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Medical Mycology, 2017, 0, 1–14
doi: 10.1093/mmy/myx081
Advance Access Publication Date: 0 2017
Original 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,∗
1
Department of Mycology, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking
Union Medical College, Nanjing 210042, Jiangsu, People’s Republic of China, 2 Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20057, USA, 3 Division
of Infectious Diseases, University of British Columbia, Vancouver, BC V6H 3Z6, Canada and 4 Demo Lab,
Shanghai AB Sciex Analytical Instrument Trading Co., Ltd, IBP, Shanghai, 200335, People’s Republic of
China
∗
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 public health. Cutaneous mucormycosis caused by Mucor irregularis (formerly Rhizomucor
variabilis) is an emerging disease in China. To survive in the human body, M. irregularis
must overcome the hypoxic (low oxygen) host microenvironment. However, the exact molecular mechanism of its pathogenicity and adaptation to low oxygen stress
environment is relatively unexplored. In this study, we used Illumina HiSeq technology (RNA-Seq) to determine and compare the transcriptome profile of M. irregularis
CBS103.93 under normal growth condition and hypoxic stress. Our analyses demonstrated a series of genes involved in TCA, glyoxylate cycle, pentose phosphate pathway,
and GABA shunt were down-regulated under hypoxic condition, while certain genes in
the lipid/fatty acid metabolism and endocytosis were up-regulated, indicating that lipid
metabolism was more active under hypoxia. Comparing the data with other important
human pathogenic fungi such as Aspergillus spp., we found that the gene expression
pattern and metabolism in responses to hypoxia in M. irregularis were unique and different. We proposed that these metabolic changes can represent a species-specific hypoxic
adaptation in M. irregularis, and we hypothesized that M. irregularis could use the intralipid pool and lipid secreted in the infection region, as an extracellular nutrient source to
support its hypoxic growth. Characterizing the significant differential gene expression in
this species could be beneficial to uncover their role in hypoxia adaptation and fungal
C The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.
All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
1
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Medical Mycology, 2017, Vol. 00, No. 00
pathogenesis and further facilitate the development of novel targets in disease diagnosis
and 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 exhibiting high rates of morbidity and mortality. The incident
rate of mucormycosis has increased by 7.4% per year (from
0.7 to 1.2 cases/million persons) in the last decade.1 Mucormycosis is most commonly caused by members of Mucorales. This fungal infection can be transmitted by spores
in the air, ingestion, or direct contact with injured skin.2,3
Based on clinical presentations, mucormycosis can be divided into two types—invasive and cutaneous mucormycosis. The former is a severe life-threating fungal infection,
commonly prevalent in individuals with impaired immunity, while the latter has mostly a milder condition which
can manifest at even immunocompetent patients.
Mucor irregularis (renamed from Rhizomucor variabilis4 ) was first isolated from a skin lesion, which had been
presented as a primary cutaneous infection in the hand of
a Chinese patient in 1991.5 Since then, approximately 30
cases of primary cutaneous infection caused by M. irregularis have been documented,6–8 of which 23 cases were
from China. In contrast to the angioinvasive mucormycosis
(commonly caused by Rhizopus oryzae), infection caused
by M. irregularis presents as a chronic disease, tending to
be limited to dermal and subcutaneous tissues without vascular invasion. Most patients with M. irregularis infections
were immunocompetent or at least had no apparent immunodeficiency, but some patients were badly disfigured
due to misdiagnoses in early stage.9–13
M. irregularis is a unique pathogen in its temperature
tolerance among the Mucorales. In general, Mucorales spp.
are highly thermotolerant or even thermophilic, with maximum growth temperatures up to 37–45◦ C. However, M.
irregularis cannot grow above 37◦ C, which may be one of
the major reasons for its inability to cause deep-tissue infections.14 Therefore, this pathogen may have special genetic
traits for environmental adaptation that differ from other
Mucorales species for high temperature tolerance. To date,
most studies on mucormycosis have focused on species that
cause invasive infections,12,13,15 but the knowledge for disease management remains limited, especially the pathogenesis/pathophysiology of M. irregularis is poorly understood.
To survive in a human host, the pathogenic fungi need to
tolerate and overcome in vivo micro-environmental stress
conditions. One of the stresses is hypoxia (low oxygen)
condition. It is well established that the oxygen levels in
most human tissues are considerably below atmospheric
level (20.9%); the oxygen concentration varies across different tissues in the body, and ranging from 2.5% in
the kidney to 9% in the lung. Furthermore, oxygen partial pressure of the skin is only 41 mmHg (∼6% oxygen
concentration).16,17 Since oxygen is a critical component
to many essential biochemical processes, the ability to survive under hypoxic conditions has been hypothesized to
be a necessary virulence attribute of human pathogenic
fungi.18–21 To understand how human pathogenic fungi
adapt and survive in low oxygen conditions, several investigations have examined the global fungal transcriptome
responses to hypoxia in Aspergillus nidulans, A. fumigatus, Candida albicans, and Cryptococcus neoformans.22–25
The results demonstrated that, pathogenic fungi possess
different mechanisms to maintain energy in order to survive and grow in oxygen-limited environments. However,
some transcriptome changes and gene regulation patterns
are 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 as
the down-regulation of genes responsible for ribosomal and
purine/pyrimidine biosynthesis.22–25
In contrast to these invasive pathogens, far less is known
about hypoxia responses of M. irregularis, which is confined to causing skin lesions. For better understanding of its
adaptation in reduced oxygen level condition and its consequential significance in the pathogenicity of mucormycosis
during skin infection, we performed transcriptome sequencing (RNA-seq) to examine the transcriptional responses of
M. irregularis to hypoxia. The RNA-Seq data of M. irregularis (CBS103.93) under atmospheric environment was
compared to the one under 6% O2 concentration. The latter was used to mimic the skin oxygen concentration.16,17
The main goal of the study is to provide an overview of
genes that are up- or down- regulated by low level of oxygen stress, particularly on genes involved in pathogenesis
and hypoxia pathway that may distinguish it from other
clinical important pathogenic fungi.
Methods
Growth comparison under normoxic and hypoxic
atmospheres
Mucor irregularis standard strain CBS103.93 was maintained at the National Fungi Strain Reserve Center (Nanjing, China). Since the fungus causes skin infection, it was
Xu et al.
cultured on solid medium instead of liquid broth, which can
be used for blood or blood vessel infection fungus. The fungus 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 filtered
through filter paper with average pore size of 40 µm. The
concentration of spores (conidia) was determined with a
hemocytometer under a microscope, and 5 µl conidial suspensions adjusted to 1 × 103 conidia/µl were spotted onto
the center of the plates. Plates were pre-incubated at 27◦ C
under normoxic condition for 12 hours, then either kept
normoxic (20.9% O2 ) or shifted to a hypoxia chamber
(HuaXi Elctronics Technetronic, Changsha, China) with
6% O2 and incubated until the mycelia covered the entire
cultivation dish. The diameter of the colony was measured
and averaged from two separate experiments.
To compare the growth of M. irregularis on medium
with or without triglycerides in normoxic and hypoxic conditions, 5 µl conidial suspensions were spotted onto the
center of the plates containing 70% MEA or 70% MEA
supplemented 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 at
27◦ C under normoxic condition. Of these, three plates were
then incubated in hypoxic condition (6% O2 ), while the remaining three plates were still kept under normoxic condition. After 6 hours of incubation, all the repeat plates were
collected, and the mycelium was ground to fine powder for
total RNA isolation using Qiagen RNeasy Plant Mini kit
(Qiagen, Hilden, Germany). Total RNA concentration was
quantified with an Ultrospec 2100 Pro (Amersham Pharmacia, Little Chalfont, England).
Messenger RNA (mRNA) was purified by polyA selection method using oligo(dT) beads and was separated
in fragmentation buffer to elute 100-bp to 400-bp fragments. Total mRNAs were then reverse-transcribed into
complementary DNAs (cDNAs) for library construction.
RNA-seq transcriptome library was prepared according
to a TruSeqTM RNA sample preparation kit from Illumina Technology (San Diego, California) using 5 µg of
RNA. Afterward, double-stranded cDNA was synthesized
using a SuperScript double-stranded cDNA synthesis kit
(Invitrogen, Carlsbad, California) with random hexamer
primers (Illumina, San Diego, California). Then the synthesized cDNA was subjected to end-repair, phosphorylation and ‘A’ base addition according to Illumina’s library
construction protocol. Libraries were size selected for tar-
3
geting fragments of 200–300 bp on 2% Low Range Ultra Agarose followed by 15 cycles of polymerase chain
reaction (PCR) amplification using Phusion DNA polymerase (NEB, Ipswich, Massachusetts). After quantification
by TBS-380 mini-fluorometer (Promega, Madison, Wisconsin), the paired-end RNA-seq sequencing library was sequenced by Illumina HiSeq platform (2 × 150 bp read
length) at Biozeron Biotech Company (Shanghai, China).
The sequencing of M. irregularis under normoxia and hypoxia were performed, respectively.
Transcriptome data processing and assembly
After Illumina sequencing, the raw reads were in FASTQ
format. The adapter sequences and reads of poor quality
were trimmed (ambiguous bases and quality value ≤5).
Reads obtained for M. irregularis under normoxia and hypoxia conditions were assembled de novo, respectively by
Trinity (http://trinityrnaseq.sourceforge.net/) using the default parameters (–min_contig length 200 –min kmer cov
1 –max reads per graph 20000 –group pairs distance 500
–path reinforcement distance 75), which had been reported
in other publications.26,27 The contigs and unigenes of less
than 200 bp were discarded due to low annotation rate.27,28
The raw data of M. irregularis under normoxia and hypoxia
were deposited in NCBI as BioProject PRJNA347489, in
which SRA483033, SRA483036, and SRA483037 were derived from normoxia, and SRA483176, SRA483180, and
SRA483179 from hypoxic condition. The assembled sequencing data were deposited in the NCBI-TSR database
(TSR: GFBC00000000).
Functional annotation
Functional annotations were performed by sequence
comparison to public databases including the NCBI
nonredundant
protein
database
(NR
database,
http://www.ncbi.nlm.nih.gov),
Swiss-Prot
database
(http://www.expasy.ch/sprot), clusters of orthologous
groups for eukaryotic complete genomes database (KOG)
(ftp://ftp.ncbi.nih.gov/pub/COG/KOG/kyva), and Kyoto
encyclopedia of genes and genomes (KEGG) pathway
database (http://www.genome.jp/kegg/) using BLASTX
alignment with an E-value of 1.0E−5 , respectively. In
addition, Blast2GO program and BlastX29 were also used
to perform GO annotation of unigenes, and then WEGO30
software was used to perform GO classification, assigning
biological processes, molecular functions, and cellular
components.
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Medical Mycology, 2017, Vol. 00, No. 00
Identification of differentially expressed genes
To better interpret the expression levels of each unigene, FPKM (Fragments Per kb per Million reads)31 was
used to eliminate the influence of differences of gene
lengths and sequencing depth. Then, the adjusted expression level can be used for direct comparison of differences between samples. The false discovery rate (FDR)
method was also introduced to determine the threshold
P-value in multiple tests using Cufflink software package. An FDR ≤ 0.05 was used as the threshold to determine the significance of gene expression differences between the two tested temperatures.32 GO functional enrichment and KEGG pathway analysis were carried out
by Goatools (https://github.com/tanghaibao/Goatools) and
KOBAS (http://kobas.cbi.pku.edu.cn/home.do).33
Confirmation of gene transcripts using
quantitative RT-PCR
The total RNA used for RNA-Seq was also employed to
verify the expression levels of selected target genes. Firststrand cDNA was synthesized from 2 µg DNase-treated
R
Q RT SuperMix for qPCR
total RNA using the HiScript
(+gDNA wiper) (Vazyme, Nanjing, China). Quantitative
real time polymerase chain reaction (qPCR) amplifications
in 20 µl volumes, containing 10 µl SYBR Green Master
Mix (Vazyme, Nanjing, China), 20 ng template, and 4
pmol of each primer, were performed using the Stratagene
Mx3000p Real-Time PCR instrument (Agilent Technologies, Hansen, Connecticut). The temperature profile was
95◦ C for 5 min, 40 cycles of 95◦ C for 15 s, 55◦ C for
25 s, and 72◦ C for 25 s. The reaction was conducted in
triplicate. The threshold values for each target gene were
normalized using the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). The relative expression was estimated by employing the 2−CT method.34 Seven predicted
genes involved in glycolysis/gluconeogenesis, GABA shun,
glycerolipid metabolism, fatty acid metabolism pathways
were selected according to the RNA-seq data. The RTqPCR primers were designed with MacVector 11 (Accelrys,
San Diego, California) (Table S1).
Results
Comparison of growth status under hypxia
and normoxia
The effect of hypoxia on the growth of pathogenic fungus
M. irregularis was tested under 6% O2 conditions in an
oxygen-controlled chamber, which was mimicking the level
of oxygen in human skin. After incubation for 3 days, the
Figure 1. Growth of Mucor irregularis CBS103.93 in normoxic and hypoxic culture. A total of 5 × 103 conidia was spot inoculated onto MEA
plates initially incubated in air at 27◦ C. After 12 h, plates were either
kept normoxic (20.9% O2 ) or shifted to a hypoxic condition (6% O2 ) and
incubated for a further 3 days. B. The diameters of the colonies were
measured over 78 h and are expressed in cm. Values represent the mean
of three biological replicates. Bar = 2 cm.
sizes of M. irregularis colonies on MEA plates were always
smaller than those kept in the normoxic incubator (Fig. 1A).
The average of colony diameters for M. irregularis mycelia
under hypoxia condition was 5.3 ± 0.2 cm after 72 hours,
while the colonies were 7.6 ± 0.3 cm in diameter under
normoxic growth condition (Fig. 1B).
Transcriptomes of M. irregularis under normoxia
and hypoxia
To better understand the transcriptomic profile of M. irregularis in low oxygen condition, total RNAs were extracted from two conditions in triplicates. A total of 126
and 205 million reads were generated from the hypoxic
and normoxic samples, respectively (Table S2), from which
variable 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 1247
bp, with N50 of 1761bp (Table S3). The variance among
the three biological replicates from normoxic and hypoxic conditions was evaluated using the scatter plots of
gene expression (Fig. S1). The correlation among the three
Xu et al.
5
replicates in each tested condition was significantly higher
than the results between the two conditions for any particular given replicate, comfirming the data were reliable for
downstream analysis.
Functional annotation of the unigenes
Figure 2. Sequence similarity and species distribution of the top BLASTx
hits against the NR database for each unigene.
A total of 26,693 (86.76%) genes had positive hits at least
once in the NR, the Swiss-Prot protein, KEGG or KOG
database, in which 85.4% of the sequences were homologous to the gene sequences listed in the NR database
when the e-value frequency distribution has fixed significant hits as 1.0E−60 (Fig. 2A). However, the blast hits
against SwissProt, KEGG and KOG databases only resulted in 58.30% (17,933 transcripts), 38.73% (11,915
transcripts) and 8.44% (2597 transcripts) of positive hits
from genomic sequences, respectively (Table S3). The closest related species to M. irregularis in the NR database was
Rhizopus delemar with a 36.98% identity between the two
species, and only 6802 (22.11%) unigenes could be annotated, by searching against the NR database (Fig. 2B), while
the remaining unigenes were either hypothetical genes or
functionally uncharacterized.
Based on the NR annotation and gene ontology
classification, 21085 unigenes were assigned with GO
terms. 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 at
least one GO term. Most consensus sequences were grouped into three major functional categories, namely biological process, cellular component,
and molecular function.
6
Medical Mycology, 2017, Vol. 00, No. 00
Figure 4. Histogram of clusters of orthologous groups (KOG) classification. All unigenes were aligned to KOG database for prediction and classification
based on possible functions.
categories in terms of biological processes, cellular components, and molecular functions clusters (Fig. 3, Table S4).
For the biological processes category, the genes related to
metabolic processes (7585, accounting for 35.97%) were
dominant, 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 and
cell (both 4594, 21.79%) were the dominant groups, followed 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 activity, 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 were
functionally classified into 25 molecular families such as
cellular structure, biochemistry metabolism, molecular processing, and signal transduction (Fig. 4). Within these broad
categories, we found a few notable groups, such as translation, ribosomal structure, and biogenesis (accounting for
334, 10.14%), followed by posttranslational modification,
protein turnover and chaperones at 10.00% (329), signal
transduction mechanisms at 7.69% (253), and transcription bringing up the rear at 5.65% (186). Apart from the
largest group of functionally uncharacterized genes (337,
10.24%), the remaining genes were involved either in nuclear structure, cell motility, extracellular structure, or defense mechanisms (5, 3, 3, and 1 unigenes, respectively)
(Fig. 4).
To further explore the molecular interaction between
genes, the KEGG database was also used to predicate
the potential pathways in which these genes might be involved. Among the 30761 annotated genes, 11,915 were
clustered to 34 processes/pathways, including signal transduction, translation, carbohydrate metabolism, endocrine
functions, and other biosynthetic pathways (data not
shown).
Differentially expressed unigenes
The differentiation of gene expression between normoxia
and hypoxia was evaluated by FPKM with FDR corrections (P < .05); genes showing at least twofold changes in
expression were considered to be differentially expressed. A
total of 1112 transcripts (Table S5) had altered expression
significantly in response to different oxygen conditions, of
which 531 transcripts were significantly up-regulated and
581 transcripts were down-regulated in response to hypoxia
(twofold changes cut off, Table S5).
The up-regulated genes were associated with the
regulation of gene expression (GO:0010468), regulation of metabolic process (GO:0080090, GO:0019222,
and GO:0031323), antioxidant activity (GO:0051920,
GO:0016209, and GO:0016684), transcription factor
activity (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 genes
were involved in hydrolases activity (GO:0016798,
GO:0004553, and GO:0016811), defense response
(GO:0006952
and
GO:0009605),
carbohydrate
metabolism (GO:0030246, GO:0005975, GO:0016810,
GO:0015926, GO:0004410, and GO:0004339) and
calcium ion binding processes (GO:0005509) (Table 1).
The biological functions of the 1112 differentially expressed 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
<Up-regulated in hypoxia>
GO:0003700 transcription factor activity, sequence-specific DNA
binding
GO:0004090 carbonyl reductase (NADPH) activity
GO:0006355 regulation of transcription, DNA-templated
GO:0010468 regulation of gene expression
GO:0016209 antioxidant activity
GO:0016684 oxidoreductase activity, acting on peroxide as acceptor
GO:0019222 regulation of metabolic process
GO:0031323 regulation of cellular metabolic process
GO:0044429 mitochondrial part
GO:0051920 peroxiredoxin activity
GO:0060590 ATPase regulator activity
GO:0080090 regulation of primary metabolic process
<Down-regulated in hypoxia>
GO:0004339 glucan 1,4-alpha-glucosidase activity
GO:0004410 homocitrate synthase activity
GO:0004553 hydrolase activity, hydrolyzing O-glycosyl compounds
GO:0005509 calcium ion binding
GO:0005975 carbohydrate metabolic process
GO:0006952 defense response
GO:0009605 response to external stimulus
GO:0015926 glucosidase activity
GO:0016798 hydrolase activity, acting on glycosyl bonds
GO:0016810 hydrolase activity, acting on carbon-nitrogen (but not
peptide) bonds
GO:0016811 hydrolase activity, acting on carbon-nitrogen (but not
peptide) bonds, in linear amides
GO:0030246 carbohydrate binding
database by pathway enrichment analysis. The pathways that were significantly enriched included starch and
sucrose metabolism (ko00500), glycerolipid metabolism
(ko00561), glycerophospholipid metabolism (ko00564),
pyruvate metabolism (ko00620), amino sugar and nucleotide sugar metabolism (ko00520), oxidative phosphorylation (ko00190), and carbon metabolism (ko04141).
Also some functional categories such as carbon metabolism,
fatty acid/lipid metabolism and endocytosis were observed
during comparison (Fig. 5). Many of the findings reported
in M. irregularis (Fig. 6, Table S6) were different from
that reported in other human pathogenic fungi (Table 2).
For example, the genes involved in glycolysis, fatty acid
metabolism, oxidative phosphorylation, steroid biosynthesis, and pentose phosphate pathway were often found to
be up-regulated in other fungi such as Cryptococcus neoformans, Candida albicans, and Aspergillus nidulans under
hypoxic conditions.35–39
Observed
Annotated
Expected
P-value
23
615
10.62
.000743
2
45
45
5
4
54
50
7
3
2
52
7
1241
1207
73
48
1825
1575
176
17
6
1591
0.12
21.42
20.84
1.26
0.83
31.50
27.19
3.04
0.29
0.10
27.46
.0059
3.29E−06
1.43E−06
.00863
.00938
1.25E−04
3.76E−05
.0337
.0029
.00426
1.08E−05
6
2
23
12
22
6
4
8
24
11
81
5
427
240
596
37
21
120
438
232
1.53
0.09
8.06
4.53
11.26
0.70
0.40
2.27
8.27
4.38
.00434
.00343
8.21E−06
2.18E−03
3.26E−03
6.25E−05
5.84E−04
2.04E−03
3.93E−06
.00488
5
61
1.15
.00589
6
62
1.17
1.11E−03
Reduced ability to degrade the carbohydrate
under hypoxia
In M. irregularis many transcripts involved in glycolysis
were reduced in response to hypoxia. For example,
acetyl-coenzyme A synthetase (Acs, GFBC01001446), one
of the most highly reduced transcripts, two fructosebisphosphatase
aldolases
(FbaA,
GFBC01011669
and GFBC01021420), which are involved into the
early steps of converting fructose 1,6-phosphate to
glyceraldehyde 3-phosphate, pyruvate kinase (Pyk,
GFBC01029099) along with aldehyde dehydrogenases
(Aldh, GFBC01005625), which converts acetaldehyde to acetate for central carbohydrate and lipid
metabolism tended to be transcriptionally reduced
(Fig. 5, 6).
Of the seven genes involved in starch and sucrose
metabolism, six decreased in the transcriptional levels
after a shift to hypoxic growth condition. Somewhat
8
Medical Mycology, 2017, Vol. 00, No. 00
Figure 5. Hypoxia decreased levels of expression for genes involved in carbon metabolism, oxidative phosphorylation, and increased the expression
of genes involved in fatty acid/lipid metabolism and endocytosis processes. Heat map showing the cluster analysis of genes differentially expressed
prior to hypoxia (0 hour) and in hypoxia (6 hours) of Mucor irregulars CBS103.93.
surprisingly, only one exception, the trehalose 6-phosphate
phosphatase (TPS, GFBC01001384), which catalyzes the
dephosphorylation of trehalose 6-phosphate to form trehalose, was up-regulated.
Altered expression in steroid biosynthesis
and GABA shunt pathway genes
In response to hypoxia, the steroid biosynthesis pathway of
M. irregularis was not significantly affected, only two sterol
reductase genes egr4 (GFBC01007963, GFBC01007636)
and a cytochrome P450 (GFBC01017808) reduced transcription, while the transcription level of sterol regulatory element-binding protein SREBP was not significantly altered. These results suggested that the steroid
biosynthesis of M. irregularis appeared to not be affected or partially decreased in response to hypoxia, which
was incongruent to the patterns of many other fungi
(Table 2).
In addition, seven differentially expressed genes were
identified to be involved in the GABA shunt of
M. irregularis, including two glutamate decarboxylase
(Gad, GFBC01008155 and GFBC01027882), one 4aminobutyrate transaminase (GatA, GFBC01030508), and
four vesicular inhibitory amino acid transporter (VGAT).
We found that the expression of Gad and GatA was reduced, but VGATs were up-regulated in response to hypoxia. In contrast to the fungi such as Aspergillus nidulans
and C. neoformans in which the genes of the GABA shunt
were up-regulated for energy metabolism, the inconsistency
of these genes alternation elicited that the GABA shunt was
inactive in M. irregularis under low oxygen (Table 2).
Bolstered catabolic potential in lipid metabolism
and activation in endocytosis
The hypoxic exposure also led to marked changes in
gene 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 during
hypoxia 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, alcohol
dehydrogenase (NADP+); PckA, phosphoenolpyruvate carboxykinase; GAD, glutamate decarboxylase; ACOX, acyl-CoA oxidase]; ACSL, long chain
fatty 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.
Glycolysis
Gluconeogenesis (PEP carboxykinase)
Pentose phosphate pathway
Oxidative phosphorylation
β-oxidation
Lipid/Fatty acid metabolism
GABA shunt/ergosterol biosynthesis
Endocytosis
M. irregularis
A. nidulans
A. niger
A. oryzae
A. fumigatus
C. albicans
C. neoformans
↓
↑
↓
↓
↑
↑
↓
↑
↑
↓
↑
NR
↑
↑
↑
NR
↑
NR
↑
NR
↑
↑
↑
NR
↑
↑
NR
NR
NR
NR
↑
NR
↑
NR
↑
↓
NR
↓
↑
NR
↑
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 neoformans.23 ,36
that in M. irregulairs, the triacylglycerol (TAG) lipase
(GFBC01029180) that degrades triacylglycerol into fatty
acid, and a member of the cytosolic phospholipase A2
group IV family (GFBC01029364), which catalyzes phosphatidylcholine to acyl-glycero-3-phosphocholine for cellular 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 involved in β-oxidation, were also up-regulated. These results
led us to speculate that the hypoxic M. irregularis might use
the intracellular lipid pool as energy source.
Along with this speculation, M. irregularis would need
an approach to maintain the lipid homeostasis under hypoxic growth conditions. Previous studies showed that
endocytosis was important for bringing nutrients into
the cell to maintain lipids and protein homeostasis in
the cell;40,41 hence, analyses on endocytosis were conducted in M. irregularis. Eight genes associated with
endocytosis were differentially expressed in hypoxia,
of which five genes (GFBC01019239, GFBC01021838,
GFBC01028777 [HSP71-like], GFBC01016923 [HSP70],
and GFBC01029894 [HSP71-like]) were up-regulated under hypoxic condition. It was noteworthy that such
10
Figure 7. Gene expression for validation in (A) qRT-PCR and (B) RNAseq assays in normoxia and hypoxia exposed strains. The data represent
the 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 genes
under hypoxia has not been reported in other pathogenic
fungi 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 using the same biological RNA samples. Seven target genes
from different functional categories that were verified as
up- or down-regulated through qRT-PCR as an independent measure of differential gene expression (Fig. 7A). All
the genes used in validation showed the same pattern of
expression as that of the RNA-seq results (Fig. 7B) demonstrating the reliability of the RNA seq data.
Discussion
The comparison of mycelial diameter between normoxia
and hypoxia clearly demonstrated that the reduced oxygen
supply was a growth limiting factor in M. irregularis. Similarly, M. plumbeus, another member of Mucorales, also
had slower growth in hypoxia than in atmospheric oxy-
Medical Mycology, 2017, Vol. 00, No. 00
gen.42 Almost all clinical cases caused by M. irregularis were
chronic cutaneous infection, except the case that reported a
facial lesion with an extraordinary pulmonary infection.13
Xia et al. observed that the patient’s symptoms subsided in
summer and were aggravated in winter, which was different
from most superficial fungal infection in human, suggesting
that different optimal temperatures for fungal growth may
be responsible for this behavior. Apart from this explanation, since the oxygen content is the highest in the lung of
human body, which could be a favorable condition for its
lung infection and invasion rather than other inner organs.
Thus, we hypothesized that the growth of M. irregularis
was retarded during the invasion of human skin in hypoxic
environment, having the ability to overcome the low oxygen
condition is essential establishment of the skin infection.
To understand the molecular mechanisms of hypoxia adaptation in this pathogenic fungi, we investigated the transcriptome profiles of M. irregulairs
using Illumina RNA-seq. Many transcripts related to
glycolysis were reduced in response to hypoxia such
as Acs (GFBC01001446), FbaA (GFBC01011669 and
GFBC01021420), Pyk (GFBC01029099), and Aldh
(GFBC01005625). However, these glycolic genes were
strongly induced in response to hypoxia in A. nidulans and
C. albicans,37,39 which was contrary to the response of
M. irregularis (Table 2). Furthermore, seven differentially
expressed unigenes were identified in starch and sucrose
metabolism, of which the expression levels of six genes
were 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 an
alternative carbon source43 and can be metabolized during
adaptive response to various stress conditions including
dehydration, oxidative stress, heat, cold, and freezing stress
in yeast and filamentous fungi.44–46 The elevated level of
TPS might played an important role in regulating carbon
utilization in respond to hypoxia stress for M. irregularis.
It will be important to further verify the role of TPS in the
infection mechanism.
Decreased expression of genes involved in the TCA
cycle and aerobic respiration has been demonstrated in
other fungi such as C. albicans and fission yeast when exposed to hypoxia.39,47 We found that three NADH-quinone
oxidoreductase (Ndh, GFBC01024053, GFBC01024863,
GFBC01007772) were also down-regulated, thus, mitochondrial respiration chain complexes I in M. irregularis
was diminished by limited oxygen supply. The underlying
mechanism to maintain energy flow in M. irregularis during
oxygen depletion could be different from other filamentous
fungi, in which mitochondrial respiration for ATP production was active under hypoxia.48 Another finding was that
Xu et al.
gluconeogenesis in M. irregularis was up-regulated, demonstrated by increased levels of phosphoenolpyruvate carboxykinase (PckA, GFBC01011023) and transcription activator of gluconeogenesis ERT1 (GFBC01017450). PckA
is a key enzyme in the reductive branch of the TCA cycle, which in other pathogens. For example, this enzyme is
important for the re-oxidation of intracellular NADH during the hypoxic growth of Mycobacterium tuberculosis49
and is activated in A. oryzae upon hypoxia (Table 2). The
induced transcripts of PckA and ERT1 suggested that M.
irregularis might use the reductive branch of the TCA cycle
to adapt to the hypoxia conditions. Taken together, these
results suggested that energy generating through carbohydrate metabolism activity was likely decreased in response
to hypoxia in M. irregularis, indicating that M. irregularis
was evolved to have different metabolism properties from
other fungi such as A. nidulans upon hypoxia.
The steroid biosynthesis pathway has been identified
as responsive to hypoxia in A. fumigatus, C. neoformans and C. albicans,18,50,51 and sterols were considered as an oxygen sensing system due to its high oxygen requirement for sterol biosynthesis. Transcripts of
steroid biosynthesis enzymes such as the C-14 sterol reductases ERG24, and the C-4 methyl sterol oxidases ERG25,
which has been shown to require oxygen, were highly
induced under hypoxia in C. neoformans.51 However,
the steroid biosynthesis pathway in M. irregularis was
not significantly affected. Only two sterol reductase genes
egr4 (GFBC01007963, GFBC01007636) and a cytochrome
P450 (GFBC01017808) were seen to be reduced, while
the transcripts of SREBP, which has been assigned as a
key transcriptional regulator for ergosterol biosynthesis in
many eukaryotes,52 were not significantly altered in response to hypoxia. This minor alternation of ergosterol
biosynthesis when compared to other fungus may be caused
by the different oxygen levels in each testing experiment.53
In terms of oxygen consumption, mitochondrial electron
transport chain for ATP formation and sterol biosynthesis are two main sinks. We speculated that the decrease
in gene expression involved in sterol biosynthesis, and the
down regulation of three Ndh in hypoxic M. irregularis
were likely to be a response to the balance shift in TAG
and fatty acids catabolism, which normally require more
O2 and generate higher amounts of NADH would be generated. This speculation could also explain the GABA shunt
response mentioning below.
Balance of NADH/NAD+ level during hypoxia may play
crucial roles for fungal survival. GABA shunt is an important contributor for energy metabolism that can prevent
the NADH accumulation in hypoxic-grown fungal cells.37
The activation of GABA shunt has been reported in sev-
11
eral fungi that were cultivated under limited oxygen conditions.18,37,54 For instance, the expressions of almost all
genes in the GABA-shunt pathway including the glutamate
decarboxylase, 4-aminobutyrate transaminase, and succinate semialdehyde dehydrogenase were induced from 1.6to 5.7-fold in A.nidulans.55 In M. irregularis, seven differentially expressed genes were identified involved in the GABA
shunt, of which that the Gad and GatA expression were
decreased but the VGATs were up-regulated in response to
hypoxia. The KEGG predicted that glutamate pathway also
showed that Gad was associated with glutamate biosynthesis, and had a wide range of functions depending on the
organism. For example, in S. cerevisiae, GAD1 is critical
for cell tolerance to oxidative stress.56 However, M. irregularis may benefit by the decreased levels of transcripts associated with the GABA shunt and glutamate biosynthesis
to regulate the intracellular redox status of cell in response
to hypoxia.
Intracellular lipid homeostasis is vital for normal membrane structure and function, as well as for cell survival in response to lipid metabolism perturbations resulting from environmental stresses.57 Triacylglycerol (TAG)
metabolism is a central core for intracellular lipid homeostasis, since the TAG is not only a source of cellular energy,
but also a key player in lipid synthesis, particularly in membrane biogenesis.58 The enzyme TAG lipase which always
degrades triacylglycerol into fatty acid can also participate
in TAG mobilization and phospholipid metabolism through
its lysophospholipid acyltransferase activity.58 We found
that TAG lipase (GFBC01029180) of M. irregularis was
observed to be up-regulated under hypoxia (Fig. 5, 6). However, this gene’s expression had not been described as differentially expressed to hypoxia in the other six pathogenic
fungi mentioned above when exposing to hypoxia. Also,
the expression level of GFBC01029364, a member of the
cytosolic phospholipase A2 group IV family catalyzing
phosphatidylcholine to acyl-glycero-3-phosphocholine for
cellular energy, was increased as well. In agreement
with an activation of lipid/fatty acid degrading process,
we found that the ACSL (GFBC01014149) and ACOX
(GFBC01011826) of M. irregulairs, encoding the proteins
for β-oxidation were also up-regulated, which was different
to the decreased ACOX level in A. fumigatus in response
to hypoxia.18 These results suggested that the hypoxic M.
irregularis rather use the intracellular lipid pool instead of
the carbohydrates as energy source.
Three HSP proteins associated with endocytosis were upregulated under the hypoxic condition. The HSP71 gene has
a high degree of homology to other Hsp70.59 The HSP70
protein can bind to the plasma membrane of macrophage,
specifically on its lipid raft-microdomain and functions as
12
Figure 8. Growth comparison of Mucor irregularis CBS103.93 on MEA
medium treated with or without triglycerides. A total of 5 × 103 conidia
were spot inoculated onto the plates, and incubated in normoxia and
hypoxia (6% O2 ) conditions at 27◦ C for 3 days. Bar = 2 cm.
Medical Mycology, 2017, Vol. 00, No. 00
In conclusion, this is the first transcriptome study to our
knowledge to provide new insights into the molecular mechanisms of pathogenesis in M. irregularis based on its adaptation to hypoxia. Major responses to hypoxia observed in
this study include: decreased gene transcription in the glycolysis, oxidative phosphorylation and carbon metabolism
in contrast to previous observations in other fungi such as
Aspergillus spp. In contrast, the levels of transcription in
genes involved in the lipid/fatty acid metabolism and endocytosis were up-regulated in response to hypoxia. We
hypothesize that M. irregularis cells may use the intra-lipid
pool and the lipid absorbed from the extracellular environment through endocytosis as energy source during its infection. This transcriptome (RNA-seq) investigation has provide significant baseline data for future clinical, molecular
and genetic studies in M. irregularis towards understanding of infection mechanism and biomarkers development in
rapid disease diagnosis and treatment.
Supplementary material
an enhancer when macrophage-mediated antigen uptake
has taken place, which in turn stimulates the phagocytosis
process.60,61 Given that the three HSP genes associated with
endocytosis in M. irregularis were induced under hypoxia,
we speculated that M. irregularis might use this mechanism
to absorb the extracellular lipid through endocytosis.
Furthermore, M. irregularis infections often present as a
destructive skin lesion at exposed surface of the skin, typically in the central face area where the sebaceous glands are
abundantly distributed.9,13,62–64 In general, the sebum of
human sebaceous glands is primarily composed of triglycerides (∼41%), wax esters (∼26%), squalene (∼12%), and
free fatty acids (∼16%). So we hypothesized that hypoxia
may increase the endocytosis of M. irregularis for extracellular nutrient sources such as triglycerides and fatty
acids. To verify this hypothesis, we investigated the hypoxic growth of M. irregularis on MEA and MEA supplemented with triglycerides. Our results showed that the
sizes of M. irregularis colonies on the medium supplemented
with triglycerides were larger after 3 days’ incubation under
hypoxia (Fig. 8). The phenotypic growth supported our hypothesis that triglycerides or fatty acids could promote the
growth of M. irregularis under hypoxia. In summary, the
hypoxic microenvironments may suppress carbohydrates
metabolism in M. irregularis during infection but accelerate fatty acid metabolism to meet energy demands. Also,
the lipid uptake from host serum may provide the extracellular nutrient source as energy/resources for the hypoxic
growth during the infection, which then defines the specific
pathogenicity of M. irregularis.
Supplementary data are available at MMYCOL online.
Acknowledgments
This work was supported by the National Natural Science Foundation of China [grant No. 81471905], the Postdoctoral Science Foundation of Chinese Academy of Medical Sciences and Peking Union
Medical College [2015], and the National Basic Research Program
of China (973 Program) [grant No. 2013CB531600].
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and the writing of the paper.
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