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Expression of recombinant antigenic proteins from Angiostrongylus cantonensis: a brief report.

ABSTRACT: Cerebral angiostrongyliasis is an acute inflammation caused by the infection of the nematode Angiostrongylus cantonensis that results in eosinophilic meningitis. The current immunological assay of choice is an immunoblot that detects antibodies to a 31 kDa protein present in crude extracts of the female worm. Recently we have identified diagnostic targets from excretion and secretion products and determined the composition of the 31 kDa antigen after 2-D gel electrophoresis and mass spectrometry. Here we cloned and expressed five proteins in prokaryotic and eukaryotic systems. Recombinant proteins were purified and analysed by Western blot assays and among them 14-3-3, Lec5 and ES7 were recognized by Angiostrongylus-specific serum, although the signal was weak.

SUBMITTER: Morassutti AL

PROVIDER: S-EPMC3689479 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Expression of recombinant antigenic proteins from Angiostrongylus cantonensis: a brief report.

Morassutti Alessandra L AL Perelygin Andrey A Levert Keith K Lin Seh-Ching SC Lee Yeuk-Mui YM da Silva Alexandre J AJ Wilkins Patricia P PP Graeff-Teixeira Carlos C

Hawai'i journal of medicine & public health : a journal of Asia Pacific Medicine & Public Health 20130601 6 Suppl 2

Cerebral angiostrongyliasis is an acute inflammation caused by the infection of the nematode Angiostrongylus cantonensis that results in eosinophilic meningitis. The current immunological assay of choice is an immunoblot that detects antibodies to a 31 kDa protein present in crude extracts of the female worm. Recently we have identified diagnostic targets from excretion and secretion products and determined the composition of the 31 kDa antigen after 2-D gel electrophoresis and mass spectrometry ...[more]

PMID: 23900614

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Project description:Human angiostrongyliasis results from accidental infection with Angiostrongylus, an intra-arterial nematode. Angiostrongylus cantonensis infections result in eosinophilic meningitis, and A. costaricensis infections cause eosinophilic enteritis. Immunological methodologies are critical to the diagnosis of both infections, since these parasites cannot be isolated from fecal matter and are rarely found in cerebrospinal fluid samples. A. costaricensis and A. cantonensis share common antigenic epitopes which elicit antibodies that recognize proteins present in either species. Detection of antibodies to a 31-kDa A. cantonensis protein present in crude adult worm extracts is a sensitive and specific method for immunodiagnosis of cerebral angiostrongyliasis. The objective of the present work was to isolate and characterize the 31-kDa proteins using soluble protein extracts derived from adult female worms using both one- (1DE) and two-dimensional (2DE) gel electrophoresis. Separated proteins were blotted onto nitrocellulose and probed using sera from infected and non-infected controls. The 31-kDa band present in 1DE gels and the 4 spots identified in 2DE gels were excised and analyzed by electrospray ionization mass spectrometry. Using the highest scores obtained following Mascot analysis, amino acid sequences were obtained that matched four unique proteins: tropomyosin, the 14-3-3 phosphoserine-binding protein, a protein containing a nascent polypeptide-associated complex domain, and the putative epsilon subunit of coatomer protein complex isoform 2. Oxidative cleavage of diols using sodium m-periodate demonstrated that carbohydrate moieties are essential for the antigenicity of all four spots of the 31-kDa antigen. In this article we describe the identification of the 31-kDa antigen, and provide DNA sequencing of the targets. In conclusion, these data suggest that reactivity to the 31-kDa proteins may represent antibody recognition of more than one protein, and recombinant protein-based assays for cerebral angiostrongyliasis diagnosis may require eukaryotic expression systems to maintain antigenicity.

Project description:BACKGROUND: Angiostrongylus cantonensis is a parasite endemic in the Southeast Asian and Pacific regions. Humans are incidentally infected either by eating uncooked intermediate hosts or by consuming vegetables containing the living third-stage larvae. The 14-3-3? protein is a cerebrospinal fluid (CSF) marker of neuronal damage during the development of Creutzfeldt-Jakob disease. In addition, increased 14-3-3? protein is also found in CSF from patients with a variety of neurological disorders. The goal of this study is to determine the roles of serum/CSF14-3-3? protein in patients with eosinophilic meningitis. METHODS: In a cohort study among nine Thai laborers with eosinophilic meningitis due to eating raw snails (Pomacea canaliculata), we examined the CSF weekly while patients were still hospitalized and followed up the serum for 6 months. The levels of 14-3-3? protein in CSF were analyzed by western blot and an in-house 14-3-3? enzyme-linked immunosorbent assay (ELISA) measurement was established and tested in an animal model of eosinophilic meningitis. RESULTS: The elevated 14-3-3? level was detected in the CSF from eight out of nine (81%) patients After 2 weeks of treatment, all patients showed a declined level or cleared of 14-3-3? protein in the CSF. By developing an in-house ELISA for measurement of 14-3-3? protein, it was found that the serum 14-3-3? level was significantly increased in patients during initial visit. . This finding was consistent to the animal experiment result in which there was severe blood brain barrier damage three weeks after infection and increased 14-3-3? protein expression in the CSF and serum by western blot and in house ELISA. After treatment, the serum 14-3-3? level in meningitis patients was rapidly returned to normal threshold. There was a correlation between initial CSF 14-3-3? level with severity of headache (r?=?0.692, p?=?0.039), CSF pleocytosis (r?=?0.807, p?=?0.009) and eosinophilia (r?=?0.798, p?=?0.01) in the CSF of patients with eosinophilic meningitis (Spearman's correlation test). CONCLUSIONS: The serum 14-3-3? concentrations may constitute a useful marker for blood brain barrier damage severity and follow up in patients with eosinophilic meningitis caused by A. cantonensis.

Dataset Information

Expression of recombinant antigenic proteins from Angiostrongylus cantonensis: a brief report.

Publications

Expression of recombinant antigenic proteins from Angiostrongylus cantonensis: a brief report.

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