[Screening of three novel antimicrobial peptides with antifungal pathogens].

OBJECTIVE: In order to discover novel antimicrobial peptides against important crop pathogens, we designed and screened a high capacity random peptide library and isolated a number of clones expressing peptides with antifungal activity. We selected 96 peptides from the library and synthesized their sequence, which were used to assay their activity against crop fungal pathogens. METHODS: Using agar diffusion assay, these peptides were assayed for their activity against pathogens that cause cotton Fusarium wilt (Fusarium f. sp, vasinfecum), cotton red rot (Fusarium moniliforme), wheat spot blotch (Bipolaris sorokiniana) and potato early blight (Alternaria solani). RESULTS: The three random peptides, A6, D4 and F10, showed the strongest activity against the above four crop fungal pathogens. Through Blastp analysis, we did not find they have homologous sequences with known antimicrobial peptides. CONCLUSION: The novel antimicrobial peptides will provide gene resources for preventing important crop pathogens.

Characterization of an abaecin-like antimicrobial peptide identified from a Pteromalus puparum cDNA clone.

Abaecin is a major antimicrobial peptide, initially identified from the honeybee. In our effort to discover new antimicrobial peptides from the endoparasitoid wasp Pteromalus puparum, we identified an antibacterial cDNA clone that codes a fragment with high amino acid sequence similarity to abaecin. The proline-rich peptide (YVPPVQKPHPNGPKFPTFP, named PP30) was chemically synthesized and characterized in this study. Antimicrobial assays indicated that the cationic peptide was active against both Gram-negative and positive bacteria, but not active against fungi tested. No hemolytic activity was observed against human erythrocytes after 1h incubation at concentration of 125 microM or below. The antibacterial activity of PP30 against Escherichia coli was attenuated in the presence of increasing concentrations of NaCl. Transmission electron microscopic (TEM) examination of PP30-treated E. coli cells showed morphological changes in the cells and extensive damage to the cell membranes. The circular dichroism (CD) spectroscopy studies indicated that PP30 formed random coil structures in phosphate buffer (pH 7.4), 50% TFE and 25 mM SDS solution. Expression analysis of the gene coding for the peptide indicated that its expression was upregulated upon bacterial infection, indicating that the gene may play a role in preventing potential infection by microorganisms during parasitization in Pieris rapae.

Novel antimicrobial peptides identified from an endoparasitic wasp cDNA library.

We screened an endoparasitic wasp (Pteromalus puparum) cDNA library for DNA sequences having antimicrobial activity using a vital dye exclusion assay. Two dozens of clones were isolated that inhibited the growth of host Escherichia coli cells due to expression of the cloned genes. Three peptides (PP13, PP102 and PP113) were synthesized chemically based on the amino acid sequences deduced from these clones and assayed for their antimicrobial activity. These peptides have net positive charges and are active against both Gram-negative and -positive bacteria, but are not active against fungi tested. Their hemolytic activity on human red blood cells was measured, and no hemolytic activity was observed after 1-h incubation at a concentration of 62.5 microM or below. A Blast search indicated that the three peptides have not been previously characterized as antimicrobial peptides (AMPs). Salt-dependency studies revealed that the biocidal activity of these peptides against E. coli decreased with increasing concentration of NaCl. Transmission electron microscopic (TEM) examination of PP13-treated E. coli cells showed extensive damage of cell membranes. The CD spectroscopy studies noted that the enhanced alpha-helical characteristics of PP13 strongly contribute to its higher antimicrobial properties. These results demonstrate the feasibility to identify novel AMPs by screening the expressional cDNA library.

Screening and cloning of antimicrobial DNA sequences using a vital staining method.

A novel method for cloning of genes coding for cytotoxic molecules based on a cell viability assay is described. The working hypothesis is that expression of DNA sequences coding for cytotoxic molecules in bacterial cells will lead to cell death or impairment, and the isolation of the impaired or dead cells could lead to identification of DNA sequences responsible for debilitating the host cells. We verified this concept by isolating the well known antimicrobial Puroindoline b gene in Escherichia coli cells. We further demonstrated the feasibility to use this approach for isolating DNA encoding for antimicrobials from cDNA expression libraries. Sequence analysis and bioassay indicated that the isolated clones encoded previously characterized antimicrobial proteins (AMPs), proteins not previously characterized as AMPs, as well as novel antimicrobial peptides. In addition, clones harboring ribosomal protein encoding cDNA were also identified. Therefore, this method could also be used to identify host genes important in maintaining bacterial cell viability.

Recombinant protein expression plasmids optimized for industrial E. coli fermentation and plant systems produce biologically active human insulin-like growth factor-1 in transgenic rice and tobacco plants.

Human insulin-like growth factor-1 (hIGF-1) is a growth factor with clinical significance in medicine. The therapeutic potential of recombinant hIGF-1 (rthIGF-1) stems from the fact that hIGF-1 resembles insulin in many aspects of physiology. The expression of hIGF-1 in transgenic tobacco and rice plants using different expression cassettes is reported here. In the present study, two coding sequences were tested, one with the original human sequence, but partially optimized for expression in E. coli and the other with a plant-codon-optimized sequence that was expected to give a higher level of expression in plant systems. Three different hIGF-1 recombinant expression constructs were generated. All expression constructs utilized the maize ubiquitin 1 promoter with or without a signal sequence. Analyses conducted using a hIGF-1 specific ELISA kit showed all transgenic plants produced hIGF-1 and the accumulated hIGF-1 increased from the E. coli codon bias to higher levels when the hIGF-1 coding sequence was codon-optimized to match that of the maize zeamatin protein--the most transcribed gene in maize endosperm suspension cells. Further analyses that compared the functionality of the bacterial signal peptide Lam B in plants showed that this leader peptide led to lower expression levels when compared to transgenic plants that did not contain this sequence. This indicated that this expression construct was functional without removal of the bacterial signal sequence. The maize ubiquitin 1 promoter was found to be more active in rice plants than tobacco plants indicating that in this case, there was a class preference that was biased towards a monocot host. Biological analyses conducted using protein extracts from transgenic plants showed that the rthIGF-1 was effective in stimulating the in vitro growth and proliferation of human SH-SY5Y neuroblastoma cells. This indicated that the plant-produced rthIGF-1 was stable and biologically active. As some plants have been reported to express an endogenous insulin-like protein, we also looked for any effect of the human growth factor in transgenic plants, but no developmental or morphological differences with wild type tobacco or rice plants were detected. Since insulin and hIGF-1 share some overlapping roles, hIGF-1 may become a substitute therapeutic agent in subjects with certain defects in their insulin receptor signaling. Hence, if the full beneficial potential of rthIGF-1 is achieved, it is expected that in the future the demand will likely increase significantly.