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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Molecular Plant Pathology Laboratory » Research » Publications at this Location » Publication #333924
Title: Antimicrobial activity of bacteriophage derived triple fusion protein against Staphylococcus aureus

Author
item Kovalskaya, Natalia
item HERNDON, ELEANOR - US Department Of Agriculture (USDA)
item Foster Frey, Juli
item Donovan, David
item Hammond, Rosemarie

Submitted to: AIMS Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/19/2019
Publication Date: 6/25/2019
Citation: Kovalskaya, N.Y., Herndon, E., Foster Frey, J.A., Donovan, D.M., Hammond, R. 2019. Antimicrobial activity of bacteriophage derived triple fusion protein against Staphylococcus aureus. AIMS Microbiology. https://doi.org/10.3934/microbiol.2019.2.158.
DOI: https://doi.org/10.3934/microbiol.2019.2.158

Interpretive Summary: The emergence of antibiotic-resistant bacteria as a result of widespread use of antibiotics in medicine and animal husbandry can lead to the spread of resistant bacteria in the environment. However, despite the availability of new vaccines and antibiotics, there is a need for the development of alternative treatments and new antimicrobials to combat bacterial pathogens. Peptidoglycan hydrolases, enzymes that degrade the peptidoglycan of the bacterial cell wall, are one of the most promising antimicrobial agents. We report the production of a functionally active triple-acting fusion protein in plants that inhibits the growth of Staphylococcus aureus, the bacterium often responsible for abscesses, respiratory infections and food poisoning. These results will be of interest to scientists who are producing pharmaceuticals in plant tissues and developing strategies to control bacterial diseases.

Technical Abstract: The increasing spread of antibiotic-resistant microorganisms has led to the necessity of developing alternative antimicrobial treatments. The use of peptidoglycan hydrolases, naturally produced by living organisms and causing bacterial cell wall lysis, is a promising approach to combat bacterial infections. In our study, we constructed a triple-acting fusion gene consisting of an N-terminal amidase-5 domain of a LambdaSA2 prophage endolysin gene (D-glutamine-L-lysin endopeptidase), a mid-protein amidase-2 domain of Staphylococcal phage 2638A endolysin gene (N-acetylmuramoyl-L-alanine amidase), and a full-length copy of the Staphylococcus simulans lysostaphin gene (glycyl-glycine endopeptidase). A histidine tag was added to facilitate purification. The resulting gene was also codon optimized for expression in tobacco plants and incorporated into the cowpea mosaic virus-based pEAQ-HT vector with subsequent introduction into Nicotiana benthamiana through agroinfiltration. Only with codon optimization were we able to produce the protein in plants. The expression level of the triple-acting fusion protein was 0.12 mg per gram fresh weight of plant tissue. Antimicrobial assays with Staphylococcus aureus 305 and Escherichia coli BL21 (DE3) revealed antibacterial activity of the purified plant-produced triple-acting fusion protein preferably against Gram-positive S. aureus 305, showing 14% of inhibition of growth. Our experiments revealed that, among the plant virus-based vectors used for expression of the 1.9 kb-triple fusion gene (the potato virus X-based vector pP2C2S and Alternanthera mosaic virus-based vectors pGD5TGB1L8823-MCS-CP3, pGDAltMV3-7) only transient expression using the pEAQ-HT vector facilitated production of the functionally active protein.