Angiostrongylus cantonensis L5, a human cerebrospinal fluid parasite, causes eosinophilic meningitis, which is associated with a tissue inflammatory response associated with a high percentage of eosinophils. Eosinophils participate in the destruction of helminths using superoxide dismutase and hydrogen peroxide (H2O2) produced by the respiratory burst. In contrast, helminths survive and attenuate tissue inflammatory responses through eosinophils. In a previous study, we demonstrated the extracellular function of Acan-Gal-1 to induce apoptosis in macrophages. Here, the intracellular function of Acan-Gal-1 was investigated to further elucidate the mechanisms involved in the ability of A. cantonensis L5 worms to overcome the inflammatory response in the human central nervous system.
In this study, the model organism Caenorhabditis elegans was used to investigate the intracellular function of Acan-Gal-1 in protecting the worm from host immune attack. First, the structural properties of Akan-Gal-1 were analyzed using bioinformatics; second, qRT-PCR was used to monitor the stage specificity of Acan-gal-1 expression in A. cantonensis. Microinjection was performed to detect the tissue specificity of lec-1 expression, a homologue of Acan-gal-1, in C. elegans. Third, microinjection was performed to develop Acan-gal-1::rfp transgenic worms. Oxidative stress assay and Oil Red O lipid staining were then used to determine the function of Acan-Gal-1 in C. elegans.
Stage-specific detection of Acan-gal-1 showed that Acan-gal-1 was up-regulated in both L5 and adult worms. Tissue specificity revealed that the C. elegans Acan-gal-1 homologue, lec-1, is ubiquitously expressed and predominantly localizes to the cuticle. Investigating the intracellular function of Acan-Gal-1 in surrogate C. elegans showed significant resistance to oxidative stress in N2 mice expressing pCe-lec-1::Acan-gal-1::rfp with reduced lipid accumulation; Lec-1 mutant mice with increased lipid accumulation were susceptible to oxidative stress, and this phenotype could be rescued by expression of pCe-lec-1::Acan-gal-1::rfp. Expression of pCe-lec-1::Acan-gal-1::rfp or lec-1 RNAi in fat-6;fat-7 double mutant worms with reduced fat reserves had no significant effect on tolerance to oxidative stress.
Akan-Gal-1, which is upregulated in C. elegans worms, protects worms from prosurvival oxidative stress-induced damage by reducing lipid accumulation. This may represent a mechanism by which the upregulated Acan-Gal-1 worm of A. cantonensis L5 overcomes eosinophilic immune attack in the human central nervous system.
Angiostrongylus cantonensis is considered the primary cause of human eosinophilic meningitis and meningoencephalitis in China, Japan, Southeast Asia, and the Pacific Islands [1,2,3]. The final host of this parasite is the rat; lives in the pulmonary artery of rats and reaches sexual maturity [4, 5]. As an atypical host, humans acquire this parasite by consuming terrestrial freshwater molluscs, such as the golden apple/canal apple snail Pomacea canaliculata, which harbor infective third-instar larvae (iL3). After entering the human small intestine, iL3 infects the central nervous system through the bloodstream and develops into 5th stage larvae (L5) causing eosinophilic meningitis [6,7,8,9,10].
A high percentage of eosinophils entering the central nervous system from the circulation is thought to enhance the inflammatory response of tissues during helminth infection, leading to the primary pathological changes of eosinophilic meningitis . Using peroxidase and hydrogen peroxide (H2O2) as eosinophil peroxidase, a high-level enzyme complex is released and secreted into the granular matrix of eosinophils [12, 13]. respiratory burst [14,15,16]. In contrast, parasites with high eosinophil content have evolved to survive by attenuating eosinophil-mediated tissue inflammatory responses [ 11 ]. Therefore, A. cantonensis L5, which resides in human cerebrospinal fluid (CSF) with a high percentage of eosinophils, is able to resist eosinophil oxidative stress damage.
In our previous study, proteomic analysis of A. cantonensis L5 and A. cantonensis iL3 using two-dimensional differential gel electrophoresis (2D-DIGE) showed that the expression level of A. cantonensis RPS-30 (Acan-RPS-30). L5 was lower than iL3 and higher than A. cantonensis Galectin-1 (Acan-Gal-1) . We recently showed that Acan-RPS-30, reduced in A. cantonensis L5, plays a protective role in oxidative stress-induced worm survival by inhibiting apoptosis.