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Proteomic analysis of the response of Trichinella spiralis muscle larvae to exogenous nitric oxide.

ABSTRACT: Trichinella spiralis mainly dwells in the muscle tissue of its host and is the main causative agent of trichinellosis in humans. Nitric oxide (NO), an important intracellular signaling molecule that may restrict pathogen growth in infected hosts, has been known for its anti-pathogenic activity, including resistance to T. spiralis. Herein, we applied label-free analysis to investigate the effect of sodium nitroprusside (SNP, a NO donor compound) on the proteome of T. spiralis muscle larvae (ML), followed by Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway cluster analyses. Of the 1,476 proteins detected in the ML, 121 proteins showed differential expression, including 50 significantly upregulated and 71 downregulated proteins. The functions of the 108 annotated proteins were primarily related to signal transduction, transcription/translation, material metabolism, protein synthesis/assembly/degradation, and stress/defense/antioxidation. Quantitative real-time polymerase chain reaction (qRT-PCR) assay verified that FRMD5 and CUT-1 gene expression levels were significantly increased, while COX2 gene expression level was significantly decreased. GO annotation and KEGG pathway analyses showed that the majority of differentially expressed proteins were mainly involved in the molecular function of the catalytic activity, biological process of the immune system process, metabolic process, cellular component organization, biological adhesion, and cellular component of the macromolecular complex. Our results demonstrate the first comprehensive protein expression profile of the ML in response to NO stress and provide novel references for understanding the potential mechanism underlying the effects of NO on trichinellosis.

SUBMITTER: Wang X

PROVIDER: S-EPMC5988324 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Proteomic analysis of the response of Trichinella spiralis muscle larvae to exogenous nitric oxide.

Wang Xiaoli X Li Liang L Wei Xing X Wang Yuanyuan Y Zhang Hui H Shi Ao A Liu Tao T Yang Xiaodi X Fang Qiang Q

PloS one 20180605 6

Trichinella spiralis mainly dwells in the muscle tissue of its host and is the main causative agent of trichinellosis in humans. Nitric oxide (NO), an important intracellular signaling molecule that may restrict pathogen growth in infected hosts, has been known for its anti-pathogenic activity, including resistance to T. spiralis. Herein, we applied label-free analysis to investigate the effect of sodium nitroprusside (SNP, a NO donor compound) on the proteome of T. spiralis muscle larvae (ML), ...[more]

PMID: 29870543

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Project description:The present study compares the immunogenic patterns of muscle larvae excretory-secretory proteins (ML E-S) from T. spiralis and T. britovi recognized by Trichinella-infected human sera. Samples were analyzed using two-dimensional electrophoresis (2-DE) coupled with 2D-immunoblot and liquid chromatography-tandem mass spectrometry LC-MS/MS analysis, two ELISA procedures and a confirmatory 1D-immunoblot test. Sera were obtained from nine patients with a history of ingestion of raw or undercooked meat who presented typical clinical manifestations of trichinellosis and from eleven healthy people. Specific anti-Trichinella IgG antibodies were detected in all samples tested with the Home-ELISA kits, but in only four samples for the commercially-available kit. The 1D-immunoblot results indicated that all nine serum samples were positive for T. spiralis ML E-S antigens, expressed as the presence of specific bands. In contrast, eight of the serum samples with T. britovi E-S ML antigens were positive, with one serum sample taken from a patient at 33dpi (days post infection) being negative. To identify immunoreactive proteins that are specifically recognized by host antibodies, both species of ML E-S proteins were subjected to 2D-immunoblotting with human serum taken at 49 dpi. The sera recognized 22 protein spots for T. spiralis and 18 for T. britovi in 2D-immunoblot analysis. Their molecular weights (MW) ranged from 50 to 60 kDa. LC-MS/MS analysis identified both common and specifically-recognized immunoreactive proteins: transmembrane serine protease 9, serine protease, antigen targeted by protective antibodies and Actin-1 partial were shared for both Trichinella species; hypothetical protein T01_7775 and P49 antigen, partial those specific to T. spiralis; deoxyribonuclease-2-alpha and hypothetical protein T03_17187/T12_7360 were specific to T. britovi. Our results demonstrate the value of 2-DE and 2D-immunblot as versatile tools for pinpointing factors contributing to the parasite-host relationship by comparing the secretomes of different Trichinella species.

Project description:BackgroundTrichinellosis is a zoonotic disease in humans caused by Trichinella spp. The present study was undertaken to discover excretory-secretory (E-S) proteins from T. spiralis and T. britovi muscle larvae (ML) that hold promise for species-specific diagnostics. To that end, the purified E-S proteins were analyzed by fluorescent two-dimensional difference gel electrophoresis (2-D DIGE) coupled with protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). To search for immunoreactive proteins that are specifically recognized by host antibodies the E-S proteins were subjected to two-dimensional (2-DE) immunoblotting with antisera derived from pigs experimentally infected with T. spiralis or T. britovi.ResultsAccording to 2-D DIGE analysis, a total of twenty-two proteins including potentially immunogenic proteins and proteins produced only by one of the two Trichinella species were subjected to LC-MS/MS for protein identification. From these proteins seventeen could be identified, of which many were identified in multiple spots, suggesting that they have undergone post-translational modification, possibly involving glycosylation and/or proteolysis. These proteins included 5'-nucleotidase, serine-type protease/proteinase, and p43 glycoprotein (gp43) as well as 49 kDa E-S protein (p49). Our findings also suggest that some of the commonly identified proteins were post-translationally modified to different extents, which in certain cases seemed to result in species-specific modification. Both commonly and specifically recognized immunoreactive proteins were identified by 2-DE immunoblotting; shared antigens were identified as gp43 and different protease variants, whereas those specific to T. britovi included multiple isoforms of the 5'-nucleotidase.ConclusionsBoth 2-D DIGE and 2-DE immunoblotting approaches indicate that T. spiralis and T. britovi produce somewhat distinctive antigen profiles, which contain E-S antigens with potential as species-specific diagnostic markers for Trichinella. Our results also demonstrate the value of 2-D DIGE as a versatile tool to compare secretomes of different Trichinella species for pinpointing factors contributing to the interaction with the host.

Dataset Information

Proteomic analysis of the response of Trichinella spiralis muscle larvae to exogenous nitric oxide.

Publications

Proteomic analysis of the response of Trichinella spiralis muscle larvae to exogenous nitric oxide.

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