This work describes the de-novo design of peptides that inhibit a broad range of plant pathogens. Four structurally different groups of peptides were developed that differ in size and position of their charged and hydrophobic clusters and were assayed for their ability to inhibit bacterial growth and fungal spore germination. Several peptides are highly active at concentrations between 0,1 and 1 µg/ml against plant pathogenic bacteria, such as Pseudomonas syringae, Pectobacterium carotovorum, and Xanthomonas vesicatoria. Importantly, no hemolytic activity could be detected for these peptides at concentrations up to 200 µg/ml. Moreover, the peptides are also active after spraying on the plant surface demonstrating a possible way of application. In sum, our designed peptides represent new antimicrobial agents and with the increasing demand for antimicrobial compounds for production of “healthy” food, these peptides might serve as templates for novel antibacterial and antifungal agents.
Antimicrobial peptides are important defense compounds of higher organisms that can be used as therapeutic agents against bacterial and/or viral infections. We designed several antimicrobial peptides containing hydrophobic and positively charged clusters that are active against plant and human pathogens. Especially peptide SP1-1 is highly active with a MIC value of 0.1 μg/ml against Xanthomonas vesicatoria, Pseudomonas corrugata and Pseudomonas syringae pv syringae. However, for commercial applications high amounts of peptide are necessary. The synthetic production of peptides is still quite expensive and, depending on the physico-chemical features, difficult. Therefore we developed a plant/tobacco mosaic virus-based production system following the 'full virus vector strategy' with the viral coat protein as fusion partner for the designed antimicrobial peptide. Infection of Nicotiana benthamiana plants with such recombinant virus resulted in production of huge amounts of virus particles presenting the peptides all over their surface. After extraction of recombinant virions, peptides were released from the coat protein by chemical cleavage. A protocol for purification of the antimicrobial peptides using high resolution chromatographic methods has been established. Finally, we yielded up to 0.025 mg of peptide per g of infected leaf biomass. Mass spectrometric and NMR analysis revealed that the in planta produced peptide differs from the synthetic version only in missing of N-terminal amidation. But its antimicrobial activity was in the range of the synthetic one. Taken together, we developed a protocol for plant-based production and purification of biologically active, hydrophobic and positively charged antimicrobial peptide.
Touching of contaminated objects and surfaces is a well-known method of virus transmission. Once they are attached to the hands, viruses can easily get adsorbed and initiate infection. Hence, disinfection of frequently touched surfaces is of major importance to prevent virus spreading. Here we studied the antiviral activity of a glucoprotamin-containing disinfectant against influenza A virus and the model virus vaccinia virus (VACV) dried on inanimate surfaces. The efficacy of the surface disinfectant on stainless steel, polyvinyl chloride, and glass coupons was investigated in a quantitative carrier test. Vacuum-dried viruses were exposed to 0.25%, 0.5%, and 1% disinfectant for 5 min, 15 min, and 30 min without agitation, and residual infectivity was determined by endpoint titration. Although glucoprotamin was highly active against both viruses in suspension, limited antiviral activity against the surface-dried viruses was detected. Even after 30 min of exposure to 1% disinfectant, VACV was not completely inactivated. Furthermore, influenza A virus inactivation was strongly affected by the surface composition during the 5-min and 15-min treatments with 0.25% and 0.5% disinfectant. The results presented in this study highlight the relevance of practical tests to assess the antiviral activity of surface disinfectants. High virucidal activity in solution is not necessarily indicative of high antiviral activity against surface-dried viruses. In addition, we want to emphasize that the mere exposure of surfaces to disinfectants might not be sufficient for virus inactivation and mechanical action should be applied to bring attached viruses into contact with virucidal compounds.
Material of bovine origin is often used in biotechnological applications. Bovine viral diarrhea virus (BVDV) is one of the major viral contaminants, and not only detection and inactivation but also quantification of the viral load in bovine starting material is required by the regulatory agencies. Here, we investigated combined virus propagation in cell culture and quantitative real-time PCR (qRT-PCR) for the applicability to detect and estimate low BVDV titers in bovine lung lavages, the source material for manufacturing pulmonary surfactant. qRT-PCR analyses of the crude lung lavages were performed and qRT-PCR calibration curves based on infective viral doses (TCID 50 /mL) were generated with a detection limit of 100 TCID 50 /mL. Lung lavages were inoculated on susceptible MDBK cells and cell culture samples were again analyzed by qRT-PCR. Immunofluorescence staining was performed to prove qRT-PCR results. Interestingly, initial BVDV contaminations in lung lavages were below qRT-PCR detection limit. An amplification step in cell culture enabled BVDV propagation to levels detectable by qRT-PCR. In comparison with the qRT-PCR calibration curve and control experiments with defined inoculation doses, the estimation of minor BVDV contaminations in lung lavages was possible. Both techniques can be successfully combined to estimate the viral load in dilute sample material.
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