Fig. 1. Purification and antimicrobial activity of hemocyte extract of M. coruscus. The extract was fractionated by the CapCell-Pak C18 reversed-phase column. Elution was performed with a linear gradient of 5-65% CH3CN in 0.1% TFA for 60 min at a flow rate of 1 ml/min. The eluate was monitored at 220 nm. Fraction of the absorbance peak (indicated by the arrow) showed antimicrobial activity against B. subtilis (inset). The elution point of the active peak was at 29% CH3CN. Scale bar indicates 5 mm.
Fig. 2. Final purification of pooled active fraction and antimicrobial activity. The active fraction was applied to a PEPTIDE XB-C18 reversed-phase column. Elution was performed with a linear gradient of 20-40% CH3CN in 0.1% TFA for 30 min at a flow rate of 0.5 ml/min. The eluate was monitored at 220 nm. The elution point of the active peak was at 33% CH3CN (indicated by the arrow). Antimicrobial activity of the purified peak (before) and proteinase K treated purified peak (after) against B. subtlis. Scale bar indicates 5 mm.
Fig. 3. The molecular weight of the purified peptide was determined using an ultraflleXtremeTM MALDI TOF/MS spectrometer equipped with a pulsed smart beam II in linear mode. The molecular weight of the purified peptide is 4041.866 Da.
Fig. 4. Multiple alignment of nucleotide sequences of mytilin B precursor and mytilin B isoforms. Signal peptide is indicated by grey box. The mature peptide and polyadenylation signal is indicated by black outlined. Conserved residues are indicated by dot.
Fig. 5. Amino acid sequence alignment of mytilin B precursor with 4 isoform of mytilin B. Signal peptide and mature peptide regions were outlined by black boxes. Conserved residues of amino acids are indicated by dot.
Fig. 6. Hemolytic activity of mytilin B1, mytilin B2 and piscidin 1 against erythrocytes of flounder (Paralichthys olivaceus). (A) The concentration of each sample was 100 μg/ml. (B) Effect of hemolytic activity depend on the concentrations of each samples (100, 50, 25, 12.5, 6.5 μg/ml).
Fig. 7. Morphological differences of HUVEC after 24 hr growth with each specimens. (A) HUVEC control (B) HUVEC treated with mytilin B1 (C) HUVEC treated with mytilin B2 (D) HUVEC treated with melittin.
Fig. 8. Cell viability of HUVEC treated with mytilin B after 24 hr growth. Error bars represent the mean ± SD of three technical replicates.
Fig. 9. Quantitative analysis of the mytilin B gene expression level from the various tissues. HEP; hepatopancreas, SIP; siphon, GIL; gill, ADD; adductor muscle, FOO; foot, HEM; hemocyte, MAN: mantle. Error bars represent the mean ± SD of three technical replicates.
Table 1. Pathogenic bacteria strains used in this study
Table 2. Antimicrobial activity of hemocyte extract of M. coruscus against pathogenic bacteria
Table 3. Antimicrobial activity and stability of mytilin B1 and mytilin B2 against pathogenic bacteria under heat & salt conditions
Table 4. Minimal effective concentrations (MECs, μg/ml) of mytilin B antimicrobial peptides