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Novel Antimicrobials from Endophytes of Northern Medicinal Plants

Periodic Report Summary - TEJAM (Novel antimicrobials from endophytes of northern medicinal plants)

Antibacterial resistance is spreading globally at an alarming rate and continues to increase. New potent antimicrobials are therefore constantly needed. Many of the pharmaceutical companies during the past decades focussed mainly on the development of antibiotics that offered variations on existing themes; therefore the flow of truly new compounds acting on new targets was diminished, whereas the bacterial resistance continued to spread. Even genomics did not provide the knowledge-based compounds that were expected, and now a new promise is being looked for in natural products from endophytic fungi.

During the TEJAM project new, high-throughput methods for screening antibacterial and antioxidant activities were tested and used for isolating antibacterial and antioxidant endophytic fungi. Fungal endophytes were isolated from two shrubs i.e. rhododendron tomentosum (RT) and calluna vulgaris (CV). All endophytic fungi were grown in two different media, enriched (MEB) and depleted (DM), and screened for antibacterial and antioxidant activities. As a result, 15 % of endophytic fungi of RT produced antibacterials and 14 % antioxidants; in total, 29 % of the isolates had biological activity. In contrast, 8 % of endophytes of CV produced antibacterials and 16 % antioxidants. The majority of antibacterial compounds were produced in DM media and antioxidants in MEB media. Therefore, it was advisable to test various alternative media for the production of antibacterials and antioxidants.

The world's biodiversity is overall underexplored. The classical microbiology was based on the study of cultured organisms, which represented an extremely low percentage of all microbial diversity. Various approaches for studying the uncultured microbes were developed in the biotechnology era, enabling scientists to access these previously untapped biological resources. In this study, fungal endophytes that were morphologically distinct were selected for identification. Eighteen and 20 isolates of endophytic fungi were identified from rhododendron tomentosum (RT) and calluna vulgaris (CV), respectively, by sequencing the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2). The unculturable endophytic fungi were identified by polymerase chain reaction (PCR) amplification, cloning and sequencing of the ITS region. Eighteen clones from CV and 11 clones from RT were sequenced and searched through the Genbank database using the basic local alignment search tool (BLAST). Moreover, all sequences were submitted to the Genbank and accession numbers were obtained.

In addition, TEJAM utilised and optimised state of the art methods, such as metagenomics on endophyte research to identify an antibacterial protein from empetrum nigrum L. The library was screened to select antibacterial clones using staphylococcus aureus as a target organism by the double agar layer method. One unique clone exhibiting antibacterial activity was selected from the metagenomic library. Based on the deduced amino acid similarity, the metagenomic gene encoded a protein without any known signal peptide, having three conserved pentatricopeptide motifs and displaying from 26 to 28 % identity to other hypothetical proteins. The predicted gene was expressed in escherichia coli and the protein was digested with trypsin. The trypsin digest was active against staphylococcus aureus. The active peptide was in the process of being identified by the time of this reporting period.