Antimicrobial activity of bacteriophage derived triple fusion protein against Staphylococcus aureus

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

doi:10.3934/microbiol.2019.2.158

Antimicrobial activity of bacteriophage derived triple fusion protein against Staphylococcus aureus

AIMS Microbiol. 2019 Jun 25;5(2):158-175. doi: 10.3934/microbiol.2019.2.158. eCollection 2019.

Authors

Natalia Y Kovalskaya¹, Eleanor E Herndon², Juli A Foster-Frey³, David M Donovan³, Rosemarie W Hammond⁴

Affiliations

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.

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.

Keywords: CPMV-based vector; Nicotiana benthamiana; antimicrobials; codon optimization; endolysins; lysostaphin; transient expression.