Restrained Wnt Signaling Pathway by Enhanced EsGSK3β Activity Facilitates the Infection of Spiroplasma and Leads to Neuropathic Diseases in Crustaceans.

Abstract

Spiroplasma eriocheiris has been identified as a lethal pathogen of Eriocheir sinensis tremor disease (TD), one neurological disease with typically paroxysmal tremors of the pereiopod. But, the pathogenic mechanism of TD was still unknown. First, in the current study, the phosphoproteomic changes of E. sinensis thoracic ganglion after S. eriocheiris infection were obtained using TMT labeling and affinity enrichment followed by high-resolution LC-MS/MS analysis. A total of 349 phosphorylation proteins are upregulated, and 331 phosphorylation proteins are downregulated when compared to the control sample with quantitative ratios above 1.5 or below 1/1.5 that are deemed significant. Bioinformatics analysis (Gene ontology, Kyoto Encyclopedia of Genes and Genomes pathway, and domain analysis, etc.) showed that Wnt signaling pathways were restrained, corresponding to many nervous system developments, and signal transmission pathway was also disrupted. Neurotransmitter metabolite analysis showed metabolic dysregulation of four key neurotransmitters (5-Hydroxy-L-tryptophan, serotonin, acetylcholine, and γ-amino-butyric acid) in thoracic ganglion of E. sinensis following S. eriocheiris infection. Second, similar to its effects on the thoracic ganglion cells, S. eriocheiris infection of hemocytes (the primary target cells and most crucial immune cells in crabs) also suppressed the host Wnt pathway through enhanced EsGSK3β activity, both in vivo and in vitro. Co-immunoprecipitation analysis showed that EsGSK3β could directly interact with Esβ-catenin. Inhibited or enhanced EsGSK3β activity of hemocytes could reduce or facilitate S. eriocheiris infection by regulating the stability and nuclear translocation of Esβ-catenin. Finally, further analysis showed that Wnt-β-catenin pathway could functionally crosstalk with Toll pathway to positively regulate hemocytes antibacterial peptides transcription. Altogether, our results suggest that S. eriocheiris could restrain the crab Wnt pathway, reduce antibacterial peptides transcription to help its infection and disorder neurotransmitters to cause neuropathic TD at last.