RESUMO
To date, few studies have been carried out aimed at characterizing the toxins synthesized by hydrocorals of the genus Millepora. The purpose of this study was to explore the toxin diversity and antibacterial activity of the "fire coral" M. complanata using a transcriptomic data mining approach. In addition, the cytolytic and antibacterial activities of the M. complanata nematocyst proteome were experimentally confirmed. Cytolysins were predicted from the transcriptome by comparing against the Animal Toxin Annotation Project database, resulting in 190 putative toxins, including metalloproteases, hemostasis-impairing toxins, phospholipases, among others. The M. complanata nematocyst proteome was analyzed by 1D and 2D electrophoresis and zymography. The zymograms showed different zones of cytolytic activity: two zones of hemolysis at ~25 and ~205 kDa, two regions corresponding to phospholipase A2 (PLA2) activity around 6 and 25 kDa, and a proteolytic zone was observed between 50 and 205 kDa. The hemolytic activity of the proteome was inhibited in the presence of PLA2 and proteases inhibitors, suggesting that PLA2s, trypsin, chymotrypsin, serine-proteases, and matrix metalloproteases are responsible for the hemolysis. On the other hand, antimicrobial peptide sequences were retrieved from their transcripts with the amPEPpy software. This analysis revealed the presence of homologs to SK84, cgUbiquitin, Ubiquicidin, TroTbeta4, SPINK9-v1, and Histone-related antimicrobials in the transcriptome of this cnidarian. Finally, by employing disk diffusion and microdilution assays, we found that the nematocyst peptidome of M. complanata showed inhibitory activity against both Gram-positive and Gram-negative bacteria including S. enteritidis, P. perfectomarina, E. coli, and C. xerosis, among others. This is the first transcriptomic data mining analysis to explore the diversity of the toxins synthesized by an organism of the genus Millepora. Undoubtedly, this work provides information that will broaden our general understanding of the structural richness of cnidarian toxins.
Assuntos
Antozoários , Hidrozoários , Toxinas Biológicas , Animais , Antibacterianos/efeitos adversos , Escherichia coli , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Hemólise , Metaloproteases , Fosfolipases A2 , ProteomaRESUMO
Coral bleaching caused by global warming has resulted in massive damage to coral reefs worldwide. Studies addressing the consequences of elevated temperature have focused on organisms of the class Anthozoa, and up to now, there is little information regarding the mechanisms by which reef forming Hydrozoans face thermal stress. In this study, we carried out a comparative analysis of the soluble proteome and the cytolytic activity of unbleached and bleached Millepora complanata ("fire coral") that inhabited reef colonies exposed to the 2015-2016 El Niño-Southern Oscillation in the Mexican Caribbean. A differential proteomic response involving proteins implicated in key cellular processes, such as glycolysis, DNA repair, stress response, calcium homeostasis, exocytosis, and cytoskeleton organization was found in bleached hydrocorals. Four of the proteins, whose levels increased in bleached specimens, displayed sequence similarity to a phospholipase A2, an astacin-like metalloprotease, and two pore forming toxins. However, a protein, which displayed sequence similarity to a calcium-independent phospholipase A2, showed lower levels in bleached cnidarians. Accordingly, the hemolytic effect of the soluble proteome of bleached hydrocorals was significantly higher, whereas the phospholipase A2 activity was significantly reduced. Our results suggest that bleached M. complanata is capable of increasing its toxins production in order to balance the lack of nutrients supplied by its symbionts.