Antimicrobial activity of bacteriophage derived triple fusion protein against <em>Staphylococcus aureus</em>

Natalia Y. Kovalskaya; Eleanor E. Herndon; Juli A. Foster-Frey; David M. Donovan; Rosemarie W. Hammond; Natalia Y. Kovalskaya; Eleanor E. Herndon; Juli A. Foster-Frey; David M. Donovan; Rosemarie W. Hammond

doi:10.3934/microbiol.2019.2.158

AIMS Microbiology

2019, Volume 5, Issue 2: 158-175. doi: 10.3934/microbiol.2019.2.158

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Research article

Antimicrobial activity of bacteriophage derived triple fusion protein against Staphylococcus aureus

1.
Floral and Nursery Plants Research Unit, U.S. National Arboretum, Agricultural Research Service, ORISE - U.S. Department of Agriculture, Beltsville, MD, USA
2.
Kerry’s Nursery, Miami, FL, USA
3.
Animal Biosciences and Biotechnology Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD, USA
4.
Molecular Plant Pathology Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD, USA

Received: 18 March 2019 Accepted: 19 June 2019 Published: 25 June 2019

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 (D-glutamine-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 non-replicating Cowpea mosaic virus (CPMV)-based vector pEAQ-HT and the replicating Alternanthera mosaic virus (AltMV)-based pGD5TGB1_L8823-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.
- endolysins,
- lysostaphin,
- antimicrobials,
- CPMV-based vector,
- transient expression,
- codon optimization,
- Nicotiana benthamiana
Citation: Natalia Y. Kovalskaya, Eleanor E. Herndon, Juli A. Foster-Frey, David M. Donovan, Rosemarie W. Hammond. Antimicrobial activity of bacteriophage derived triple fusion protein against Staphylococcus aureus[J]. AIMS Microbiology, 2019, 5(2): 158-175. doi: 10.3934/microbiol.2019.2.158

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Abstract

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 (D-glutamine-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 non-replicating Cowpea mosaic virus (CPMV)-based vector pEAQ-HT and the replicating Alternanthera mosaic virus (AltMV)-based pGD5TGB1_L8823-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.

Abbreviation TF: triple-acting fusion; PGH: peptidoglycan hydrolases; IB: inclusion body; SDS-PAGE: sodium dodecyl sulfate–polyacrylamide gel electrophoresis; DNA: deoxyribonucleic acid; RNA: ribonucleic acid; PCR: polymerase chain reaction; RT-PCR: reverse transcription polymerase chain reaction; Ni-NTA: nickel-nitrilotriacetic acid; IPTG: Isopropyl β-D-1-thiogalactopyranoside; DTT: dithiothreitol; CFU: colony-forming unit; IC: 50% inhibitory concentration; TSA: Tryptic Soy Agar; TSB: Tryptic Soy Broth; CP: coat protein; AMV: ;
Acknowledgments

We thank Dr. George Lomonossoff (John Innes Centre, Norwich, UK) and Plant Bioscience Ltd., Norwich, UK for providing the pEAQ-HT vector, and Dr. John Hammond (USDA ARS NEA USNA FNPRU) for providing the pGD5TGB1_L8823-MCS-CP3 vector.
This research was supported in part by an appointment to the Agricultural Research Service (ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-SC0014664. All opinions expressed in this paper are the author's and do not necessarily reflect the policies and views of USDA, ARS, DOE, or ORAU/ORISE.
Mention of trade name or commercial products in this publication is solely to provide specific information and does not imply recommendation or endorsement by the U. S. Department of Agriculture. USDA is an equal opportunity provider and employer.

Conflict of interest

All authors declare no conflicts of interest in this paper.

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