ORIGINAL RESEARCH PAPER

Differentially-expressed genes in Candida albicans exposedto e-poly-L-lysine

Ruo-Song Ye • Zhi-Hong Zhang • Heng-Yi Xu •

Feng Xu • Zoraida P. Aguilar • Yong-Hua Xiong •

Zhe-Ling Zeng • Hua Wei

Received: 9 May 2013 / Accepted: 29 July 2013 / Published online: 24 August 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Candida albicans is an opportunistic

human pathogen whose disinfection is a challenge.

e-Poly-L-lysine (e-PL), an antagonistic agent, can

disrupt cell membranes and inhibit the growth of

C. albicans. Genes that were differentially-expressed

in response to e-PL were isolated from C. albicans and

identified by suppression subtractive hybridization.

Ten subtracted clones, that share [98 % homology

with known genes of C. albicans, were isolated.

Among these, four genes encoded cell wall-associated

proteins. Real-time quantitative PCR and northern blot

hybridization suggest that these genes are involved in

the response to e-PL. These findings will help to

determine the mechanism of the antimicrobial activity

of e-PL against C. albicans.

Keywords Candida albicans � Cell wall

proteins � Northern blot hybridization �e-Poly-L-lysine � Suppression subtractive

hybridization

Introduction

e-Poly-L-lysine (e-PL) is an antimicrobial peptide

originally isolated from Streptomyces albulus ssp.

lysinopolymerus strain 346. It contains 25–30 L-lysine

residues. With its broad antimicrobial activity

Ruo-Song Ye and Zhi-Hong Zhang have contributed equally to

the study.

Electronic supplementary material The online version ofthis article (doi:10.1007/s10529-013-1319-y) contains supple-mentary material, which is available to authorized users.

R.-S. Ye � Z.-H. Zhang � H.-Y. Xu � H. Wei (&)

State Key Laboratory of Food Science and Technology,

Nanchang University, 235 Nanjing East Road,

Nanchang 330047, Jiangxi, People’s Republic of China

e-mail: weihua114@live.cn

R.-S. Ye

e-mail: yeruosong@163.com

H.-Y. Xu

e-mail: kidyxu@163.com

F. Xu � Y.-H. Xiong

Jiangxi-OAI Joint Research Institute,

Nanchang University, Nanchang 330047,

People’s Republic of China

Z. P. Aguilar

OceanNanoTech, LLC, Springdale, AR 72764, USA

Z.-L. Zeng (&)

Department of Environment and Chemical Engineering,

Nanchang University, Xuefu Road 999,

Nanchang 330031, Jiangxi, People’s Republic of China

e-mail: zlzengjx@163.com

123

Biotechnol Lett (2013) 35:2147–2153

DOI 10.1007/s10529-013-1319-y

(including Gram-negative and Gram-positive bacteria,

yeasts and molds) (Geornaras et al. 2007), e-PL is

widely used to inhibit pathogens and to preserve

packaged food in certain countries. To our knowledge,

very few studies have been conducted on resistance

fungi or yeasts to e-PL.

Candida albicans is an important opportunistic

human pathogen. It causes local and superficial

mucosal infections, as well as inducing systemic

infections in patients with weakened immune systems.

Antimicrobial agents that inhibit C. albicans often do

so by disrupting the outer cell surface structure (Tsai

et al. 2011), thereby inducing changes in gene

expression (Khodavandi et al. 2011). In Escherichia

coli, e-PL possibly disrupts the outer membrane and

causes an abnormal distribution of the cytoplasm by

cationic adsorption (Shima et al. 1984). However, the

mechanism of e-PL’s antimicrobial activity has not

been determined at the molecular level. In the present

study, C. albicans was used as a model microorganism

to provide clues to the mechanism of e-PL’s antimi-

crobial effects by identifying molecules that it may

interact with.

Suppression subtractive hybridization (SSH) is

based on suppression PCR and can be used to isolate

and identify complementary DNAs from differen-

tially-expressed genes in eukaryotic cells. In this

study, SSH was used to identify differentially-

expressed genes in C. albicans in response to e-PL

exposure. The differentially-expressed genes were

subsequently confirmed by dot blot hybridization. The

expression levels of these genes were determined by

real-time quantitative PCR (RT-qPCR) and northern

blot hybridization. The differentially-expressed genes

in e-PL-treated C. albicans suggested that induced

changes in metabolism could be an important mech-

anism of the antimicrobial activity of e-PL.

Materials and methods

Yeast and e-poly-L-lysine (e-PL) treatment

Candida albicans ATCC 14053 was used as the model

strain. e-PL (average MW 4700, Bainafo, Zhengzhou,

China) was added to C. albicans cultures at 0–150 lg/

ml. A decrease in yeast growth rate, as calculated by

plate count, was observed after treatment. The tests

were performed in triplicate.

RNA extraction and cDNA synthesis

A suspension containing 106 cells/ml treated with 0 or

50 lg e-PL/ml was prepared for RNA extraction.

Total RNA was extracted according to the method

described by Khodavandi et al. (2011), dissolved in

RNase-free water and then quantified using a Nano-

Drop spectrophotometer. Poly(A)? RNA required for

SSH was isolated from total RNA using an Oligotex-

dT30 mRNA purification kit.

mRNA from each sample was used as a template to

synthesize first and second strand cDNA, using an

M-MLV reverse transcriptase cDNA synthesis kit,

according to the manufacturer’s protocol. Double-

stranded cDNAs were digested with RsaI to obtain

blunt ends for adaptor ligation.

Construction of subtracted cDNA libraries

cDNA subtraction was performed in two hybridiza-

tions to produce two subtracted cDNA libraries,

according to the method described by Chang et al.

(2012). The forward-subtracted library was obtained

using the control cell culture as the driver sample and

the e-PL-exposed cells as the tester. The reverse

library was constructed using the e-PL-exposed cells

as the driver sample and control cells as the tester. The

forward and reverse reaction libraries were designed

to produce clones that were upregulated and down-

regulated in e-PL-treated cells, respectively.

Two rounds of PCR were conducted to amplify the

tester-specific sequences using adaptor-specific prim-

ers (listed in Supplementary Table 1) and to enrich the

desired differentially-expressed cDNAs containing

both adaptors. The purified PCR products were ligated

into vector pMD18-T and transformed into E. coli

JM109 competent cells.

Dot blot hybridization

The resulting PCR products were spotted onto a nylon

membrane. Dot blotting was performed using a

digoxigenin (DIG)-high prime DNA labeling and

detection starter kit I, according to the manufacturer’s

instructions. cDNAs of e-PL treated and control

groups were labeled using the DIG-high prime

supplied in the kit and used as a probe. Clones

showing a significantly increased hybridization signal

(300 % above control levels), which was detected

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using the corresponding tester cDNA probe rather than

the driver cDNA probe, were selected for sequencing.

Sequencing of the subtracted cDNA clones

and bioinformatics analysis

The cDNA clones from the two SSH libraries were

sequenced, and repetitive and false-positive sequences

were removed. Nucleic acid homologies were identi-

fied by searching the nucleotide databases at the

National Center for Biotechnology Information

(NCBI) using the Basic Local Alignment Search Tool

(BLAST). Homologies showing [98 % sequence

identity over more than 100 nucleotides were consid-

ered significant.

Real-time quantitative PCR

Differentially-expressed genes identified by SSH were

chosen for specific analysis using RT-qPCR. The first

strand was synthesized from e-PL-treated and control

C. albicans genes using a primescript first-strand

cDNA synthesis kit. PCR primers were designed based

on gene sequences from GenBank (Supplementary

Table 2). b-Actin (a housekeeping gene) (Khodavandi

et al. 2011) and a cell wall-associated protein trans-

lation elongation factor, EF-1a (Frost and Nilsen

2003), were used as internal controls to normalize

dissimilar RNA concentrations during RNA

extraction.

Northern blot hybridization

The change in gene expression during exposure to

e-PL was examined by northern blotting. RNA isolates

from C. albicans samples were treated with e-PL for 0,

1 and 3 h. Northern blotting was conducted using

either ENO1 or ASI3 PCR products as the hybridiza-

tion probe. ACT was used as a control probe (PCR

primers are listed in Supplementary Table 2).

Statistical analysis

Statistical analysis was performed using SigmaPlot 11

(Systat Software, San Jose, CA, USA) and SPSS 13.0

(SPSS Inc., Chicago, IL, USA) software by means of

independent one-way ANOVA tests for each sampling

point. The differences among the three groups were

assessed by means of the least significant difference

(LSD) multiple comparison test. Statistical signifi-

cance was set at p \ 0.05.

Results

C. albicans grown in various concentrations

of e-PL

Candida albicans was treated with up to 150 lg e-PL/

ml. The antimicrobial effect reached 95 % mortality

with e-PL at 150 lg/ml (Fig. 1). An optimal concen-

tration of 50 lg/ml of e-PL was selected for

subsequent SSH, whereby e-PL present at sub-lethal

concentrations may activate cell gene expression.

Evaluation of subtraction efficiency

and differential screening of SSH clones

The forward and reverse-subtracted cDNA libraries

obtained from C. albicans mRNA subtracted from e-

PL-exposed and control cells were subjected to PCR,

which showed a smear of products with a size range of

250–750 bp (Fig. 2a), indicating that the SSH cDNA

library was successfully constructed.

One hundred subtracted clones from each library

were isolated. The inserts were amplified by PCR and

Fig. 1 Effect of concentration on the antimicrobial activity of

e-poly-L-lysine (e-PL) against Candida albicans. Cultures were

grown in YPD at 37 �C to OD600 = 0.2 and then treated with e-

PL at 50, 100 and 150 lg/ml. Cultures continued to grow at the

same rate until the control culture reached a final OD600 = 0.8.

Yeast growth rate was calculated after treatment with e-PL in

comparison with the number of untreated cells by a plate count.

Error bars indicate standard deviations for three replicates

Biotechnol Lett (2013) 35:2147–2153 2149

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their sizes ranged from 200 to 750 bp (Fig. 2b). Dot

blotting was used to screen positive clones identified

by PCR amplification (Fig. 2c).

Differential gene transcription in C. albicans

exposed to e-PL

After sequence assembly and BLASTx and BLASTn

analysis, three genes obtained from the forward library

were found to be homologous to C. albicans ASI3 (a

ubiquitin protein ligase), GCN4 translational activa-

tor, GCN1, and arginyl tRNA synthetase, AGRS

(Table 1). Seven genes obtained in the reverse library

were involved in glycolysis, lipid metabolism, and

protein synthesis (Table 1). Among these genes, four

genes [enolase (ENO1), fatty acid synthetase beta

subunit (FAS1), hexokinase II (HXK), and hexose

transporter (HXT61)] are cell wall-associated pro-

teins. The DNA segments obtained from the two

libraries were 99–100 % similar to the corresponding

sequences in C. albicans SC5314.

Validation of differential transcription using

RT-qPCR

The transcription levels of ten genes obtained by SSH

were assessed (Fig. 3). The results indicated that the

ASI3, GCN1, and AGRS genes from the forward-

subtracted cDNA library were upregulated by 175,

400 and 480 %, respectively. By contrast, the seven

genes from the reverse-subtracted cDNA library were

downregulated. Thus, the RT-qPCR results were

consistent with those of SSH. TEF1, another cell

wall-associated protein EF-1a gene, was used as a

control gene to demonstrate that the expressions of the

four cell wall-associated protein genes were specifi-

cally downregulated by e-PL stress.

Northern blot analysis

We performed northern blotting to analyze the

expressions of ENO1 and ASI3 during exposure to

e-PL from 0 to 3 h (Fig. 4a). The intensity the ENO1

Fig. 2 Second PCR products of subtractive hybridization and

detection of inserted fragments by colony PCR and dot blot

analysis. a Second PCR products from subtractive hybridiza-

tion. Lane 1 PCR products of forward-subtracted hybridization

library of C. albicans. Lane 2 PCR products of reverse-

subtracted hybridization library of C. albicans. b Insets of 17

(Lanes 1–17) randomly selected clones in the forward-sub-

tracted library and seven (Lanes 18–24) randomly selected

clones in the reverse-subtracted library of C. albicans. c Dot blot

analysis. C1, forward-subtracted probe of C. albicans. C2,

reverse-subtracted probe of C. albicans. M 2,000 bp DNA

marker

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and ASI3 bands were compared with the intensity of

the ACT band to show their relative gene expression

levels at different treatment times (Fig. 4b). The

expression level of ENO1 gene in C. albicans

decreased in response to e-PL during 0–3 h. By

contrast, ASI3 gene accumulated. Thus, these data

demonstrated that ASI3 expression was activated by

e-PL in a time-dependent manner, but ENO1 was

inhibited.

Discussion

SSH was used to identify C. albicans genes that may

be involved in the mechanism of e-PL’s antimicrobial

effect; three genes were upregulated in response to e-

PL and seven genes were downregulated. RT-qPCR

and northern blotting confirmed that these changes in

gene expressions were a response to e-PL stress.

Enolase is a glycolytic enzyme in the cell wall of

C. albicans. It binds to the plasminogen system and

enhances the invasion of C. albicans into the tissue

barrier (Jong et al. 2003), indicating that it is one of the

key factors in pathogenesis (Ebanks et al. 2006). In

this study, e-PL-induced down-regulation of the

ENO1 gene may decrease the pathogenicity of

Table 1 Differentially-expressed genes identified in Candida

albicans by suppression subtractive hybridization

Gene Encoding products Genebank

access no.

Genes of cell wall associated proteins

ENO1 Enolase L10290.1

FAS1 Fatty acid synthetase beta

subunit

XM_711784.1

HXK Hexokinase II XM_712405.1

HXT61 Hexose transporter XM_714505.1

Other proteins

DAK Dihydroxyacetone kinase XM_718181.1

PFK1 Phosphofructokinase alpha

subunit

XM_716943.1

ASI3 Ubiquitin protein ligase XM_711185.1

ARGS Arginyl tRNA synthetase XM_713338.1

GCN1 GCN4 translational activator XM_712038.1

PEX28 Peroxisomal integral membrane

protein

XM_718062.1

Fig. 3 Relative gene expression levels of C. albicans treated

with and without e-PL. Transcripts of the relative genes were

quantified by real-time quantitative PCR. Data were analyzed

using the comparative critical threshold 2-DDCT method. The

relative expression ratios are presented as log2 values in the

histogram. Ratios[0 indicate upregulation of gene expression.

Ratios\0 indicate downregulation. Error bars indicate standard

deviations for three replicates. *p \ 0.05 compared with the

control group. The designated genes are FAS1 fatty acid

synthetase beta subunit, HXK hexokinase II, PFK1 phospho-

fructokinase alpha subunit, HXT61 hexose transporter, PEX28

peroxisomal integral membrane protein, ENO1 enolase, DAK

dihydroxyacetone kinase, ASI3 ubiquitin protein ligase, GCN1

GCN4 translational activator, ARGS arginyl tRNA synthetase,

TEF1 translation elongation factor EF-1a, ACT b-actin

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C. albicans. Other genes obtained from reverse-

subtracted cDNA libraries have important functions

in glycolysis, such as those encoding phosphofructo-

kinase-1 (PFK1), HXK, HXT61, and dihydroxyace-

tone kinase (DAK). PFK is a key rate-controlling

enzyme in glycolysis. The b-subunit of phosphofruc-

tokinase (PFK1) is specific to, and important in,

glycolysis (Schmidt et al. 2008). The HXK is enzyme

involved in a highly conserved glycolytic pathway and

is upregulated in the transition of C. albicans from the

yeast to the hyphal state (Ebanks et al. 2006). In

addition, glucose metabolism transporter and hexose

transporter (HXT61) are downregulated in response to

e-PL treatment. The expression and function of HXT

genes are regulated by varying glucose levels (Ozcan

and Johnston 1999). DAK is an enzyme that converts

glycerol to dihydroxyacetone phosphate, an interme-

diate molecule in glycolysis (Yu et al. 2010).

Decreased expression of these genes would have an

important impact on glycolysis. Thus, e-PL may

inhibit carbohydrate metabolism in C. albicans by

down-regulating the expressions of glycolysis-associ-

ated genes.

In addition to glycolytic genes, genes involved in

fatty acid and amino acid metabolism were obtained

from subtracted cDNA libraries. The FAS1 gene

encodes the b-subunit of fatty acid synthase (FAS).

FAS is associated with cell membrane synthesis, and

fatty acids are used to produce phospholipids, the

major components of the cell membrane (Tamano

et al. 2013). Decreased FAS expression could signif-

icantly decrease the production of these compounds.

The peroxisome is also important in lipid metabolism

and free radical detoxification in yeast. PEX28 is a

peroxin protein that controls peroxisome size and

peroxisome proliferation (Vizeacoumar et al. 2004).

Decreased expression of PEX28 gene after e-PL

treatment may lead to disordered peroxisome biogen-

esis in C. albicans.

ASI3, and its homologous protein ASI1, contain a

conserved and cytoplasmically oriented ubiquitin

ligase-like RING domains at their extreme C-termini

(Forsberg et al. 2008). These proteins also regulate

ubiquitin-mediated processing activity in the ubiqui-

tin–proteasome degradation pathway (Weissman et al.

2011). Ubiquitin protein ligases are specific compo-

nents of the ubiquitin–proteasome pathway. Upregu-

lation of ASI3 indicated that the e-PL antimicrobial

activity might involve enhanced protein degradation.

AGRS is an aminoacyl-tRNA synthetase (AARS)

Fig. 4 Northern blot hybridization analysis of the expression of

ENO1, ASI3 and ACT in C. albicans during exposure to e-PL

from 0 to 3 h using specific DIG-High-labeled PCR products as

probes. a Northern blot analysis. b Band intensity analysis.

Lane 1 RNA from Candida albicans. Lane 2 RNA from C.

albicans treated with e-PL for 1 h. Lane 3 RNA from C. albicans

treated with e-PL for 3 h. The designated genes are ENO1

enolase, ASI3 ubiquitin protein ligase, ACT b-actin

2152 Biotechnol Lett (2013) 35:2147–2153

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involved in the first step of protein synthesis, which

produces aminoacyl-tRNAs as building blocks (Zhou

et al. 2013). Transcription factor GCN4 is involved in

coordinating metabolic and morphogenetic responses

to amino acid starvation induced by activated eIF2a

kinase (GCN2) and GCN1 (Kubota et al. 2000).

Increased expression of these three genes suggested

that the cell structures of C. albicans were destabilized

and that amino acid starvation was induced during e-

PL exposure.

Antimicrobial peptides function by inhibiting cell

adhesion and aggregation during C. albicans infection

by interacting with cell wall components (Tsai et al.

2011). As a classic cationic antimicrobial agent, e-PL

binds to the cell surface and interacts with proteins

(Shima et al. (1984)). The response of the cell surface

proteins may affect the transduction of nutrient-

(ligand)-induced signals, which are localized in the

plasma membrane and transduce information regard-

ing the presence of extracellular glucose, amino acids

and other factors (Forsberg and Ljungdahl 2001). In

conclusion, our results demonstrated that the antimi-

crobial activity of e-PL caused a series of changes in

genes’ expressions and affected various metabolic

processes in C. albicans.

Acknowledgments This work was supported by ‘‘Twelfth

Five-Year Plan’’ for National Science and Technology Support

Program (2011BAK10B06, 2011BAK10B01, 2011BAK10B02),

and State Key Laboratory of Food Science and Technology,

Nanchang University (SKLF-TS-200916, SKLF-MB-201002).

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