Defensio PhD Thesis Marcus Seibold Experimental and Theoretical Investigations of Penicillium brevicompactum Dierckx. introduction. fungus.

Transcription

1 Defensio PhD Thesis Marcus Seibold Experimental and Theoretical Investigations of Penicillium brevicompactum Dierckx introduction fungus metabolite University Vienna, Institute of Theoretical Chemistry, Fr page 1 of 33

2 introduction Experimental and Theoretical Investigations of Penicillium brevicompactum Dierckx introduction fungus metabolite University Vienna, Institute of Theoretical Chemistry, Fr page 2 of 33

3 introduction antimicrobial peptides - part of the innate immune system of virtually all forms of life - appeared very early in the evolution - common properties: small, high ratio of cationic amino acids four groups of antimicrobial peptides: (1) linear α-helical peptides generally antibacterial activities (2) multiple cysteine-bridge-containing peptides (defensins) antibacterial and antifungal activities (3) proline-rich peptides against bacteria and filamentous fungi (4) the glycine-rich peptides against gram-negative/positive bacteria Gordon et al, Curr Eye Res July ; 30(7): University Vienna, Institute of Theoretical Chemistry, Fr page 3 of 33

4 introduction.. growing interest in antimicrobial peptides and defensins.. published articles per year with key word antimicrobial peptides published articles per year with key word defensin source: NCBI University Vienna, Institute of Theoretical Chemistry, Fr page 4 of 33

5 introduction.. antimicrobial peptides are potential drug candidates in human therapy.. Hancock et al, Nat Biotechnol Dec;24(12): University Vienna, Institute of Theoretical Chemistry, Fr page 5 of 33

6 introduction.. mechanisms of action of antimicrobial peptides.. disruption of cell membrane toroidal pore carpet-like inhibition respiration induction of barrel-like inhibition nucleotide synthesis inhibition folding inhibition ribosome interference with membrane structure / biosynthesis modified from Peters et al; PLoS Pathog October; 6(10): e University Vienna, Institute of Theoretical Chemistry, Fr page 6 of 33

7 introduction my work area: antimicrobial peptides defensins fungal defensins fungi mammals defensins defensins insects defensins against bacteria against fungi plants defensins defensins invertebrates University Vienna, Institute of Theoretical Chemistry, Fr page 7 of 33

8 introduction.. fungal defensins are current topics of several research groups.. Prof. Marx, Innsbruck P. chrysogenum, defensin PAF Prof. Theis, Berlin A. gigangteus, defensin AFP Prof. Nasri, Tunis A. clavatus, defensin AcAFP my contribution: P. brevicompactum, defensin BP University Vienna, Institute of Theoretical Chemistry, Fr page 8 of 33

9 introduction contribution of my PhD study characterisation of a new defensin, the bubble first time shown co-expression of a defensin with a clinically relevant small molecule first time shown that P. chrysogenum encodes two defensins University Vienna, Institute of Theoretical Chemistry, Fr page 9 of 33

10 introduction.. once upon a time there was a fungal contamination of experimental barley cultures in a greenhouse.... growing the contamination itself was successful and due to yellow-green fluorescence of exudate bubbles the fungus evoked some interest.. eventually leading to this defensio today.. University Vienna, Institute of Theoretical Chemistry, Fr page 10 of 33

11 introduction bubbles under UV light at 20 magnification.. bubbles on the surface of Penicillium brevicompatcum contain mainly two substances; they are separated by gel chromatography.. gel chromatography bubble (BP) mycophenolic acid University Vienna, Institute of Theoretical Chemistry, Fr page 11 of 33

12 fungus Experimental and Theoretical Investigations of Penicillium brevicompactum Dierckx introduction fungus metabolite University Vienna, Institute of Theoretical Chemistry, Fr page 12 of 33

13 fungus overview fungus identification inhibition studies University Vienna, Institute of Theoretical Chemistry, Fr page 13 of 33

14 fungus identification.. after several attempts to extract genomic DNA from the fungus a protocol optimized for plant DNA was successful.. genetic identification - amplification of internal transcribed spacer region of 5.8 rdna - data base comparison at NCBI result - Penicillium brevicompactum Dierckx University Vienna, Institute of Theoretical Chemistry, Fr page 14 of 33

15 fungus inhibition studies.. since the fungus is a kind of Penicillium, basic experiments for hints of a potential antibiotic effect were performed.. inhibition studies - co-cultivation with bacteria - Enterococcus faecium - Lactococcus lactis - Escherichia coli - 20 C, 37 C - spores of the fungus - bubbles of the fungus result - neither the spores of the fungus nor the bubbles inhibit the growth of the bacteria.. later experiments concerning yeast told a different story.. (slide 26) University Vienna, Institute of Theoretical Chemistry, Fr page 15 of 33

16 Experimental and Theoretical Investigations of Penicillium brevicompactum Dierckx introduction fungus metabolite University Vienna, Institute of Theoretical Chemistry, Fr page 16 of 33

17 overview primary sequence crystal structure gdna, exons, introns similarity to other s structure prediction of other s BP homolog in P. chrysogenum inhibition of yeast University Vienna, Institute of Theoretical Chemistry, Fr page 17 of 33

18 primary sequence.. due to the high content and purity of the dissolved in the bubbles, a significant part of the primary sequence of the bubble was revealed by Edman degradation straight ahead from the Petri dish and allowed primer design for subsequent PCR experiments for getting the cdna.. the resulting sequence shows - high content of cysteins - relatively basic amino acids - relatively small size DTCGSGYNVDQRRTNSGCKAGNGDRHFCGCDRTGVVECKGGKWTEVQDCGSSSCKGTSNGGATC University Vienna, Institute of Theoretical Chemistry, Fr page 18 of 33

19 crystal structure.. crystallization of the bubble was straight forward and revealed important properties: - 4 disulfide-bridges - for defensins typical twisted anti-parallel beta-sheet Olsen et al. Solving the structure of the bubble using the anomalous sulfur signal from single-crystal in-house Cu K diffraction data only. Acta Crystallogr D Biol Crystallogr Feb;60 (Pt 2): Epub 2004 Jan 23. PDB Code: 1UOY University Vienna, Institute of Theoretical Chemistry, Fr page 19 of 33

20 gdna, exons, introns.. the architecture of the gene was revealed by genome walking using thermal asymmetric interlaced PCR bp BP gdna intron exon 1 exon 2 signal peptide propeptide mature secreted University Vienna, Institute of Theoretical Chemistry, Fr page 20 of 33

21 similarity to other s signal peptide propeptide mature.. the alignments shows two clusters: the BP cluster on top and the PAF cluster below University Vienna, Institute of Theoretical Chemistry, Fr page 21 of 33

22 similarity to other s.. very high homology in the BP cluster.. BP P. brevicompactum chr P.chrysogenum the M. thermophila glo C. globosum fis N. fischeri fum A.fumigatus University Vienna, Institute of Theoretical Chemistry, Fr page 22 of 33

23 structure prediction of other s.. successful structure prediction of hypothetical s from whole genome sequences of similar fungi.. black = bubble BP P. brevicompactum chr P.chrysogenum colored = hypothetical s glo C. globosum fis N. fischeri fum A.fumigatus University Vienna, Institute of Theoretical Chemistry, Fr page 23 of 33

24 BP homolog in P. chrysogenum.. whole genome search revealed that P.chrysogenum has a BP homolog in addition to PAF.. Query = BP sequence Sbjct = translated P. chrysogenum gene sequence.. therefore the genome of P. chrysogenum encodes for two defensins and penicillin.. University Vienna, Institute of Theoretical Chemistry, Fr page 24 of 33

25 BP homolog in P. chrysogenum.. P. chrysogenum Wisconsin harbours the PAF gene which establishes the PAF cluster on chromosome 24; on chromosome 21 also the BP analogue is present and establishes the BP cluster.. University Vienna, Institute of Theoretical Chemistry, Fr page 25 of 33

26 inhibition of yeast.. spiking studies demonstrated a concentration dependent inhibition of yeast cultures by the bubble.. viability of yeast was reduced by about 30 % University Vienna, Institute of Theoretical Chemistry, Fr page 26 of 33

27 metabolite Experimental and Theoretical Investigations of Penicillium brevicompactum Dierckx introduction fungus metabolite University Vienna, Institute of Theoretical Chemistry, Fr page 27 of 33

28 metabolite overview metabolite isolation identification NMR calculation application University Vienna, Institute of Theoretical Chemistry, Fr page 28 of 33

29 metabolite isolation.. about 100 ml bubbles were collected and standard procedures yielded pure crystals.. processing of the bubbles - organic extraction (tert-butylmethylether) - concentration of organic phase - solubilisation in MeOH:H 2 O - gel chromatography - concentration of eluate - one week crystallisation on room temperature picture of MPA crystals University Vienna, Institute of Theoretical Chemistry, Fr page 29 of 33

30 metabolite identification 1 H NMR 13 C NMR.. crystals were dissolved in DCCl 3 and NMR experiments identified mycophenolic acid.. University Vienna, Institute of Theoretical Chemistry, Fr page 30 of 33

31 metabolite NMR calculation.. further verification was established by quantum chemical calculations and comparison with the SPECINFO data base.. University Vienna, Institute of Theoretical Chemistry, Fr page 31 of 33

32 metabolite application mycophenolic acid - first antibiotic derived from a fungus (Cosio 1896) - antibacterial, antifungal, antiviral, antitumor effects - used in human therapy as an immunosuppressive University Vienna, Institute of Theoretical Chemistry, Fr page 32 of 33

33 summary.. summary of the PhD study.. promoter sequence intron exons characterisation of the bubble 3D structure signal peptide propeptide size alignment with similar s absence of glycans pi structure prediction of other s first time shown co-expression of a defensin with a clinically relevant small molecule BP secreted together with mycophenolic acid first time shown that P. chrysogenum encodes two defensins University Vienna, Institute of Theoretical Chemistry, Fr page 33 of 33

34 acknowledgment There are many people I have to thank and especially the colleagues from the Institute of Theoretical Chemistry Institute of Structural Biology Department of Pharmaceutical Technology and Biopharmaceutics University Vienna, Institute of Theoretical Chemistry, Fr page 34 of 33