DIPLOMARBEIT Titel der Diplomarbeit Biocidal activity and biochemistry of Leptodactylus pentadactylus frog foam nests – an analysis with insights into N-glycosylation Verfasserin Sylvia Tippl angestrebter akademischer Grad Magistra der Naturwissenschaften (Mag. rer. nat.) Wien, im Februar 2011 Studienkennzahl lt. Studienblatt: A 441 Studienrichtung lt. Studienblatt: Diplomstudium Genetik – Mikrobiologie (Stzw.) Betreuerin / Betreuer: Univ.-Doz. Mag. Dr. Julia Walochnik Contents CONTENTS ABBREVIATIONS .......................................................................................................... VII 1 INTRODUCTION ......................................................................................................... 1 1.1 BIOFOAMS IN NATURE ............................................................................................. 1 1.2 FOAM NESTS OF LEPTODACTYLUS PENTADACTYLUS ................................................ 3 1.3 OTHER FOAM NESTING FROGS ................................................................................ 4 1.4 FUNCTIONS OF FROG FOAMS ................................................................................... 8 1.4.1 Biocidal activity ........................................................................................................ 11 1.5 BIOCHEMICAL PROPERTIES OF FROG FOAMS ....................................................... 12 1.5.1 Glycoproteins/ Glycosylation.................................................................................... 14 1.5.1.1 O-Glycosylation ........................................................................................... 15 1.5.1.2 N-Glycosylation ........................................................................................... 16 1.5.1.3 Glycan structures of frogs ............................................................................ 19 1.6 LEPTODACTYLUS PENTADACTYLUS ......................................................................... 20 1.6.1 Systematics................................................................................................................ 20 1.6.2 Geographical distribution and habitat ....................................................................... 23 1.6.3 Morphology ............................................................................................................... 23 1.6.4 Feeding ecology ........................................................................................................ 24 1.6.5 Skin secretions .......................................................................................................... 24 1.7 TEST ORGANISMS ................................................................................................... 26 1.7.1 Protozoa .................................................................................................................... 26 1.7.1.1 Trypanosoma cruzi ....................................................................................... 26 1.7.1.1 Leishmania donovani and L. infantum ......................................................... 28 1.7.1.2 Acanthamoeba .............................................................................................. 30 1.8 AIMS OF THE STUDY ............................................................................................... 32 2 MATERIAL AND METHODS .................................................................................. 33 2.1 FROG FOAM............................................................................................................ 33 2.2 PURIFICATION ........................................................................................................ 33 2.3 SOLUBILITY ........................................................................................................... 33 2.4 HOMOGENISATION ................................................................................................ 34 III Contents 2.4.1 Sonicator ................................................................................................................... 34 2.4.2 Mortar and pestle ....................................................................................................... 34 2.4.3 Bead Beater ............................................................................................................... 34 2.5 PROTEIN CHARACTERISATION .............................................................................. 35 2.5.1 Protein concentration ................................................................................................ 35 2.5.2 SDS-PAGE ................................................................................................................ 36 2.5.2.1 Coomassie Brilliant Blue staining ................................................................ 37 2.6 N-GLYCAN ANALYSIS ............................................................................................ 37 2.6.1 Blots .......................................................................................................................... 37 2.6.1.1 Tank-blot ...................................................................................................... 38 2.6.1.2 Semi-dry blot ................................................................................................ 38 2.6.1.3 Incubation with lectins and antibodies ......................................................... 38 2.6.1.4 Development with alkaline phosphatase and BCIP®/NBT.......................... 40 2.6.2 “In-gel release method” for N-glycan analysis .......................................................... 40 2.6.2.1 SDS-PAGE and Coomassie staining ............................................................ 40 2.6.2.2 Washing of the gel pieces ............................................................................. 41 2.6.2.3 Tryptic and N-glycosidase F digestion ......................................................... 42 2.6.2.4 Purification of the released N-glycans .......................................................... 42 2.6.2.5 MALDI-TOF-MS analysis of N-glycans ...................................................... 42 2.6.3 Preparation of 2-aminopyridine derivatised N-glycans ............................................. 43 2.6.3.1 Pepsin digestion ............................................................................................ 43 2.6.3.2 Binding to a cation exchanger ...................................................................... 43 2.6.3.3 Desalting by gel filtration ............................................................................. 44 2.6.3.4 N-Glycosidase F digestion and purification of the N-glycans ...................... 44 2.6.3.5 Derivatisation with aminopyridine ............................................................... 45 2.6.3.6 Separation of pyridylaminated N-glycans by HPLC .................................... 46 2.7 ANALYSIS OF BIOCIDAL ACTIVITY ........................................................................ 46 2.7.1 Cell culture ................................................................................................................ 47 2.7.1.1 Trypanosoma cruzi ....................................................................................... 47 2.7.1.2 Leishmania spp. ............................................................................................ 47 2.7.1.3 Acanthamoeba .............................................................................................. 48 2.7.1.4 Bacteria and fungi ........................................................................................ 49 2.7.2 Microtiter plate assays ............................................................................................... 50 2.7.2.1 Hemocytometer after Bürker ........................................................................ 51 2.7.3 Plate diffusion assays ................................................................................................ 51 2.7.3.1 Plate diffusion assays with filter paper ......................................................... 51 IV Contents 2.7.3.2 Plate diffusion assays without filter paper ................................................... 52 2.8 MICROORGANISMS ASSOCIATED WITH THE FOAM ............................................... 52 2.8.1 Isolation ..................................................................................................................... 52 2.8.2 Screening of bacteria for in vitro antibiosis .............................................................. 53 2.8.2.1 Staining of the mycelium of T. mentagrophytes .......................................... 53 2.8.3 Characterization of the antibiotic bacteria ................................................................ 53 2.8.3.1 API ............................................................................................................... 53 2.8.3.2 MALDI-Biotyper ......................................................................................... 54 3 RESULTS ..................................................................................................................... 55 3.1 SOLUBILITY AND HOMOGENISATION .................................................................... 55 3.2 PROTEIN CHARACTERISATION .............................................................................. 57 3.3 GLYCOSYLATION ................................................................................................... 58 3.3.1 N-Glycome ...............................................................................................................