Isolation, identification, and omics analysis of extracellular vesicles from Trichinella spiralis newborn larvae

doi:10.12140/j.issn.1000-7423.2024.02.014

Abstract

Abstract:

Objective To isolate and identify the main components of extracellular vesicles (EV) secreted by Trichinella spiralis newborn larvae and elucidate the function of these EVs. Methods T. spiralis muscle larvae were sourced from the muscles of mice, which had been utilized for T. spiralis preservation. The small intestine was excised 5 days after infected with muscle larvae, and T. spiralis adults were obtained through mesh screening. These adults were then cultured in RPMI 1640 medium for 48 hours to yield newborn larvae. EVs were collected by differential ultracentrifugation from newborn larvae culture fluid of T. spiralis, and were observed morphologically under transmission electron microscope, and the diameter of the newborn larvae EVs was measured by nanoparticle tracking analysis. The EV protein components were verified using Western blotting, and analyzed by proteomic mass spectrometry. RNA sequencing technology was applied to analyze the miRNA sequences of the newborn larvae and their secreted EVs. The differences of protein components and differential expression between newborn larvae and their EVs were compared using t-test. The differentially expressed proteins and miRNA target genes were analyzed by gene ontology (GO) functional item enrichment and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment. WoLFPSORT was used to analyze the subcellular localization of proteins. Results Transmission electron microscopy and nanoparticle tracking analysis revealed that T. spiralis newborn larvae produce cup-shaped, membranous vesicles with a diameter of approximately (101.7 ± 1.8) nm. Western blotting demonstrated that the newborn larvae-derived EVs generate specific bands when reacted with sera from mice infected with Trichinella spiralis, with a approximate molecular weight of 65 000. Through proteomics mass spectrometry, 1 424 and 231 proteins were detected in the newborn larvae and EV samples, respectively. Among these, 220 proteins were expressed in both groups, with 116 displaying differential expression patterns (31 upregulated and 85 downregulated in EVs). The subcellular localization analysis revealed that the differentially expressed proteins were predominantly localized in the cytoplasm (30.2%, 35/116), nucleus (19.0%, 22/116), and cell membrane (17.2%, 20/116). miRNA sequencing data showed that 183 and 117 miRNAs were detected in newborn larvae and EV samples, respectively; 46 miRNAs were found in both groups, with 40 showing differential expression patterns (10 upregulated and 30 downregulated in EVs). Notably, let-7-5p, miR-125-5p, miR-10, and miR-92 exhibited higher levels of upregulation in newborn larvae-derived EVs. The GO functional enrichment analysis indicated that the differentially expressed proteins could be categorized into three main groups: cell composition (57), molecular function (73), and biological processes (63). The target genes of differentially expressed miRNA were primarily associated with autophagy-related genes such as transferase activation of autophagy-related 12 and ubiquitin linkage enzyme complex. KEGG pathway enrichment analysis suggested that the differentially expressed proteins were significantly enriched in metabolic pathways and protein processing in endoplasmic reticulum-related pathways, whereas the target genes of differentially expressed miRNA were only involved in the autophagy-other pathway. Conclusion This study successfully isolated and identified the EVs secreted by newborn larvae of T. spiralis. 116 differentially expressed proteins and 40 miRNAs were identified between the newborn larvae and their EVs, predominantly participating in metabolic, autophagic and other pathways, which inferring that newborn larvae EVs may play important roles in evading host immune defence at early Trichinella infection.

Key words: Trichinella spiralis, Newborn larvae, Extracellular vesicles, Proteomics, miRNA sequencing

CLC Number:

R383.15

LIU Yi, CAI Yuchun, CHEN Jiaxu, CHEN Shaohong, YU Yingfang. Isolation, identification, and omics analysis of extracellular vesicles from Trichinella spiralis newborn larvae[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2024, 42(2): 225-233.

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Uniprot 登录号 Uniprot ID	蛋白质名称 Protein name	亚细胞定位 Subcellular location	MISEV 2023分类 MISEV 2023 categories
A0A0V1BH03	镁转运蛋白1 Magnesium transporter protein 1	细胞膜 Plasma membrane	1a
A0A0V1BT16	代谢型谷氨酸受体2 Metabotropic glutamate receptor 2	细胞膜 Plasma membrane	1a
A0A0V1BZ62	P型铜离子转运蛋白 P-type Cu(+) transporter	细胞膜 Plasma membrane	1a
A0A0V1BT87	烯醇化酶 Phosphopyruvate hydratase	线粒体 Mitochondria	2b
A0A0V1AYF7	α-肌动蛋白 Alpha-actinin	细胞质 Cytoplasm	2b
E5SLU1	肌动蛋白5C Actin-5C	细胞骨架 Cytoskeleton	2b
A0A0V1C0Z2	微管蛋白α链 Tubulin alpha chain	线粒体 Mitochondria	2b
A0A0V1BAN8	微管蛋白α-3链 Tubulin alpha-3 chain	线粒体 Mitochondria	2b
E5SWF2	微管β链 Tubulin beta chain	细胞质 Cytoplasm	2b

miRNA名称 miRNA_name	miRNA序列（5'→3'） miRNA sequence (5'→3')	新生幼虫组平均表达量 Average expression level of newborn larvae	EV组平均表达量 Average expression level of EVs	t值 t value	P值 P value
ngi-miR-92a_L + 1_1ss21AG	TATTGCACTTGTCCCGGCCTGT	25	13 582	6.978	< 0.05
mle-miR-92b-3p_1ss11TG	TATTGCACTTGTCCCGGCCTGT	25	13 582	6.978	< 0.05
tca-mir-3885-p3_1ss3AG	GAGGGCGGCGGCGGCGGC	0	623	5.228	< 0.05
tca-miR-10-5p_1ss12TA	TACCCTGTAGAACCGAATTTGT	32	11 644	5.026	< 0.05
lgi-miR-10_1ss12TA	TACCCTGTAGAACCGAATTTGT	32	11 644	5.026	< 0.05
tca-miR-125-5p	TCCCTGAGACCCTAACTTGTGA	208	17 769	4.880	< 0.05
lgi-miR-100_R + 1_1ss16AT	AACCCGTAGATCCGATCTTGTGA	80	4 597	4.625	< 0.05
cel-let-7-5p	TGAGGTAGTAGGTTGTATAGTT	116	85 725	4.501	< 0.05
tca-mir-3885-p5_1ss16CG	GGCGGCGGCGGCGGCGGA	0	31	4.429	< 0.05
tca-mir-3885-p3_1ss16CG	GGCGGCGGCGGCGGCGGA	0	31	4.429	< 0.05