Antimicrobial activity of bacteriophage derived triple fusion protein against Staphylococcus aureus
- US Dept. of Agriculture (USDA), Beltsville, MD (United States). Oak Ridge Inst. for Science and Education (ORISE). Agricultural Research Service (ARS). U.S. National Arboretum. Floral and Nursery Plants Research Unit
- Kerry’s Nursery, Miami, FL (United States)
- US Dept. of Agriculture (USDA), Beltsville, MD (United States). Agricultural Research Service (ARS). Animal Biosciences and Biotechnology Lab.
- US Dept. of Agriculture (USDA), Beltsville, MD (United States). Agricultural Research Service (ARS). Animal Biosciences and Biotechnology Lab.
- US Dept. of Agriculture (USDA), Beltsville, MD (United States). Agricultural Research Service (ARS). Molecular Plant Pathology Lab.
The increasing spread of antibiotic-resistant microorganisms has led to the necessity of developing alternative antimicrobial treatments. The use of peptidoglycan hydrolases is a promising approach to combat bacterial infections. In our study, we constructed a 2 kb-triple-acting fusion gene (TF) encoding the N-terminal amidase-5 domain of streptococcal LambdaSA2 prophage endolysin (Dglutamine-L-lysin endopeptidase), a mid-protein amidase-2 domain derived from the staphylococcal phage 2638A endolysin (N-acetylmuramoyl-L-alanine amidase) and the mature version (246 residues) of the Staphylococcus simulans Lysostaphin bacteriocin (glycyl-glycine endopeptidase) at the C-terminus. The TF gene was expressed in Nicotiana benthamiana plants using the nonreplicating Cowpea mosaic virus (CPMV)-based vector pEAQ-HT and the replicating Alternanthera mosaic virus (AltMV)-based pGD5TGB1L8823-MCS-CP3 vector, and in Escherichia coli using pET expression vectors pET26b+ and pET28a+. The resulting poor expression of this fusion protein in plants prompted the construction of a TF gene codon-optimized for expression in tobacco plants, resulting in an improved codon adaptation index (CAI) from 0.79 (TF gene) to 0.93 (TFnt gene). Incorporation of the TFnt gene into the pEAQ-HT vector, followed by transient expression in N. benthamiana, led to accumulation of TFnt to an approximate level of 0.12 mg/g of fresh leaf weight. Antimicrobial activity of purified plant- and bacterial-produced TFnt proteins was assessed against two strains of Gram-positive Staphylococcus aureus 305 and Newman. The results showed that plant-produced TFnt protein was preferentially active against S. aureus 305, showing 14% of growth inhibition, while the bacterial-produced TFnt revealed significant antimicrobial activity against both strains, showing 68 (IC50 25 µg/ml) and 60% (IC50 71 µg/ml) growth inhibition against S. aureus 305 and Newman, respectively. Although the combination of codon optimization and transient expression using the non-replicating pEAQ-HT expression vector facilitated production of the TFnt protein in plants, the most functionally active antimicrobial protein was obtained using the prokaryotic expression system.
- Research Organization:
- US Dept. of Agriculture (USDA), Beltsville, MD (United States). Agricultural Research Service (ARS)
- Sponsoring Organization:
- USDOE Office of Science (SC)
- Contributing Organization:
- US Dept. of Agriculture (USDA), Beltsville, MD (United States). Oak Ridge Inst. for Science and Education (ORISE). Agricultural Research Service (ARS). U.S. National Arboretum. Floral and Nursery Plants Research Unit
- Grant/Contract Number:
- SC0014664
- OSTI ID:
- 1629709
- Journal Information:
- AIMS Microbiology, Vol. 5, Issue 2; ISSN 2471-1888
- Publisher:
- AIMS PressCopyright Statement
- Country of Publication:
- United States
- Language:
- English
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