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Vilnius, Lithuania

Starkevic U.,Nomads UAB | Starkevic U.,Vilnius University | Bortesi L.,Nomad Bioscience GmbH | Virgailis M.,LUHS Veterinary Academy | And 3 more authors.
Journal of Biotechnology | Year: 2015

Streptococcus pneumoniae is the causative agent of several serious infectious diseases. It is becoming increasingly antibiotic resistant worldwide, and thus new antimicrobials are needed. One alternative to antibiotics may be the use of peptidoglycan hydrolases, the bacteriophage lytic enzymes.In this study, we demonstrated high level expression of the S. pneumoniae bacteriophage lysin Pal in Nicotiana benthamiana - TMV (Tobacco Mosaic Virus) transient expression system. The protein was purified to homogeneity and tested for streptococci killing activity in vitro and in vivo. In vitro, Pal was able to lyse three tested S. pneumoniae strains: NCTC12695, NCTC12977 and NCTC11888. The treatment of BALB/c mice with 100. μg, 200. μg and 400. μg of Pal 1. h post-challenge with double lethal dose of S. pneumoniae NCTC12695 strain showed a clear dose response and protected from lethal sepsis 30%, 40% and 50% of mice, respectively. The improved mice survival correlated with decreased blood bacterial titers. In conclusion, these results suggest that plant-expressed bacteriophage lysins may have potential use as antimicrobial agents. © 2015 Elsevier B.V.

Denkovskiene E.,Nomads UAB | Denkovskiene E.,Vilnius University | Paskevicius S.,Nomads UAB | Werner S.,Nomad Bioscience GmbH | And 2 more authors.
Molecular Plant-Microbe Interactions | Year: 2015

Agrotransfection with viral vectors is an effective solution for the transient production of valuable proteins in plants grown in contained facilities. Transfection methods suitable for field applications are desirable for the production of high-volume products and for the transient molecular reprogramming of plants. The use of genetically modified (GM) Agrobacterium strains for plant transfections faces substantial biosafety issues. The environmental biosafety of GM Agrobacterium strains could be improved by regulating their T-DNA transfer via chemically inducible expression of virE2, one of the essential Agrobacterium virulence genes. In order to identify strong and stringently regulated promoters in Agrobacterium strains, we evaluated isopropyl-b-D-thiogalactoside-inducible promoters Plac, Ptac, PT7/lacO, and PT5/lacOlacO and cumic acid-inducible promoters PlacUV5/CuO, Ptac/CuO, PT5/CuO, and PvirE/CuO. Nicotiana benthamiana plants were transfected with a virE2-deficient A. tumefaciens strain containing transient expression vectors harboring inducible virE2 expression cassettes and containing a marker green fluorescent protein (GFP) gene in their T-DNA region. Evaluation of T-DNA transfer was achieved by counting GFP expression foci on plant leaves. The virE2 expression from cumic acid-induced promoters resulted in 47 to 72% of wild-type T-DNA transfer. Here, we present efficient and tightly regulated promoters for gene expression in A. tumefaciens and a novel approach to address environmental biosafety concerns in agrobiotechnology. © 2015 The American Phytopathological Society.

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