刚地弓形虫入侵人包皮成纤维细胞前后转录组差异分析

doi:10.12140/j.issn.1000-7423.2021.04.010

中国寄生虫学与寄生虫病杂志 ›› 2021, Vol. 39 ›› Issue (4): 480-486.doi: 10.12140/j.issn.1000-7423.2021.04.010

刚地弓形虫入侵人包皮成纤维细胞前后转录组差异分析

王龙江(), 李瑾, 尹昆, 徐超, 刘功振, 黄炳成, 魏庆宽, 孙慧^*()

山东省寄生虫病防治研究所,山东第一医科大学&山东省医学科学院,济宁 272033

收稿日期:2020-12-08 修回日期:2021-02-23 出版日期:2021-08-30 发布日期:2021-08-06
通讯作者: 孙慧
作者简介:王龙江（1988-）,男,硕士研究生,从事寄生虫病防治研究。E-mail： ljw880108@163.com
基金资助:
山东省重点研发项目(2019GSF107054);国家自然科学基金(81501770);山东省医药卫生科技发展计划项目(2018WSA303);山东省医药卫生科技发展计划项目(2017WS103);山东省医学科学院青年项目(2018-26);山东第一医科大学学术提升计划(2019QL005)

Comparative analysis of transcriptomes in Toxoplasma gondii before and after invasion in human foreskin fibroblasts

WANG Long-jiang(), LI Jin, YIN Kun, XU Chao, LIU Gong-zhen, HUANG Bing-cheng, WEI Qing-kuan, SUN Hui^*()

Shandong Institute of Parasitic Diseases, Shandong First Medical University & Shandong Academy of Medical Sciences, Jining 272033, China

Received:2020-12-08 Revised:2021-02-23 Online:2021-08-30 Published:2021-08-06
Contact: SUN Hui
Supported by:
Key Research and Development Program of Shandong Province(2019GSF107054);National Natural Science Foundation of China(81501770);Projects of Medical and Health Technology Development Program in Shandong Province(2018WSA303);Projects of Medical and Health Technology Development Program in Shandong Province(2017WS103);Project of Shandong Academy of Medical Sciences(2018-26);Academic Promotion Programme of Shandong First Medical University(2019QL005)

摘要/Abstract

摘要：

目的利用RNA-seq技术分析刚地弓形虫入侵人包皮成纤维（HFF）细胞前后转录组的差异。方法将纯化的刚地弓形虫RH株速殖子与HFF细胞按3 : 1的比例共培养,37 ℃、5% CO₂培养24 h后收集HFF细胞和弓形虫共培养物（感染组）,以纯化弓形虫RH株速殖子作为对照组。提取感染组和对照组总RNA,纯化mRNA并构建转录组文库,利用BGISEQ-500平台进行高通量测序,测序获得的质控数据与NCBI数据库中的弓形虫基因组（GCF_000006565.2_TGA4_ncbi）进行比对,对感染组与对照组相比差异倍数 ≥ 2.0且P < 0.05的差异表达基因进行功能注释、基因本体（GO）功能富集分类、富集和京都基因与基因组百科全书(KEGG)通路分析以及蛋白相互作用网络分析。结果共测序获得5 888个基因,差异基因986个,其中上调基因533个,下调基因453个。GO分析结果显示,富集的GO条目分别为膜、膜的组成部分、膜的固有成分、膜相关组分等。KEGG富集通路主要为胃癌、脂肪酸生物合成、钙离子信号等通路。蛋白相互作用网络分析结果显示,相互作用蛋白数最多的6个蛋白分别是TGME49_282200、TGME49_305980（PDHE3I）、TGME49_316310（SOD）、TGME49_310440（MORN1）、TGME49_237110和TGME49_222020（PGKII）。结论弓形虫RH株速殖子入侵HFF细胞前后转录组存在差异,差异表达基因KEGG富集通路主要为胃癌、脂肪酸生物合成、钙离子信号等通路,筛选出6个相互作用蛋白数量最多的蛋白基因。

关键词: 刚地弓形虫, 转录组, 差异表达基因

Abstract:

Objective To investigate transcriptome changes in Toxoplasma gondii before and after invasion in human foreskin fibroblasts (HFF) by RNA-seq. Methods Tachyzoites of T. gondii RH strain were added to monolayered HFF cells at a parasite-cell infection ratio of 3 : 1 and incubated at 37 ℃ with 5% CO₂ for 24 h. Following the incubation, the cells (infection group) were harvested and RNA extracted, using tachyzoites of RH strain as a blank control (control group). The mRNA was purified for high-throughput sequencing using the BGISEQ-500 platform. The eligible sequence data obtained were aligned with T. gondii genome data (GCF_000006565.2_TGA4_ncbi） in NCBI database. Differentially expressed genes (2.0-fold change with a P-value of < 0.05 in the infection group in comparison with the control group) were selected for Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and protein interaction network analysis. Results A total of 5 888 genes were detected, 986 differentially expressed genes were identified, in which 533 upregulated and 453 downregulated. GO analysis showed that the GO items significantly enriched were membrane, integral component of membrane, intrinsic component of membrane, and membrane part. KEGG enrichment pathways mainly involving gastric cancer, fatty acid biosynthesis, and calcium signaling pathway. In protein-protein interaction network analysis, the top six interacting proteins were TGME49_282200, TGME49_305980 (PDHE3I), TGME49_316310 (SOD), TGME49_310440 (MORN1), TGME49_237110 and TGME49_222020 (PGKII). Conclusion There are significant differences in transcriptome in T. gondii before and after invasion in HFF cells. The KEGG enrichment pathways mainly involve gastric cancer, fatty acid biosynthesis, and calcium signaling pathway, and six top genes with regard to the number of interaction proteins are screened.

Key words: Toxoplasma gondii, Transcriptome, Differentially expressed gene

中图分类号:

R382.5

王龙江, 李瑾, 尹昆, 徐超, 刘功振, 黄炳成, 魏庆宽, 孙慧. 刚地弓形虫入侵人包皮成纤维细胞前后转录组差异分析[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(4): 480-486.

WANG Long-jiang, LI Jin, YIN Kun, XU Chao, LIU Gong-zhen, HUANG Bing-cheng, WEI Qing-kuan, SUN Hui. Comparative analysis of transcriptomes in Toxoplasma gondii before and after invasion in human foreskin fibroblasts[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2021, 39(4): 480-486.

图/表 4

图1

图2

图3

表1

弓形虫入侵HFF细胞前后差异表达基因 KEGG 通路分析

序号 No.	通路序号 Pathway ID	代谢通路 Metabolism pathway	差异基因数量 No. of differentially expressed proteins	基因名称 Gene name
1	ko05226	胃癌 Gastric cancer	22	ROP18、GAP50、ALV12、ALV4、GRA15、SUB7、TGME49_208370、TGME49_209050、TGME49_220240、TGME49_220280、TGME49_220640、TGME49_224150、TGME49_226950、TGME49_239020、TGME49_240060、TGME49_254630、TGME49_267450、TGME49_278090、TGME49_278370、TGME49_287960、TGME49_301690、TGME49_320020
2	ko00061	脂肪酸生物合成 Fatty acid biosynthesis	4	ACC1、TGME49_225990、TGME49_231890、TGME49_293590
3	ko04020	钙离子信号通路 Calcium signaling pathway	12	ROP19A、CAM1、PMCAA1、TGME49_209985、TGME49_220420、TGME49_226540、TGME49_297670、TGME49_300360、TGME49_311260、TGME49_318675、TGME49_322000、TGME49_322010
4	ko04024	cAMP信号通路 cAMP signaling pathway	11	IMC2A、CAM1、PMCAA1、TGME49_209050、TGME49_209985、TGME49_220420、TGME49_226540、TGME49_293000、TGME49_294190、TGME49_297670、TGME49_311260
5	ko04144	内吞作用 Endocytosis	13	TGME49_209985、TGME49_212300、TGME49_220460、TGME49_239580、TGME49_242860、TGME49_251170、TGME49_252490、TGME49_262950、TGME49_291800、TGME49_305270、TGME49_313450、TGME49_315210、TGME49_315350
6	ko04657	IL-17信号通路 IL-17 signaling pathway	14	ROP18、IMC6、TGME49_209050、TGME49_212810、TGME49_217540、TGME49_219270、TGME49_220640、TGME49_224150、TGME49_240810、TGME49_250115、TGME49_254630、TGME49_268870、TGME49_271970、TGME49_320150
7	ko04927	皮质醇合成与分泌Cortisol synthesis and secretion	4	TGME49_209985、TGME49_224840、TGME49_293000、TGME49_297670
8	ko04540	间隙连接 Gap junction	5	TUBA1、TGME49_209985、TGME49_212240、TGME49_221620、TGME49_297670
9	ko00300	赖氨酸生物合成 Lysine biosynthesis	2	TGME49_227090、TGME49_234440
10	ko03420	核苷酸切除修复Nucleotide excision repair	6	GTF2H3、PCNA2、TGME49_203170、TGME49_237110、TGME49_254640、TGME49_319860

表1

参考文献 24

[1]	Black MW, Boothroyd JC. Lytic cycle of Toxoplasma gondii[J]. Microbiol Mol Biol Rev, 2000, 64(3):607-623. doi: 10.1128/MMBR.64.3.607-623.2000
[2]	Flegr J. How and why Toxoplasma makes us crazy[J]. Trends Parasitol, 2013, 29(4):156-163. doi: 10.1016/j.pt.2013.01.007
[3]	Pan M, Lyu C, Zhao J, et al. Sixty years (1957—2017) of research on toxoplasmosis in China: an overview[J]. Front Microbiol, 2017, 8:1825. doi: 10.3389/fmicb.2017.01825
[4]	Hsu PC, Groer M, Beckie T. New findings: depression, suicide, and Toxoplasma gondii infection[J]. J Am Assoc Nurse Pract, 2014, 26(11):629-637. doi: 10.1002/2327-6924.12129
[5]	Liang ZH. Research progress of Toxoplasma gondii invading host cells mechanism[J]. Chin J Zoonoses, 1995, 11(5):34-36. (in Chinese)
	(梁志慧. 弓形虫速殖子侵入宿主细胞机理的研究进展[J]. 中国人兽共患病杂志, 1995, 11(5):34-36)
[6]	Peng HJ. Formation mechanism and the function of parasitophorous vacuole of Toxoplasma gondii[J]. Chin J Parasitol Parasit Dis, 2010, 28(5):382-386. (in Chinese)
	(彭鸿娟. 刚地弓形虫纳虫泡的形成机制及其作用[J]. 中国寄生虫学与寄生虫病杂志, 2010, 28(5):382-386.)
[7]	Gajria B, Bahl A, Brestelli J, et al. ToxoDB: an integrated Toxoplasma gondii database resource[J]. Nucleic Acids Res, 2008, 36:D553-D556. doi: 10.1093/nar/gkm981
[8]	Xia D, Sanderson SJ, Jones AR, et al. The proteome of Toxoplasma gondii: integration with the genome provides novel insights into gene expression and annotation[J]. Genome Biol, 2008, 9(7):R116. doi: 10.1186/gb-2008-9-7-r116
[9]	Li X, Yan JH, Chen HL, et al. Research advances on the transcriptome of mosquitoes[J]. Acta Parasitol et Med Entomol Sin, 2019, 26(3):199-208. (in Chinese)
	(李许桑妮, 闫冀焕, 陈翰林, 等. 蚊虫转录组学研究进展[J]. 寄生虫与医学昆虫学报, 2019, 26(3):199-208.)
[10]	Swierzy IJ, Händel U, Kaever A, et al. Divergent co-transcriptomes of different host cells infected with Toxoplasma gondii reveal cell type-specific host-parasite interactions[J]. Sci Rep, 2017, 7(1):7229. doi: 10.1038/s41598-017-07838-w
[11]	Song LD. Transcriptional analysis of porcine macrophages infected with different genotypes of Toxoplasma gondii[D]. Wuhan: Huazhong Agricultural University, 2018: 32-43. (in Chinese)
	(宋林栋. 猪巨噬细胞感染不同基因型弓形虫后的转录组研究[D]. 武汉: 华中农业大学, 2018: 32-43.)
[12]	Zhou CX, Elsheikha HM, Zhou DH, et al. Dual identification and analysis of differentially expressed transcripts of porcine PK-15 cells and Toxoplasma gondii during in vitro infection[J]. Front Microbiol, 2016, 7:721.
[13]	Xu LF, Yang QL, Zhang YK, et al. In vitro cultivation of Toxoplasma gondii tachyzoites RH strain in human foreskin fibroblasts[J]. Chin J Parasit Dis Control, 2004, 22(5):268-269. (in Chinese)
	(许丽芳, 杨秋林, 张愉快, 等. 用包皮成纤维细胞培养弓形虫速殖子的研究[J]. 中国寄生虫病防治杂志, 2004, 22(5):268-269)
[14]	Wu L, Zhang QX, Li TT, et al. In vitro culture of Toxoplasma gondii tachyzoites in HFF and Hela cells[J]. Chin J Parasitol Parasit Dis, 2009, 27(3):229-231. (in Chinese)
	(吴亮, 章秋霞, 李婷婷, 等. 人包皮成纤维细胞和人子宫颈癌细胞体外培养弓形虫速殖子[J]. 中国寄生虫学与寄生虫病杂志, 2009, 27(3):229-231.)
[15]	Mortazavi A, Williams BA, McCue K, et al. Mapping and quantifying mammalian transcriptomes by RNA-Seq[J]. Nat Methods, 2008, 5(7):621-628. doi: 10.1038/nmeth.1226 pmid: 18516045
[16]	Wang CB, Lu WH, Lin Y, et al. Development and application of transcriptome sequencing[J]. Eucalypt Sci Technol, 2018, 35(4):20-26. (in Chinese)
	(王楚彪, 卢万鸿, 林彦, 等. 转录组测序的发展和应用[J]. 桉树科技, 2018, 35(4):20-26.)
[17]	Wilhelm BT, Marguerat S, Watt S, et al. Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution[J]. Nature, 2008, 453(7199):1239-1243. doi: 10.1038/nature07002
[18]	Chen MX, Chen JX. Application of DNA microarray technology in human trypanosomiasis research[J]. Chin J Parasitol Parasit Dis, 2016, 34(4):377-381. (in Chinese)
	(陈木新, 陈家旭. 基因芯片技术在人体锥虫生物学特性研究方面的进展[J]. 中国寄生虫学与寄生虫病杂志, 2016, 34(4):377-381.)
[19]	Soldati D, Dubremetz JF, Lebrun M. Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii[J]. Int J Parasitol, 2001, 31(12):1293-1302. pmid: 11566297
[20]	Kato K. How does Toxoplama gondii invade host cells?[J]. J Vet Med Sci, 2018, 80(11):1702-1706. doi: 10.1292/jvms.18-0344
[21]	Zhang Y, Lai BS, Juhas M, et al. Toxoplasma gondii secretory proteins and their role in invasion and pathogenesis[J]. Microbiol Res, 2019, 227:126293. doi: S0944-5013(19)30471-9 pmid: 31421715
[22]	Robinson SA, Smith JE, Millner PA. Toxoplasma gondii major surface antigen (SAG1): in vitro analysis of host cell binding[J]. Parasitology, 2004, 128(pt 4):391-396. pmid: 15151144
[23]	Tomavo S. The major surface proteins of Toxoplasma gondii: structures and functions[J]. Curr Top Microbiol Immunol, 1996, 219:45-54. pmid: 8791687
[24]	Engelberg K, Ivey FD, Lin A, et al. A MORN1-associated HAD phosphatase in the basal complex is essential for Toxoplasma gondii daughter budding[J]. Cell Microbiol, 2016, 18(8):1153-1171. doi: 10.1111/cmi.12574 pmid: 26840427

刚地弓形虫入侵人包皮成纤维细胞前后转录组差异分析

Comparative analysis of transcriptomes in Toxoplasma gondii before and after invasion in human foreskin fibroblasts

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