Gene cloning and expression of a partial sequence of Hirudomacin, an antimicrobial protein that is increased in leech (Hirudo nipponica Whitman) after a blood meal.

Abstract

The novel antimicrobial gene Hirudomacin (Hmc), with a 249-bp cDNA, encodes a mature protein of 61 amino acids and a 22-amino acid signal peptide. Hmc exhibits the highest similarity, at 90.1%, with macin family members found in the salivary gland of the leech Hirudo nipponica Whitman. A mature Hmc protein concentration of 219 μg/mL was detected using the Bradford method. The mature Hmc protein is 6862.82 Da and contains 8 cysteine residues. Antimicrobial assays showed a minimum bactericidal concentration and 50% lethal dose of 1.56 μg/mL and 0.78 μg/mL, respectively, for Staphylococcus aureus and 0.39 μg/mL and 0.195 μg/mL, respectively, for Bacillus subtilis. Transmission electron microscopy revealed membrane integrity disruption in S. aureus and B. subtilis, which resulted in bacterial lysis. The level of Hmc mRNA in the salivary gland during three blood meal stages indicated a remarkable trend of increase (P < .05), and western blotting demonstrated that among the three blood meal stages, expression of the mature Hmc protein was highest in both the salivary gland and intestine at the fed stage (P < .05). Immunofluorescence further showed the mature Hmc protein to be localized outside the cell nucleus, with the signal intensity in the salivary gland peaking at the fed stage (P < .05). In conclusion, the mature Hmc protein exhibits broad-spectrum antimicrobial effects against gram-positive and gram-negative bacteria, and a blood meal upregulates Hmc gene and protein expression in H. nipponica.