Antimicrobial activity of a honeybee (Apis cerana) venom Kazal-type serine protease inhibitor

• Published in: Toxicon (Oxford) 2013-12, Vol.76, p.110-117
• Main Authors: Kim, Bo Yeon, Lee, Kwang Sik, Zou, Feng Ming, Wan, Hu, Choi, Yong Soo, Yoon, Hyung Joo, Kwon, Hyung Wook, Je, Yeon Ho, Jin, Byung Rae
• Format: Article
• Language: English
• Subjects: Animals; Anti-Infective Agents - chemistry; Anti-Infective Agents - isolation & purification; Anti-Infective Agents - pharmacology; antibacterial properties; antifungal properties; Antiinfectives and antibacterials; Antimicrobial activity; API; Apis cerana; Apis mellifera; Bacillus subtilis; Bacillus thuringiensis; Bacteria; Beauveria bassiana; Bee Venoms - chemistry; Bees - enzymology; Blotting, Northern; Cloning, Molecular; cysteine; Elastase; entomopathogenic fungi; Escherichia coli; Fusarium graminearum; Gram-positive bacteria; honey bees; Honeybee; Inhibitors; Insect Proteins - chemistry; Insect Proteins - isolation & purification; Insect Proteins - physiology; Kazal-type serine protease inhibitor; Microbial serine protease inhibitor; Microorganisms; Molecular Sequence Data; peptidase K; plasmin; Protease inhibitors; Proteinase; proteinase inhibitors; Sequence Alignment; Sequence Analysis, DNA; Sequence Analysis, Protein; Serine Proteinase Inhibitors - chemistry; Serine Proteinase Inhibitors - isolation & purification; Serine Proteinase Inhibitors - pharmacology; subtilisin; t-plasminogen activator; threonine; Thrombin; trypsin; Venom; venoms
• ISSN: 0041-0101; 1879-3150
• DOI: 10.1016/j.toxicon.2013.09.017

Insect-derived Kazal-type serine protease inhibitors exhibit thrombin, elastase, plasmin, proteinase K, or subtilisin A inhibition activity, but so far, no functional roles for bee-derived Kazal-type serine protease inhibitors have been identified. In this study, a bee (Apis cerana) venom Kazal-type serine protease inhibitor (AcKTSPI) that acts as a microbial serine protease inhibitor was identified. AcKTSPI contained a single Kazal domain that displayed six conserved cysteine residues and a P1 threonine residue. AcKTSPI was expressed in the venom gland and was present as a 10-kDa peptide in bee venom. Recombinant AcKTSPI Kazal domain (AcKTSPI-Kd) expressed in baculovirus-infected insect cells demonstrated inhibitory activity against subtilisin A (Ki 67.03 nM) and proteinase K (Ki 91.53 nM), but not against α-chymotrypsin or trypsin, which implies a role for AcKTSPI as a microbial serine protease inhibitor. However, AcKTSPI-Kd exhibited no detectable inhibitory effects on factor Xa, thrombin, tissue plasminogen activator, or elastase. Additionally, AcKTSPI-Kd bound directly to Bacillus subtilis, Bacillus thuringiensis, Beauveria bassiana, and Fusarium graminearum but not to Escherichia coli. Consistent with these findings, AcKTSPI-Kd showed antibacterial activity against Gram-positive bacteria and antifungal activity against both plant-pathogenic and entomopathogenic fungi. These findings constitute molecular evidence that AcKTSPI acts as an inhibitor of microbial serine proteases. This paper provides a novel view of the antimicrobial functions of a bee venom Kazal-type serine protease inhibitor. •AcKTSPI is a honeybee (Apis cerana) venom Kazal-type serine protease inhibitor.•AcKTSPI contains a single Kazal domain.•AcKTSPI inhibits microbial serine proteases such as subtilisin A and proteinase K.•AcKTSPI shows antimicrobial activity against Gram-positive bacteria and fungi.•AcKTSPI acts as an antimicrobial factor.