ABSTRACT
In this work, we explore if Gram-positive bacteria as Staphylococcus aureus or Gram-negative bacteria components as LPS, can induce the expression of seven antimicrobial peptides (AP) in an immortalized bovine umbilical vein endothelial cell line (BUVEC). By qPCR we determined the constitutive expression of all the AP evaluated. The stimulation with S. aureus or LPS induced the expression of lingual antimicrobial peptide (LAP), bovine ß-defensin 1 (DEFB1) and bovine neutrophil ß-defensin 4 (BNBD4). This expression was regulated by the autocrine production of tumor necrosis factor-α (TNF-α), indicating that bovine endothelial cells (EC) can play a more active role during infection.
Subject(s)
Endothelial Cells/metabolism , beta-Defensins/biosynthesis , Animals , Antimicrobial Cationic Peptides/biosynthesis , Antimicrobial Cationic Peptides/genetics , Autocrine Communication , Cattle , Cell Line, Transformed/drug effects , Cell Line, Transformed/metabolism , Endothelial Cells/drug effects , Female , Gene Expression Regulation , Lipopolysaccharides/pharmacology , Mastitis, Bovine/microbiology , Staphylococcus aureus/immunology , Staphylococcus aureus/isolation & purification , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/genetics , Umbilical Veins , beta-Defensins/geneticsABSTRACT
Thionins are plant antimicrobial peptides with antibacterial and antifungal activities. Thionin Thi2.1 cDNA from Arabidopsis thaliana was expressed in BVE-E6E7 bovine endothelial cell line and its activity was evaluated against Escherichia coli, Staphylococcus aureus, Candida albicans and different mammal cell lines. Total protein (2.5 microg) from conditioned medium (CM) of clone EC-Thi2.1 inhibited the growth of E. coli, S. aureus (>90%) and C. albicans strains (>80%) in relation to the CM from control cells. Also, CM of EC-Thi2.1 inhibited the viability of several transformed and normal mammal cell lines (38-95%). These results suggest that thionin Thi2.1 is an antimicrobial peptide that could be use in the treatment of mammalian infectious diseases.
Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Arabidopsis Proteins/pharmacology , Arabidopsis/metabolism , Candida albicans/drug effects , Endothelium/drug effects , Escherichia coli/drug effects , Animals , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Blotting, Western , Cattle , Culture Media, Conditioned , Endothelium/cytology , Immunoprecipitation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Staphylococcus aureus/drug effectsABSTRACT
Bovine mastitis is mainly caused by Staphylococcus aureus and antimicrobial therapy, commonly used for its control, has resulted in an increase in the frequency of resistant staphylococci in recent years. Thus, alternative therapies are desirable and the antimicrobial peptides represent attractive control agents. In this work, we expressed the antimicrobial peptide thionin Thi2.1 cDNA from Arabidopsis thaliana in the bovine endothelial cell line BVE-E6E7 and evaluated its activity against bovine mastitis S. aureus isolates. A polyclonal population from BVE-E6E7 cells transfected with the pThi2.1 construct was obtained and thionin Thi2.1 expression was confirmed by RT-PCR. From this population, eight stably transfected cell clones were obtained and their conditioned media (CM) were evaluated against the S. aureus ATCC 27543 strain. Clones showed high antibacterial activity (>95%) relative to the activity of the polyclonal population. The C8 clone showed the highest antibacterial activity (>99%) and its CM was evaluated against eleven bovine mastitis S. aureus isolates. A 2.5microg aliquot of total protein from the C8 clone's CM inhibited the growth of S. aureus isolates (>40%) relative to the CM from BVE-E6E7 cells used as control. Growth inhibition of S. aureus isolates was dose-dependent, showing a total inhibition at concentrations higher than 3.12microg/ml. These results suggest that thionin Thi2.1 antimicrobial peptide could be use in the treatment of bovine mastitis.
