New Insights into the Toxin Diversity and Antimicrobial Activity of the "Fire Coral" Millepora complanata

Abstract

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.

Keywords: Millepora complanata; antibacterial peptides; cytolysins; toxin diversity; transcriptomics.

Figure 1

(A) Overrepresented gene ontology (GO) terms across the molecular function (MF) subontology and (B) toxin distribution from the M. complanata transcriptome.

Figure 2

(A) Multiple sequence alignment between a hemolysin-like protein from M. complanata (Mc_hemolisyn-like) and four centipede toxins (TX14A_SCODE or Scoloptoxin SSD14, A0A646QER6_9MYRI, A0A646QI04_9MYRI, and A0A646QD69_9MYRI) (Amino acid conservation is highlighted in blue). (B) Multiple sequence alignment between a M. complanata hydralysin-like (Mc_hydralysin-like) toxin with four hydralysins (HLYS_HYDVU, HLYS1_HYDVU, HLYS2_HYDVU, and HLYS3_HYDVU). Annotations: Upper flanking motif between positions 80–115 (gray line); region responsible for transmembrane pore formation at positions 116–145 (black line); and lower franking motif within positions 146–190 (blue line). (Amino acid conservation is highlighted in blue).

Figure 3

(A) SDS-PAGE electrophoretic profile (Mc), and (B) Two-dimensional electrophoretic profile of the nematocyst proteome of M. complanata. MW—molecular mass (kDa). IP—isoelectric point (pH).

Figure 4

Zymographic analysis. (A) Hemolytic activity, two zones of hemolysis at ~25 and ~205 kDa. (B) Regions corresponding to PLA2 activity around 6 and 25 kDa. (C) Proteolytic activity observed within a molecular mass range of 50 to 205 kDa.

Figure 5

Percentage of the inhibitory effect of enzymatic inhibitors on the hemolytic activity of the proteome of M. complanata nematocysts. (A) Hemolytic activity (control), (B) 44.2% for EDTA, (C) 19.68% for Batimastat, (D) 2.36% for TLCK, (E) 11.59% for TPCK, (F) 37.59% for PMSF, and (G) Varespladib with 19.66% inhibition.