title: Antimicrobial activity of recombinant mature bovine neutrophil β-defensin 4 on mycobacterial infection.
abstract: 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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: State Key Lab of Agrobiotechnology, National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, China; Department of Veterinary Medicine, Henan University of Animal Husbandry and Economy (Longzihu Campus), Zhengzhou, China.
authors.forename: J-J
authors.initials: JJ
authors.lastname: Kang
authors.affiliation: State Key Lab of Agrobiotechnology, National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Lyu
authors.affiliation: State Key Lab of Agrobiotechnology, National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: D-M
authors.initials: DM
authors.lastname: Zhao
authors.affiliation: State Key Lab of Agrobiotechnology, National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, China; College of Wildlife Resource, Northeast Forestry University, Harbin, China.
authors.forename: L-H
authors.initials: LH
authors.lastname: Tian
authors.affiliation: State Key Lab of Agrobiotechnology, National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: X-M
authors.initials: XM
authors.lastname: Yin
authors.affiliation: State Key Lab of Agrobiotechnology, National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: L-F
authors.initials: LF
authors.lastname: Yang
authors.affiliation: State Key Lab of Agrobiotechnology, National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: K-D
authors.initials: KD
authors.lastname: Teng
authors.affiliation: State Key Lab of Agrobiotechnology, National Animal Transmissible Spongiform Encephalopathy Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, China.
authors.forename: X-M
authors.initials: XM
authors.lastname: Zhou
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002417:Cattle; D002473:Cell Wall / Q000187:drug effects / Q000648:ultrastructure; D015169:Colony Count, Microbial; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008264:Macrophages / Q000187:drug effects / Q000382:microbiology; D051379:Mice; D050296:Microbial Viability / Q000187:drug effects; D008855:Microscopy, Electron, Scanning; D009163:Mycobacterium bovis / Q000187:drug effects* / Q000254:growth & development / Q000648:ultrastructure; D020102:Mycobacterium smegmatis / Q000187:drug effects* / Q000254:growth & development / Q000648:ultrastructure; D000067996:RAW 264.7 Cells; D011994:Recombinant Proteins / Q000494:pharmacology; D013997:Time Factors; D023083:beta-Defensins / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; D023083:beta-Defensins