular functions, 48.35%(10195) of the unigenes was assigned to catalytic activ-ity, followed by in descending order 42.86% for binding(9038), 5.94% for transporter activity (1253), and 3.11%for molecular function regulator (655) (Fig. 3).

Based on the KOG database, the putative proteins werefunctionally classified into 25 molecular families such ascellular structure, biochemistry metabolism, molecular pro-cessing, and signal transduction (Fig. 4). Within these broadcategories, we found a few notable groups, such as trans-lation, ribosomal structure, and biogenesis (accounting for334, 10.14%), followed by posttranslational modification,protein turnover and chaperones at 10.00% (329), signaltransduction mechanisms at 7.69% (253), and transcrip-tion bringing up the rear at 5.65% (186). Apart from thelargest group of functionally uncharacterized genes (337,10.24%), the remaining genes were involved either in nu-clear structure, cell motility, extracellular structure, or de-fense mechanisms (5, 3, 3, and 1 unigenes, respectively)(Fig. 4).

To further explore the molecular interaction betweengenes, the KEGG database was also used to predicatethe potential pathways in which these genes might be in-volved. Among the 30761 annotated genes, 11,915 were

clustered to 34 processes/pathways, including signal trans-duction, translation, carbohydrate metabolism, endocrinefunctions, and other biosynthetic pathways (data notshown).

Differentially expressed unigenes

The differentiation of gene expression between normoxiaand hypoxia was evaluated by FPKM with FDR correc-tions (P < .05); genes showing at least twofold changes inexpression were considered to be differentially expressed. Atotal of 1112 transcripts (Table S5) had altered expressionsignificantly in response to different oxygen conditions, ofwhich 531 transcripts were significantly up-regulated and581 transcripts were down-regulated in response to hypoxia(twofold changes cut off, Table S5).

The up-regulated genes were associated with theregulation of gene expression (GO:0010468), regula-tion of metabolic process (GO:0080090, GO:0019222,and GO:0031323), antioxidant activity (GO:0051920,GO:0016209, and GO:0016684), transcription factoractivity (GO:0003700, GO:0006355, and GO:0001071),energy metabolism related process (GO:0060590,GO:0004090, and GO:0003959) and mitochondrion(GO:0044429) (Table 1). The down-regulated geneswere involved in hydrolases activity (GO:0016798,GO:0004553, and GO:0016811), defense response(GO:0006952 and GO:0009605), carbohydratemetabolism (GO:0030246, GO:0005975, GO:0016810,GO:0015926, GO:0004410, and GO:0004339) andcalcium ion binding processes (GO:0005509) (Table 1).

The biological functions of the 1112 differentially ex-pressed genes (DEGs) were also evaluated in the KEGG

Xu et al. 7

Table 1. Significant GO terms in response to hypoxia.

GO term counts

GO IDs Terms Observed Annotated Expected P-value

<Up-regulated in hypoxia>

GO:0003700 transcription factor activity, sequence-specific DNAbinding

23 615 10.62 .000743

GO:0004090 carbonyl reductase (NADPH) activity 2 7 0.12 .0059GO:0006355 regulation of transcription, DNA-templated 45 1241 21.42 3.29E−06

GO:0010468 regulation of gene expression 45 1207 20.84 1.43E−06

GO:0016209 antioxidant activity 5 73 1.26 .00863GO:0016684 oxidoreductase activity, acting on peroxide as acceptor 4 48 0.83 .00938GO:0019222 regulation of metabolic process 54 1825 31.50 1.25E−04

GO:0031323 regulation of cellular metabolic process 50 1575 27.19 3.76E−05

GO:0044429 mitochondrial part 7 176 3.04 .0337GO:0051920 peroxiredoxin activity 3 17 0.29 .0029GO:0060590 ATPase regulator activity 2 6 0.10 .00426GO:0080090 regulation of primary metabolic process 52 1591 27.46 1.08E−05

<Down-regulated in hypoxia>

GO:0004339 glucan 1,4-alpha-glucosidase activity 6 81 1.53 .00434GO:0004410 homocitrate synthase activity 2 5 0.09 .00343GO:0004553 hydrolase activity, hydrolyzing O-glycosyl compounds 23 427 8.06 8.21E−06

GO:0005509 calcium ion binding 12 240 4.53 2.18E−03

GO:0005975 carbohydrate metabolic process 22 596 11.26 3.26E−03

GO:0006952 defense response 6 37 0.70 6.25E−05

GO:0009605 response to external stimulus 4 21 0.40 5.84E−04

GO:0015926 glucosidase activity 8 120 2.27 2.04E−03

GO:0016798 hydrolase activity, acting on glycosyl bonds 24 438 8.27 3.93E−06

GO:0016810 hydrolase activity, acting on carbon-nitrogen (but notpeptide) bonds

11 232 4.38 .00488

GO:0016811 hydrolase activity, acting on carbon-nitrogen (but notpeptide) bonds, in linear amides

5 61 1.15 .00589

GO:0030246 carbohydrate binding 6 62 1.17 1.11E−03

database by pathway enrichment analysis. The path-ways that were significantly enriched included starch andsucrose metabolism (ko00500), glycerolipid metabolism(ko00561), glycerophospholipid metabolism (ko00564),pyruvate metabolism (ko00620), amino sugar and nu-cleotide sugar metabolism (ko00520), oxidative phospho-rylation (ko00190), and carbon metabolism (ko04141).Also some functional categories such as carbon metabolism,fatty acid/lipid metabolism and endocytosis were observedduring comparison (Fig. 5). Many of the findings reportedin M. irregularis (Fig. 6, Table S6) were different fromthat reported in other human pathogenic fungi (Table 2).For example, the genes involved in glycolysis, fatty acidmetabolism, oxidative phosphorylation, steroid biosynthe-sis, and pentose phosphate pathway were often found tobe up-regulated in other fungi such as Cryptococcus neo-formans, Candida albicans, and Aspergillus nidulans underhypoxic conditions.35–39

Reduced ability to degrade the carbohydrateunder hypoxia

In M. irregularis many transcripts involved in glycolysiswere reduced in response to hypoxia. For example,acetyl-coenzyme A synthetase (Acs, GFBC01001446), oneof the most highly reduced transcripts, two fructose-bisphosphatase aldolases (FbaA, GFBC01011669and GFBC01021420), which are involved into theearly steps of converting fructose 1,6-phosphate toglyceraldehyde 3-phosphate, pyruvate kinase (Pyk,GFBC01029099) along with aldehyde dehydrogenases(Aldh, GFBC01005625), which converts acetalde-hyde to acetate for central carbohydrate and lipidmetabolism tended to be transcriptionally reduced(Fig. 5, 6).

Of the seven genes involved in starch and sucrosemetabolism, six decreased in the transcriptional levelsafter a shift to hypoxic growth condition. Somewhat

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Figure 5. Hypoxia decreased levels of expression for genes involved in carbon metabolism, oxidative phosphorylation, and increased the expressionof genes involved in fatty acid/lipid metabolism and endocytosis processes. Heat map showing the cluster analysis of genes differentially expressedprior to hypoxia (0 hour) and in hypoxia (6 hours) of Mucor irregulars CBS103.93.

surprisingly, only one exception, the trehalose 6-phosphatephosphatase (TPS, GFBC01001384), which catalyzes thedephosphorylation of trehalose 6-phosphate to form tre-halose, was up-regulated.

Altered expression in steroid biosynthesisand GABA shunt pathway genes

In response to hypoxia, the steroid biosynthesis pathway ofM. irregularis was not significantly affected, only two sterolreductase genes egr4 (GFBC01007963, GFBC01007636)and a cytochrome P450 (GFBC01017808) reduced tran-scription, while the transcription level of sterol regu-latory element-binding protein SREBP was not signifi-cantly altered. These results suggested that the steroidbiosynthesis of M. irregularis appeared to not be af-fected or partially decreased in response to hypoxia, whichwas incongruent to the patterns of many other fungi(Table 2).

In addition, seven differentially expressed genes wereidentified to be involved in the GABA shunt ofM. irregularis, including two glutamate decarboxylase(Gad, GFBC01008155 and GFBC01027882), one 4-aminobutyrate transaminase (GatA, GFBC01030508), andfour vesicular inhibitory amino acid transporter (VGAT).We found that the expression of Gad and GatA was re-duced, but VGATs were up-regulated in response to hy-poxia. In contrast to the fungi such as Aspergillus nidulansand C. neoformans in which the genes of the GABA shuntwere up-regulated for energy metabolism, the inconsistencyof these genes alternation elicited that the GABA shunt wasinactive in M. irregularis under low oxygen (Table 2).

Bolstered catabolic potential in lipid metabolismand activation in endocytosis

The hypoxic exposure also led to marked changes ingene expression involved in lipid metabolism. We found

Xu et al. 9

Figure 6. Overview of metabolic responses of Mucor irregularis in hypoxia. The figure showed the major changes in M. irregularis metabolism duringhypoxia for 6 h. Up-regulated genes are highlighted in black, while down-regulated genes are colored in gray. Genes are abbreviated in capital letters:PYK, pyruvate kinase; FbaA, Fructose-bisphosphatase aldolase; ACS, Acetyl-coenzyme A synthetase; ALDH, aldehyde dehydrogenase; ADH, alcoholdehydrogenase (NADP+); PckA, phosphoenolpyruvate carboxykinase; GAD, glutamate decarboxylase; ACOX, acyl-CoA oxidase]; ACSL, long chainfatty acyl-CoA synthetas; TAG lipase, Triacylglycerol lipase.

Table 2. Comparison of metabolic pathways (predicted by KEGG) involved in hypoxic adaptation in Mucor irregularis and six

other fungi.

M. irregularis A. nidulans A. niger A. oryzae A. fumigatus C. albicans C. neoformans

Glycolysis ↓ ↑ ↑ ↑ ↑ ↑ −Gluconeogenesis (PEP carboxykinase) ↑ ↓ NR ↑ NR NR NRPentose phosphate pathway ↓ ↑ ↑ NR ↑ ↑ NROxidative phosphorylation ↓ NR NR NR ↓ ↓ NRβ-oxidation ↑ ↑ ↑ NR NR ↑ ↑Lipid/Fatty acid metabolism ↑ ↑ ↑ NR ↓ ↑ ↑GABA shunt/ergosterol biosynthesis ↓ ↑ ↑ ↑ ↑ ↑ ↑Endocytosis ↑ NR NR NR NR NR NR

↑, up-regulated;↓, down- regulated;−, a general lack of changes;NR, Not reported;References: Aspergillus nidulans,25,37,65 Aspergillus niger,54 Aspergillus oryzae,25 Aspergillus fumigatus,18,19,48 Candida albicans,24,39 Cryptococcus neofor-mans.23,36

that in M. irregulairs, the triacylglycerol (TAG) lipase(GFBC01029180) that degrades triacylglycerol into fattyacid, and a member of the cytosolic phospholipase A2group IV family (GFBC01029364), which catalyzes phos-phatidylcholine to acyl-glycero-3-phosphocholine for cel-lular energy, were up-regulated under hypoxia (Fig. 5, 6).In addition, both the long chain fatty acyl-CoA synthase(ACSL, GFBC01014149) and acyl-CoA oxidase (ACOX,GFBC01011826) of M. irregulairs, which were both in-volved in β-oxidation, were also up-regulated. These resultsled us to speculate that the hypoxic M. irregularis might usethe intracellular lipid pool as energy source.

Along with this speculation, M. irregularis would needan approach to maintain the lipid homeostasis under hy-poxic growth conditions. Previous studies showed thatendocytosis was important for bringing nutrients intothe cell to maintain lipids and protein homeostasis inthe cell;40,41 hence, analyses on endocytosis were con-ducted in M. irregularis. Eight genes associated withendocytosis were differentially expressed in hypoxia,of which five genes (GFBC01019239, GFBC01021838,GFBC01028777 [HSP71-like], GFBC01016923 [HSP70],and GFBC01029894 [HSP71-like]) were up-regulated un-der hypoxic condition. It was noteworthy that such

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Figure 7. Gene expression for validation in (A) qRT-PCR and (B) RNA-seq assays in normoxia and hypoxia exposed strains. The data representthe mean ± standard deviation from three biological replicates. ACS(GFBC01001446), ALDH (GFBC01005625), GAD (GFBC01008155), SREBP(GFBC01022405), TAG lipase (GFBC01029180), ACOX (GFBC01001611),ACSL (GFBC01014149).

induction of endocytosis process including three HSP genesunder hypoxia has not been reported in other pathogenicfungi such as A. nidulans (Table 2).

Validation of RNA-seq findings with real-time PCR

The RNA-seq results were validated using qRT-PCR by us-ing the same biological RNA samples. Seven target genesfrom different functional categories that were verified asup- or down-regulated through qRT-PCR as an indepen-dent measure of differential gene expression (Fig. 7A). Allthe genes used in validation showed the same pattern ofexpression as that of the RNA-seq results (Fig. 7B) demon-strating the reliability of the RNA seq data.

Discussion

The comparison of mycelial diameter between normoxiaand hypoxia clearly demonstrated that the reduced oxygensupply was a growth limiting factor in M. irregularis. Sim-ilarly, M. plumbeus, another member of Mucorales, alsohad slower growth in hypoxia than in atmospheric oxy-

gen.42 Almost all clinical cases caused by M. irregularis werechronic cutaneous infection, except the case that reported afacial lesion with an extraordinary pulmonary infection.13

Xia et al. observed that the patient’s symptoms subsided insummer and were aggravated in winter, which was differentfrom most superficial fungal infection in human, suggestingthat different optimal temperatures for fungal growth maybe responsible for this behavior. Apart from this explana-tion, since the oxygen content is the highest in the lung ofhuman body, which could be a favorable condition for itslung infection and invasion rather than other inner organs.Thus, we hypothesized that the growth of M. irregulariswas retarded during the invasion of human skin in hypoxicenvironment, having the ability to overcome the low oxygencondition is essential establishment of the skin infection.

To understand the molecular mechanisms of hy-poxia adaptation in this pathogenic fungi, we inves-tigated the transcriptome profiles of M. irregulairsusing Illumina RNA-seq. Many transcripts related toglycolysis were reduced in response to hypoxia suchas Acs (GFBC01001446), FbaA (GFBC01011669 andGFBC01021420), Pyk (GFBC01029099), and Aldh(GFBC01005625). However, these glycolic genes werestrongly induced in response to hypoxia in A. nidulans andC. albicans,37,39 which was contrary to the response ofM. irregularis (Table 2). Furthermore, seven differentiallyexpressed unigenes were identified in starch and sucrosemetabolism, of which the expression levels of six geneswere decreased after a shift to the hypoxic conditions,except TPS (GFBC01001384) which was up-regulated.Previous studies had shown that trehalose can be used as analternative carbon source43 and can be metabolized duringadaptive response to various stress conditions includingdehydration, oxidative stress, heat, cold, and freezing stressin yeast and filamentous fungi.44–46 The elevated level ofTPS might played an important role in regulating carbonutilization in respond to hypoxia stress for M. irregularis.It will be important to further verify the role of TPS in theinfection mechanism.

Decreased expression of genes involved in the TCAcycle and aerobic respiration has been demonstrated inother fungi such as C. albicans and fission yeast when ex-posed to hypoxia.39,47 We found that three NADH-quinoneoxidoreductase (Ndh, GFBC01024053, GFBC01024863,GFBC01007772) were also down-regulated, thus, mito-chondrial respiration chain complexes I in M. irregulariswas diminished by limited oxygen supply. The underlyingmechanism to maintain energy flow in M. irregularis duringoxygen depletion could be different from other filamentousfungi, in which mitochondrial respiration for ATP produc-tion was active under hypoxia.48 Another finding was that

Xu et al. 11

gluconeogenesis in M. irregularis was up-regulated, demon-strated by increased levels of phosphoenolpyruvate car-boxykinase (PckA, GFBC01011023) and transcription ac-tivator of gluconeogenesis ERT1 (GFBC01017450). PckAis a key enzyme in the reductive branch of the TCA cy-cle, which in other pathogens. For example, this enzyme isimportant for the re-oxidation of intracellular NADH dur-ing the hypoxic growth of Mycobacterium tuberculosis49

and is activated in A. oryzae upon hypoxia (Table 2). Theinduced transcripts of PckA and ERT1 suggested that M.irregularis might use the reductive branch of the TCA cycleto adapt to the hypoxia conditions. Taken together, theseresults suggested that energy generating through carbohy-drate metabolism activity was likely decreased in responseto hypoxia in M. irregularis, indicating that M. irregulariswas evolved to have different metabolism properties fromother fungi such as A. nidulans upon hypoxia.

The steroid biosynthesis pathway has been identifiedas responsive to hypoxia in A. fumigatus, C. neofor-mans and C. albicans,18,50,51 and sterols were consid-ered as an oxygen sensing system due to its high oxy-gen requirement for sterol biosynthesis. Transcripts ofsteroid biosynthesis enzymes such as the C-14 sterol reduc-tases ERG24, and the C-4 methyl sterol oxidases ERG25,which has been shown to require oxygen, were highlyinduced under hypoxia in C. neoformans.51 However,the steroid biosynthesis pathway in M. irregularis wasnot significantly affected. Only two sterol reductase genesegr4 (GFBC01007963, GFBC01007636) and a cytochromeP450 (GFBC01017808) were seen to be reduced, whilethe transcripts of SREBP, which has been assigned as akey transcriptional regulator for ergosterol biosynthesis inmany eukaryotes,52 were not significantly altered in re-sponse to hypoxia. This minor alternation of ergosterolbiosynthesis when compared to other fungus may be causedby the different oxygen levels in each testing experiment.53

In terms of oxygen consumption, mitochondrial electrontransport chain for ATP formation and sterol biosynthe-sis are two main sinks. We speculated that the decreasein gene expression involved in sterol biosynthesis, and thedown regulation of three Ndh in hypoxic M. irregulariswere likely to be a response to the balance shift in TAGand fatty acids catabolism, which normally require moreO2 and generate higher amounts of NADH would be gen-erated. This speculation could also explain the GABA shuntresponse mentioning below.

Balance of NADH/NAD+ level during hypoxia may playcrucial roles for fungal survival. GABA shunt is an impor-tant contributor for energy metabolism that can preventthe NADH accumulation in hypoxic-grown fungal cells.37

The activation of GABA shunt has been reported in sev-

eral fungi that were cultivated under limited oxygen con-ditions.18,37,54 For instance, the expressions of almost allgenes in the GABA-shunt pathway including the glutamatedecarboxylase, 4-aminobutyrate transaminase, and succi-nate semialdehyde dehydrogenase were induced from 1.6-to 5.7-fold in A.nidulans.55 In M. irregularis, seven differen-tially expressed genes were identified involved in the GABAshunt, of which that the Gad and GatA expression weredecreased but the VGATs were up-regulated in response tohypoxia. The KEGG predicted that glutamate pathway alsoshowed that Gad was associated with glutamate biosynthe-sis, and had a wide range of functions depending on theorganism. For example, in S. cerevisiae, GAD1 is criticalfor cell tolerance to oxidative stress.56 However, M. irreg-ularis may benefit by the decreased levels of transcripts as-sociated with the GABA shunt and glutamate biosynthesisto regulate the intracellular redox status of cell in responseto hypoxia.

Intracellular lipid homeostasis is vital for normal mem-brane structure and function, as well as for cell sur-vival in response to lipid metabolism perturbations result-ing from environmental stresses.57 Triacylglycerol (TAG)metabolism is a central core for intracellular lipid home-ostasis, since the TAG is not only a source of cellular energy,but also a key player in lipid synthesis, particularly in mem-brane biogenesis.58 The enzyme TAG lipase which alwaysdegrades triacylglycerol into fatty acid can also participatein TAG mobilization and phospholipid metabolism throughits lysophospholipid acyltransferase activity.58 We foundthat TAG lipase (GFBC01029180) of M. irregularis wasobserved to be up-regulated under hypoxia (Fig. 5, 6). How-ever, this gene’s expression had not been described as dif-ferentially expressed to hypoxia in the other six pathogenicfungi mentioned above when exposing to hypoxia. Also,the expression level of GFBC01029364, a member of thecytosolic phospholipase A2 group IV family catalyzingphosphatidylcholine to acyl-glycero-3-phosphocholine forcellular energy, was increased as well. In agreementwith an activation of lipid/fatty acid degrading process,we found that the ACSL (GFBC01014149) and ACOX(GFBC01011826) of M. irregulairs, encoding the proteinsfor β-oxidation were also up-regulated, which was differentto the decreased ACOX level in A. fumigatus in responseto hypoxia.18 These results suggested that the hypoxic M.irregularis rather use the intracellular lipid pool instead ofthe carbohydrates as energy source.

Three HSP proteins associated with endocytosis were up-regulated under the hypoxic condition. The HSP71 gene hasa high degree of homology to other Hsp70.59 The HSP70protein can bind to the plasma membrane of macrophage,specifically on its lipid raft-microdomain and functions as

12 Medical Mycology, 2017, Vol. 00, No. 00

Figure 8. Growth comparison of Mucor irregularis CBS103.93 on MEAmedium treated with or without triglycerides. A total of 5 × 103 conidiawere spot inoculated onto the plates, and incubated in normoxia andhypoxia (6% O2) conditions at 27◦C for 3 days. Bar = 2 cm.

an enhancer when macrophage-mediated antigen uptakehas taken place, which in turn stimulates the phagocytosisprocess.60,61 Given that the three HSP genes associated withendocytosis in M. irregularis were induced under hypoxia,we speculated that M. irregularis might use this mechanismto absorb the extracellular lipid through endocytosis.

Furthermore, M. irregularis infections often present as adestructive skin lesion at exposed surface of the skin, typi-cally in the central face area where the sebaceous glands areabundantly distributed.9,13,62–64 In general, the sebum ofhuman sebaceous glands is primarily composed of triglyc-erides (∼41%), wax esters (∼26%), squalene (∼12%), andfree fatty acids (∼16%). So we hypothesized that hypoxiamay increase the endocytosis of M. irregularis for extra-cellular nutrient sources such as triglycerides and fattyacids. To verify this hypothesis, we investigated the hy-poxic growth of M. irregularis on MEA and MEA sup-plemented with triglycerides. Our results showed that thesizes of M. irregularis colonies on the medium supplementedwith triglycerides were larger after 3 days’ incubation underhypoxia (Fig. 8). The phenotypic growth supported our hy-pothesis that triglycerides or fatty acids could promote thegrowth of M. irregularis under hypoxia. In summary, thehypoxic microenvironments may suppress carbohydratesmetabolism in M. irregularis during infection but acceler-ate fatty acid metabolism to meet energy demands. Also,the lipid uptake from host serum may provide the extra-cellular nutrient source as energy/resources for the hypoxicgrowth during the infection, which then defines the specificpathogenicity of M. irregularis.

In conclusion, this is the first transcriptome study to ourknowledge to provide new insights into the molecular mech-anisms of pathogenesis in M. irregularis based on its adap-tation to hypoxia. Major responses to hypoxia observed inthis study include: decreased gene transcription in the gly-colysis, oxidative phosphorylation and carbon metabolismin contrast to previous observations in other fungi such asAspergillus spp. In contrast, the levels of transcription ingenes involved in the lipid/fatty acid metabolism and en-docytosis were up-regulated in response to hypoxia. Wehypothesize that M. irregularis cells may use the intra-lipidpool and the lipid absorbed from the extracellular environ-ment through endocytosis as energy source during its infec-tion. This transcriptome (RNA-seq) investigation has pro-vide significant baseline data for future clinical, molecularand genetic studies in M. irregularis towards understand-ing of infection mechanism and biomarkers development inrapid disease diagnosis and treatment.

Supplementary material

Supplementary data are available at MMYCOL online.

Acknowledgments

This work was supported by the National Natural Science Founda-tion of China [grant No. 81471905], the Postdoctoral Science Foun-dation of Chinese Academy of Medical Sciences and Peking UnionMedical College [2015], and the National Basic Research Programof China (973 Program) [grant No. 2013CB531600].

Declaration of interest

The authors report no conflicts of interest. The authors alone areresponsible for the content and the writing of the paper.

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