中国球形孢子丝菌双相cDNA消减文库构建及其差异基因的筛选和验证

Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes 中国球形孢子丝菌双相cDNA消减文库构建及其差异基因的筛选和验证 1 first-author 1 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zhouxun123@sina.com †E-mail:zhouxun123@sina.com zhouxun123@sina.com Department of Dermatology, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China Department of Dermatology, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China Department of Dermatology and Cosmetology, Chongqing Traditional Chinese Medicine Hospital, Chongqing 400021, China Department of Dermatology and Cosmetology, Chongqing Traditional Chinese Medicine Hospital, Chongqing 400021, China Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis , S. globosa , S. mexicana , S. pallida , S. schenckii , and S. lurei . S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa , suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China. 目的 2 为进一步研究中国球形孢子丝菌的双向转换机制，明确目的差异基因对我国球形孢子丝菌形态和双相转换的影响，探讨可能参与的信号传导通路，以期较全面地探讨孢子丝菌双相转换相关的分子机制，也为筛选抗真菌药物研究提供新的靶位和切入点。 创新点 2 以中国球形孢子丝菌双向转换为立足点，应用抑制性消减文库成功构建的高特异性的中国球形孢子丝菌酵母相（Y）和菌丝相（M）的正反cDNA基础上，对中国球形孢子丝菌双相转换相关基因进行生物信息学分析并对部分有意义的差异表达基因进行验证。 方法 2 本实验通过抑制性消减杂交技术构建中国球形孢子丝菌酵母相和菌丝相的正反cDNA消减文库，并对差异基因进行生物信息学分析，获得部分有意义的差异表达基因，再通过实时荧光聚合酶链反应（PCR）技术验证目的基因在双相中的差异表达。 结论 2 通过抑制性消减杂交技术对中国球形孢子丝菌的正向和反向两个文库进行筛选，获得可能参与球形孢子丝菌细胞壁合成、信号转到、新陈代谢类等相关的片段重叠群（contigs），结果表明中国球形孢子丝菌在双相型转换中存在明显表达差异基因，同时利用相对定量的实时荧光定量技术对其中两个基因（细胞表面分子类基因细胞周期检测点激酶CHK和结构基因类核糖体RNA基因）进行验证，认为它们可能与双相转换有关。 Sporothrix globosa Dimorphism Suppression subtractive hybridization (SSH) Real-time PCR 球形孢子丝菌 双相 抑制性消减杂交技术（SSH） 实时荧光PCR技术 Introduction 1 Species included in the Sporothrix schenckii complex are the etiological agents of sporotrichosis, which is characterized by acute or chronic lymphocutaneous lesions that can develop into disseminated disease in immunocompromised individuals (Yap, 2011 2011 ). The S. schenckii complex includes S. brasiliensis , S. globosa , S. mexicana , S. pallida , S. schenckii , and S. schenckii luriei (Marimon et al., 2006 2006 ; 2007 2007 ; Madrid et al., 2010 2010 ). Different species of Sporothrix are distributed in various regions of the world (Marimon et al., 2007 2007 ; Zhou et al., 2013 2013 ). For example, S. brasiliensis is found only in Brazil, and S. schenckii is present mainly in the Americas, Africa, and parts of Asia. Different clinical forms of sporotrichosis are related to the condition of the host, route of infection, and virulence of the pathogen, and virulence may be associated with different Sporothrix species (Arrillaga-Moncrieff et al., 2009 2009 ; Fernandes et al., 2013 2013 ). Previous studies have shown that S. globosa is the causal agent of sporotrichosis in China (Tan et al., 2013 2013 ; Yu et al., 2013 2013 ; Liu et al., 2014 2014 ). Sporothrix includes pathogenic fungi that undergo dimorphic growth from mycelial to yeast phases in response to environmental conditions, such as temperature. In particular, the fungi grow in the filamentous form at 25 °C and in the yeast form at 37 °C. Sporotrichosis is caused by the multiplication of the yeast form (Li et al., 2007 2007 ). Its pathogenicity appears to be closely related to the dimorphic transition from the mycelial to the yeast phase in humans through an unknown mechanism. Several genes that are differentially expressed during the transition from yeast to mycelial forms have been identified. For example, protein kinase C (PKC) (Aquino-Pinero et al., 1997 1997 ), Pho85s (de Jesus-Berrios et al., 2002 2002 ), and calcium/calmodulin (Valle-Aviles et al., 2007 2007 ) may be involved in the regulation of dimorphism. Further, the cPLA2 homolog (Valentin-Berrios et al., 2009 2009 ) and a 45-kDa protein (Han-Yaku et al., 1996 1996 ) are involved in the dimorphic transition of S. schenckii . However, the mechanism of dimorphic transition is complicated and unclear. Although signal transduction modulated by factors such as mitogen-activated protein kinases, cyclic adenosine monophosphate (cAMP), and pH-responsive modules may regulate this transition, the mechanism of the dimorphic transition remains undetermined. Further, the species included in the S. schenckii complex may harbor different genes that mediate the dimorphic transition. Therefore, studies of the mechanism of the dimorphic transition are required to understand the mechanism of the pathogenesis of S. globosa . To identify differentially expressed genes during the dimorphic transition of S. globosa , suppression subtractive hybridization (SSH) was used. This is a powerful method for identifying differentially expressed genes in fungi (Marques et al., 2004 2004 ), plants (Zeng et al., 2006 2006 ), and tumors (Ma et al., 2005 2005 ). In the present study, differentially expressed sequence tags (ESTs) were isolated using SSH and bioinformatics analysis, and two genes were selected and identified using a relative quantitative real-time reverse transcriptase polymerase chain reaction (qRTPCR). These two genes reside within the mycelial (M)+yeast (Y)contig63 encoding structural genes and the M+Ycontig5 encoding cell-surface molecules. Materials and methods 1 Strains and culture conditions 2 The standard S. globosa strain (C26471) was acquired from the Dermatology Department of the First Affiliated Hospital, Chongqing Medical University (Chongqing, China). The strain was identified as S. globosa according to its morphology and internal transcribed spacer (ITS) region (GenBank No. JX997727) (Liu et al., 2014 2014 ). The cells were cultured in the mycelial phase on a freshly prepared potato dextrose agar (PDA) plating medium at 25 °C, and yeast-phase cells were obtained by culturing mycelial colonies for 7 d at 37 °C on a freshly prepared brain heart infusion (BHI) solid medium supplemented with 5 g/L glucose. Continuous culture for three generations insured stable and pure yeast cells. Construction of a complementary DNA (cDNA) subtraction library 2 Approximately 200 mg of mycelia (M) and yeast (Y) cells were extracted and purified directly using an Easy Spin Total Plant RNA Extraction Kit (Aidlab, China) according to the manufacturer’s instructions. The quality and quantity of total RNA preparations were evaluated using 1% (0.01 g/ml) agarose gel electrophoresis. The cDNA was synthesized and amplified using the SMART cDNA Amplification Kit (Clontech, USA) with 1.0 μg total RNA prepared from mycelial and yeast forms. The SSH assay was performed using the PCR-Select cDNA Subtraction Kit (Clontech, USA) following the manufacturer’s instructions. Briefly, the mycelial and yeast cDNAs were digested with Rsa I to generate cDNAs with blunt ends. Adaptors were ligated to tester cDNAs followed by two rounds of subtractive hybridization, two rounds each of PCR amplification, and product purification. Each product designated “M+Y” (cDNAs from yeast as tester and cDNAs from mycelium as driver) and “Y+M” (mycelium as tester and yeast as driver) was isolated using SSH according to the method of Diatchenko et al. ( 1996 1996 ). The “M+Y” and “Y+M” cDNAs were inserted into the pGEM (Promega, USA) plasmid vector and used to transform Escherichia coli TOP10, which was cultured on Luria broth (LB) agar plates with ampicillin, isopropyl-β- D -thiogalacopyranoside, and X-gal (Autolab, China). Recombinant bacteria were selected from white colonies, which were inoculated in 50 μl of LB containing 100 mg/L ampicillin and incubated at 37 °C for 5 h. The DNA of 1 μl of each LB culture was amplified in 25 μl of PCR reaction mixture using nested PCR primers T7 and SP6 (Clonetech PCR-Select cDNA Subtraction Kit) in a 96-well plate. PCR was performed as follows: 75 °C for 7 min, 20 cycles of 91 °C for 30 s, 68 °C for 30 s, and 72 °C for 2.5 min. The PCR product (10 μl) was analyzed using 2% (0.02 g/ml) agarose gel electrophoresis. The subtractive cDNA libraries were constructed and named the “M+Y” and “Y+M” libraries. DNA sequence analysis 2 Positive colonies with inserts > 250 bp and a single strip from two subtraction libraries were selected for sequence analysis to identify ESTs using forward primers T7 or SP6 and an ABI-9600 DNA sequencing system. Data processing of ESTs was performed by masking vector and repetitive sequences and by eliminating chimeric, contaminating, and poly(A) sequences. Consensus sequences were obtained by assembling ESTs and performing cluster analysis. We used the longest predicted open reading frame (ORF) of each unique gene. The differentially expressed sequences were used to query the National Center for Biotechnology Information (NCBI) databases using BLASTN ( http://www.ncbi.nlm.nih.gov/BLASTN http://www.ncbi.nlm.nih.gov/BLASTN ), BLASTP ( http://www.ncbi.nim.gov/BLASTP http://www.ncbi.nim.gov/BLASTP ), SwissPort ( http://www.expasy.org/sport/BLASTX http://www.expasy.org/sport/BLASTX ), Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/BLASTP http://www.genome.jp/kegg/BLASTP ), Clusters of Orthologous of proteins (COG; http://www.ncbi.nlm.nih.gov/COG/BLASTX http://www.ncbi.nlm.nih.gov/COG/BLASTX ), InterPro ( http://www.ebi.ac.uk/interpro/Interproscan http://www.ebi.ac.uk/interpro/Interproscan ), and Gene Ontology (GO; http://www.geneontology.org http://www.geneontology.org or http://www.ebi.ac.uk/GO/index.html http://www.ebi.ac.uk/GO/index.html ). Differentially expressed genes were annotated and classified according to the function. Relative quantization of mRNA expression using real-time RT-PCR 2 According to the results of the bioinformatics analyses described above, two genes were selected for further analysis using real-time RT-PCR. The mRNA encoding β-tubulin was selected as the internal control. Primers for target genes were designed and synthesized by Beijing Genomics Institute (BGI, China) as shown in Table 1 1 , and 700 ng of total RNA from mycelial and yeast phases of S. globosa were reverse-transcribed using the Prime Script RT Kit (TaKaRa, Japan). cDNAs (5 μl) were diluted 1:3 (v/v) with sterile water for use as the targets of RT-PCR. Sixteen nanograms of cDNAs were assayed from the mycelial and yeast phases of S. globosa in duplicate using SYBR Premix EX Taq II (TaKaRa, Japan). Control reactions without a template were performed in duplicate. Amplification was performed using a C1000 Thermal Cycler Dice Real-time system (Bio-Rad, USA) as follows: 95 °C, 30 s; 40 cycles at 95 °C for 5 s and 60 °C for 60 s. Melting of the PCR products was performed at 60 °C. T m : annealing temperature used in real-time RT-PCR with each primer pair Target cDNA Primer Primer sequence (5'→3') T m (°C) PCR product size (bp) 2 β-tubulin BTI GGTAACCAAATCGGTGCTGCTTTC 60.40 2 120 BTII ACCCTCAGTGTAGTGACCCTTGGC 63.82 2 M+Ycontig63 63I CCCATCAAACTGCTCAGAAA 60.00 2 150 63II GCATGCTACTTGTCCGTTTG 58.00 2 M+Ycontig5 5I AGCCGTCGAAAGTTGATAGG 60.00 2 143 5II CGACTCGGAAGGGATGTAT 60.00 Results 1 Sequence analysis of differentially expressed genes 2 Colonies with inserts ranging from 400 to 1000 bp were sequenced. We obtained 751 ESTs from the “M+Y” library (average 690.98 bp) and 875 ESTs were obtained from the “Y+M” library (average 575.9 bp). After deleting ESTs, 101 unique genes, including 52 contigs and 49 singlets, were obtained from the “M+Y” library and 249 unique genes, including 109 contigs and 140 singlets, were obtained from the “Y+M” library. There were 35 and 91 gene sequences in the “M+Y” and “Y+M” libraries, respectively, that are present in the NCBI nucleotide sequence database. Their putative identities, nucleotide sequence similarities with other sequences, and insert sizes are shown in Tables 2 2 and 3 3 . The distribution of differentially expressed genes varied with the type of dimorphic transition. Upregulated genes were divided into those encoding structural proteins, metabolic enzymes, cell-surface proteins, and molecules with unknown function. Approximately 40% of the EST sequences were similar to the coding regions of proteins involved in protein synthesis, including several ribosomal proteins. The remaining ESTs were related to genes involved in transcription (about 20%), metabolism (20%), and signaling (20%). Two genes were selected and identified using relative quantification real-time RT-PCR. These genes were present in M+Ycontig63 and M+Ycontig5 and encoded structural and cell-surface molecules, respectively. Clone name Size (bp) BLAST database match Identity (%) Score E -value M+Ycontig5 569 CHK1 checkpoint-like protein ( Helicoverpa armigera ) 98 959 0 M+Ycontig12 431 Sporothrix schenckii isolate CDM 18 28S ribosomal RNA gene 100 272 10 −69 M+Ycontig44 855 Leptographium brachiatum internal transcribed spacer 2 and 28S ribosomal RNA gene, partial sequence 99 422 10 −114 M+Ycontig46 609 Paenibacillus polymyxa strain PKB1 glutamine amidotransferase 97 1019 0 M+Ycontig63 905 Ophiostoma stenoceras 18S small subunit ribosomal RNA gene 100 1130 0 M+Ycontig68 638 O. stenoceras 12S small subunit ribosomal RNA gene 98 153 10 −33 Clone name Size (bp) BLAST database match Identity (%) Score E -value Y+Mcontig4 352 Bacillus polymyxa strain PW2 large subunit 23S ribosomal RNA gene 98 571 10 −160 Y+Mcontig7 492 pG1 protein ( Lactobacillus jensenii 269-3) 99 842 0 Y+Mcontig19 247 Paenibacillus pabuli partial 16S rRNA gene, partial 23S rRNA gene and ITS1, strain US132 98 321 10 −84 Y+Mcontig20 562 pG1 protein ( L. jensenii 269-3) 98 920 0 Y+Mcontig26 715 Uncultured bacterium 16S ribosomal RNA gene 99 1273 0 Y+Mcontig119 496 Paenibacillus 16S ribosomal RNA gene, partial sequence 99 860 0 Y+Mcontig156 559 Paenibacillus sp. Fars-87 16S ribosomal RNA gene, partial sequence 100 793 0 Y+Mcontig161 625 P. polymyxa strain PKB1 glutamine amidotransferase 97 1007 0 Relative quantification of gene expression using real-time RT-PCR 2 To enhance the reliability of SSH findings, the expression levels of two genes in two forms were analyzed using real-time RT-PCR. The results are shown in Table 4 4 . Two genes (M+Ycontig5, M+Ycontig63) present in the “M+Y” library were expressed at higher levels in the yeast phase. M and Y represent the mycelial and yeast forms, respectively. Δ C T =target transcript C T –β-tubulin C T (normalization of C T for target gene relative to β-tubulin C T ). Statistical analysis of normalized expression levels between mycelial and yeast forms. The levels of each target genes differed significantly ( U -test, P < 0.05). ΔΔ C T =mean YΔ C T −mean MΔ C T : the mean value for the MΔ C T was used as a calibrator to set the baseline for comparing the mean differences in the Δ C T values of the yeast form. * Normalized target amount relative to mycelial form. The relative abundance of the corresponding mRNA expressed by the yeast form compared with that of the mycelial form. “↑”and “↓” indicate up-regulation and down-regulation of gene expression by the yeast form compared with that of the mycelial form, respectively Clone name Phase a a Target C T Tublin C T Δ C T b b ΔΔ C T c c 2 −ΔΔ C T* RA d d M+Ycontig63 M 27.58±0.16 25.48±0.11 2.10±0.04 0.00±0.04 1 Y 22.48±0.23 24.42±0.01 −1.94±0.23 −4.04±0.23 16.45 ↑16.45 M+Ycontig5 M 24.89±0.08 25.48±0.11 −0.59±0.03 0.00±0.03 1 Y 12.77±0.48 24.42±0.01 −11.65±0.49 −11.06±0.49 2134.97 ↑2134.97 Discussion 1 Sporotrichosis is one of the most common diseases caused by deep fungal infection. The symptoms of sporotrichosis include localized nodular lesions, bumps, and chronic ulcers at the point of entry through small cuts and abrasions in the skin as well as along lymph nodes and vessels (Schubach et al., 2008 2008 ). The incidence of sporotrichosis is increasing worldwide, particularly in regions with tropical and temperate climates (Schubach et al., 2008 2008 ). For example, the incidence of sporotrichosis in the South Central Highlands of Peru ranges from 48 to 60 cases per 100 000 persons and is the highest among children aged 7–14 years, approaching one case per 1000 persons (Pappas et al., 2000 2000 ). The annual incidence of sporotrichosis (6.2%) in Jilin Province in China is relatively high (Wang et al., 1998 1998 ), and sporotrichosis represents approximately 2.2% of 10 982 cases of dermatomycosis in the Chengdu area of China (Rang et al., 1999 1999 ). The genotypes of S. schenckii are diverse (MesaArango et al., 2002 2002 ). For example, de Beer (2003) discovered that S. schenckii represents more than one species, and Marimond (2007) identified at least six putative species, including four clinically relevant species as follows: S. schenckii sensu stricto , S. brasiliensis , S. globosa , and S. lurei. S. globosa causes sporotrichosis in China, and genes encoding proteins such as SSCMK1 and PhoSs differ from those identified using a cDNA subtraction library. Therefore, to address this urgent public health issue in China, in particular, it is important to identify the genes that determine pathogenicity during the transition from the mycelial to the yeast form of S. globosa . To address this issue, the SSH technique was employed to identify genes differentially expressed during the transition from the hyphal to the yeast form. The M+Ycontig5 was the most highly represented SSH sequence and was differentially expressed by a factor of 2134.97 by the yeast form. The predicted amino acid sequence of the M+Ycontig5 is 98% similar to that of the cell cycle checkpoint kinase (CHK1)-like protein of Helicoverpa armigera . CHK1 is a serine/threonine protein kinase, evolutionarily conserved, and plays pivotal roles in DNA repair, cell cycle arrest, and in inducing cell death (Chen et al., 2004 2004 ). For example, entry into mitosis is inhibited by the activation of CHK1 kinase in response to damaged DNA in late S and G2 phases of the cell cycle, which protects fungi against attack from macrophages or neutrophils (Martinho et al., 1998 1998 ; Furuya et al., 2010 2010 ). Furuya and NiKi (2010) found that activation of CHK1 is sufficient to induce hyphal differentiation of S. japonicus , which promotes survival in the continuously changing nutritional environment and imparts motility upon this otherwise nonmotile organism (Sipiczki et al., 1998 1998 ). Thus, the CHK1 signaling pathway arrests the cell cycle in response to checkpoint activation and promotes hyphal differentiation to avoid or mitigate sources of stress. Su and Cheng ( 2013 2013 ) studied Candida albicans and found that CHK1 affects the reproduction and formation of chlamydospores and hyphae and mediates tolerance to environmental stressors. The present findings support the hypothesis that the M+Ycontig5 might encode a specific entry point required for the dimorphic transition. The sequence of the M+Ycontig63 is identical to that of the gene encoding the 18S small subunit ribosomal RNA of Ophiostoma stenoceras , and its expression was increased by a factor of 16.45 in the yeast form of S. schenckii . O. stenoceras is an ophiostomatoid fungus, which is a teleomorph of secondary conidia on substrate mycelium, and is included in the S. schenckii–O. stenoceras complex (de Meyer et al., 2008 2008 ). Moreover, evidence indicates that 18S and 28S RNAs (Fleischmann et al., 2004 2004 ) mediate the transient upregulation of RNA polyadenylation, suggesting a possible role in regulating the polymorphic phenotype of C. albicans . The data presented here suggest that 18S and 28S rRNA genes may be expressed in the mycelial and yeast forms of S. schenckii . In summary, the results of the present study suggest that the dimorphic transition of S. schenckii is mediated through complex mechanisms. The expression levels of differentially expressed mRNAs identified here may be required for the dimorphic transition and pathogenicity. The transcription of these genes might be induced by HSP70 (Huang et al., 2007 2007 ). Quantitative comparisons using β-tubulin to normalize expression levels were difficult. One disadvantage is the dependence on a standard that is expressed at variable levels. 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1500151 Project supported by the National Natural Science Foundation of China (No. 31270062) 2015-06-21 2015-11-18 2015-11-09 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2015 2015 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 12 16