Antimicrobial activity of recombinant mature bovine neutrophil β-defensin 4 on mycobacterial infection.

BACKGROUND: Previous studies have shown that human cathelicidin and defensins have effective antimicrobial activity against Mycobacterium spp. OBJECTIVE: To investigate the antimycobacterial effect of mature bovine neutrophil β-defensin (mBNBD) 4 against Mycobacterium spp. infection for the first time. DESIGN: mBNBD4 protein was expressed in Pichia pastoris GS115. We used immunofluorescent assay to detect whether the recombinant mBNBD4 had entered the macrophages. The antimycobacterial activity of mBNBD4 was tested through colony-forming unit (cfu) assay. Morphological changes in the cell wall of M. bovis treated with mBNBD4 were observed by scanning electron microscope. RESULTS: mBNBD4 was expressed and successfully purified from P. pastoris with intact antimicrobial activity. The recombinant protein was able to enter Raw 264.7 macrophages and exhibited potent in vitro bactericidal activity against M. smegmatis and M. bovis. The cell wall of M. bovis was disrupted after interaction with mBNBD4. Exogenous addition of mBNBD4 to both Raw 264.7 and THP-1 derived macrophages reduced the intracellular survival of M. bovis and M. tuberculosis relative to control cells. CONCLUSION: Our data show that mBNBD4 plays an important role in inhibiting mycobacterial growth and in controlling intracellular survival of mycobacteria. mBNBD4 could therefore an effective antimycobacterial molecule in combination with other measures.