Proteomics analysis of toxins-producing dinoflagellates and toxins-contaminated marine organisms

Abstract

Paralytic shellfish poisoning (PSP) and ciguatera fish poisoning (CFP) are the two major contributors to illnesses caused by dinoflagellate toxins. Paralytic shellfish poisoning toxins (PSTs) are produced by dinoflagellates in the genera Alexandrium, Gymnodinium, and Pyrodinium while ciguatera fish poisoning toxins, such as ciguatoxins (CTXs), are originated from benthic toxic dinoflagellates (Gambierdiscus, Prorocentrum, Ostreopsis, and Coolia species). These toxins are responsible for human intoxication syndromes to the nervous system and muscles.

This study optimized the protein extraction for proteomics analysis of dinoflagellate. The protein and toxin profiles of Alexandrium tamarense CI01 at different toxin biosynthesis phases were compared; differentially expressed proteins in highly toxic algae were identified using matrix-assisted laser desorption/ionization time-of–flight (MALDI-TOF) spectrometry. Some potential proteins involved in the toxin biosynthesis of A. tamarense CI01 were also identified. The protein preparation method from dinoflagellates for proteomics is selective for proteins with different pIs and molecular weights. The Lysis method may cause variation of the target proteins, but Trizol and Tris do not. Trizol method is good at analyzing hydrophobic, high molecular weights or binding proteins on the membrane. These PST synthesis-related enzymes are common in dinoflagellates, thereby 2-DIGE and Trizol are the best staining methods for dinoflagellates toxin synthase proteomics research. Through a comparative study in A. tamarense CI01 under different nutrient conditions and growth phase, the potential toxin synthesis-related proteins were investigated. Based on proteomics results, methionine, ornithine, arginine metabolism-related proteins and photosynthesis-related proteins may be related to the PSTs biosynthesis. Results also identified a similar pathway of PST biosynthesis in both dinoflagellate and cyanobacteria.

A comparative proteomics study was applied to identify proteins of biomarkers for CTX accumulation in hepatic tissue of grouper, Cephalopholis argus, and PSTs accumulation in shellfish, Saxidomus giganteus. In C. argus with elevated CTXs, ATP synthase subunit beta and cytochrome c, ubiquitin enzymes, ATP related enzymes, and telomerase reverse transcriptase were greatly reduced. The comparative proteomic analysis revealed that PSTs and CTXs induced influx/efflux of Na+ or Ca2+ disorders in fish and shellfish. ATP synthase can control the concentration and influx/efflux of Na+. Alterations of Na+/K+ adenosine triphosphatase, liagand gated ion channel and sarcoplasmic calcium-binding protein in toxic shellfish was believed to reduce damage that PSTs (sodium channel blockers) bring to shellfish by controlling the concentration and influx/efflux of Na+ or Ca2+. Two identified resistant mechanisms to Na+ channel toxins are amino acid mutation and toxin affinity-binding proteins. However, neither of them was confirmed in the present study, but the proteins controlling the concentration and influx/efflux of Na+ or Ca2+ in this study may be due to new mechanisms. In conclusion, our comparative proteomic analysis revealed that CTXs and PSTs induced influx/efflux of Na+ or Ca2+ changes in toxic samples with a concomitant interference with signal transduction, metabolomics processes, detoxification, and anti-apoptosis. The physiological roles of ion concentration controlling and ion signal-related proteins in toxic fish and shellfish species should be further studied for their potential roles in resistance mechanisms to CTXs and PSTs.