Molecular methods for identifying food and airborne fungi

Transcription

1 Molecular methods for identifying food and airborne fungi An overview Jos Houbraken CBS-KNAW Fungal Biodiversity Centre Utrecht, the Netherlands Overview presentation Introduction Material & Methods: from culture to sequence... Sequence based identification The ribosomal operon Protein coding genes Molecular taxonomy Phylogenetic species concept Detection of fungi Real time PCR assays Hybridization based assays 2 From culture to sequence 1. Cultivation 2. DNA extraction 3. PCR 4. Gel electrophoresis 5. Sequence PCR & Sequencing 6. Comparison in databases Yes Select locus (see Table 8) No Single spore isolation Perform tentative morphological based identification Identity known on genus level? Perform PCR of selected locus Gel electrophoresis Purification of PCR product needed? Yes Clean up PCR product No Comparative ITS sequence identification Sequence PCR product BLAST search against Genbank and/ or special database 3 1

2 Material and methods Cultivation & DNA extraction Pure cultures DNA extraction; various methods: Commercial kits: e.g. MoBio UltraClean, Masterpure Yeast DNA Purification kit (EPICENTRE) Home made; based on Möller et al, 1992 and many others Material and methods PCR Amplification of wanted piece of DNA in an exponential fashion; after 35 cycles, one starting copy of a gene would yield theoretically 2 36 copies of that DNA fragment Needed: primers flanking the region, DNA polymerase (+ buffer), dntps, template DNA Detection of amplified products; usually by (agarose) gel electrophoresis with ethidiumbromide Other less toxic dyes: MegaFluor, SYBR Safe, GelRed, GelGreen Material and methods Electrophoresis 2

3 Sequence PCR followed by sequencing (Sanger) Material and methods Sequencing & BLAST search BLAST search in NCBI database (or others) Material and methods BLAST results Which locus to sequence? Locus depends on genus/species! Target sequences Common Ribosomal DNA cluster Protein coding genes nuclear mitochondrial Less common Mycotoxin biosynthetic genes; random sequences; microsatellite loci; etc 3

4 Identification using Ribosomal DNA 18S 5.8S 25-28S IGS ITS1 ITS2 IGS Division Order Family?Genus?Family Genus?Species?Division?Order Family Genus?Species?infraspecific 10 18S (Small SubUnit) and 28S (Large SubUnit) Sequencing Comparable in some way to 16S sequencing in bacteria Advantages Primers are reliable Large available dataset Higher level taxonomy Problems Weak resolution of the phylogenetic backbone for some taxonomic groups Introns in 3 end Rarely more than one strain per species sequenced Not suitable for species identification 11 Species Level Taxonomy: ITS (Internal Transcribed Spacer) Sequencing Advantages Primers are generally reliable Large available dataset Often more than one strain per species sequenced BARCODE GENE, ITS data in public databases will increase in near future Problems Differences between copies (Fusarium, Zygomycetes, Basidiomycetes) Can be difficult to align above genus or family Often lack of resolution at species level 4

5 Barcode of Life initiative (BOLD): barcodinglife.com; barcodeoflife.org Indoor Fungi: Species Level Taxonomy: Protein Coding Genes Advantages More variable than ITS, especially in introns Usually multiple strains for each species (haplotypes) Problems No standard gene Universal primers usually not possible Not (yet) applied to broad range of fungi Alignments can be difficult 14 Species Level Taxonomy: Protein Coding Genes Fairly universal beta tubulin calmodulin elongation factor 1 alpha (=TEF) actin ribosomal polymerase B2 (= RPB2) Less universal chitinase histones glucose 6 phosphate dehydrogenase hydrophobins toxin genes Less variety now than 5 years ago but no standardization yet!! 15 5

6 Trees of Aspergillus section Cervini based on calmodulin and ITS sequences Calmodulin A. parvulus CBS A. parvulus CBS A. parvulus CBS A. parvulus CBS A. parvulus CBS T A. parvulus CBS A. cervinus CBS T A. cervinus CBS Aspergillus sp. nov. CBS Aspergillus sp. nov. CBS Aspergillus sp. nov. CBS A. nutans CBS T A. nutans CBS A. kanagawaensis CBS T Aspergillus sp. nov. CBS Aspergillus sp. nov. CBS Aspergillus sp. nov. CBS Aspergillus sp. nov. CBS A. clavatus CBS T ITS ITS and β tubulin gene trees of Penicillium subgenus Penicillium ITS β-tubulin Skouboe et al., 1999, Mycol. Res. 103: Samson et al., 2004, Stud. Mycol. 49: Which region to sequence? Known genus Penicillium: subgenus Penicillium tubulin ( other subgenera tubulin or ITS (GenBank good luck!) Trichoderma: ITS, TEF1 ( Fusarium: TEF1 (RPB1, RPB2); Aspergillus: ITS, tubulin, calmodulin (Genbank) Cladosporium: Actin, TEF1 (Genbank) Yeasts: ITS and partial 28S (D1/D2 region); GenBank & CBS website 6

7 IGS Which region to sequence? General strategy If fungus completely unknown; amplify 5 end of 18S to middle of 28S at once If genus or species is known or suspected, first check GenBank for comparative sequences, especially ITS or protein coding sequences Rules of thumb for ITS If > 3 bp differences, unlikely to be the same species If < 3 bp differences (including 100%) may or may not be same species 18S 5.8S 25-28S ITS1 ITS2 IGS 19 BLAST! Beware! Learn how to interpret BLAST searches E values: the lower the E value, the more "significant" the match is. Keep in mind that virtually identical short alignments have relatively high E values. For complete ITS sequence, E = 0 will be about 97% similarity, but good ID should be > 99% BLAST computes similarity based on the query sequence sequences shorter to but identical with the query will often not be recovered There are many misidentified sequences in GenBank Many known fungi are not yet sequenced 20 Overview presentation Introduction Material and methods (DNA extraction, PCR, Sequencing) Sequence based identification The ribosomal operon Protein coding genes Molecular taxonomy Phylogenetic species concept Detection of fungi Real time PCR assays Hybridization based assays 7

8 The Age of Species Discovery Estimated fungal species (2001): million Described species (2001): 320,000 Species presently recognized (2001): 80,000 Species in culture collections (1991): 11,500 Species in GenBank (July 2001): 6,300 Species in GenBank (June 2004): 11,000 Unidentified fungal species in GenBank (June 2004): 6,600 WHERE ARE THE MISSING FUNGI? Hyde, K.D (ed.). Mycological Research 105 (12): Evolution of Taxonomic Methods 18 th and 19 th century no microscopes About 1830 to present light microscopy About 1950 electron microscopy About 1970 isoenzymes About 1985 DNA based methods All these methods are used in modern taxonomy In the literature, individual genera or species may be characterized by one, a few, or all of these methods Within one genus there will be morphospecies, biological species, and phylogenetic species Some selected species concepts Morphological species concept A population or group of populations that differs morphologically from other populations (phenotype) Biological species concept A set of actually or potentially interbreeding populations (problematic for organisms that do not reproduce sexually, like the Deuteromycetes ) Phylogenetic species concept Phylogenetics through the boundary of genetically isolated groups into interbreeding individuals. 8

9 Species concepts and DNA How different must two DNA sequences be for them to represent two different species? Enough ( enough best defined by a separate measure indicating a species level distinctiveness. ) Robust species concepts Morphological Biological give same answer (polyphasic) Phylogenetic Phylogenetic Species Concept Geneological Concordance Phylogenetic Species Recognition = GCPSR The smallest group of populations for which a unique profile of character states, including apomorphies and synapomorphies, are fixed within the population. Nixon & Wheeler 1990 Taylor et al Fungal Genetics and Biology 31: Phylogenetic Species Concept Concordance B1 D F amds12 B2 1 of 2 MP trees P E CI = I RI = J K RC = L M 100 O C A. oryzae A 77 G H 100 N A. parasiticus CA1-05 A. parasiticus CA3-01 B1 B2 peca12 O I 1 of 30 MP trees M K CI = C RI = D RC = E J 73 A. oryzae 469 P F L 100 A H G N A. parasiticus CA1-05 A. parasiticus CA3-01 B1 D bena56 E B2 1 of 6 MP trees 87 I K CI = M RI = O RC = P A G 100 H N J L F C A. oryzae 469 A. parasiticus CA1-05 A. parasiticus CA3-01 B1 E B2 86 omt12 L C 1 of 4 MP trees 96 F J CI = O RI = P RC = D I 100 K 98 M A. oryzae 447, 448, 449, 469 A G 100 H N A. parasiticus CA1-05 A. parasiticus CA3-01 B1 D B2 trpc13 L 1 MP tree M C CI = E RI = F J RC = K 95 O A. flavus P I 100 A. oryzae 469 A A. parvisclerotigenus G H N A. parasiticus CA1-05 Courtesy of David Geiser, A. parasiticus CA3-01 Pennsylvania State University 27 9

10 Three gene phylogeny of Stachybotrys chartarum complex S. chartarum S. chlorohalonata Cruse et al., Mycologia 94: , Andersen et al., Mycologia 95: , Phylogenetic Species: The F. graminearum Complex O Donnell, K., H.C. Kistler, B.K. Tacke, and H.H. Casper PNAS 97: O'Donnell, K., Ward, T. J., Geiser, D. M., Kistler, H. C. and Aoki, T Fungal Geneticsand Biology 41: Starkey et al FGB 44: Diagnosis of F. boothii morphologically similar to F. graminearum, but has slightly different conidial features Sporodochial conidia formed on SNA under black light are less than 4.5 μm wide and their dorsal and ventral lines are often parallel and gradually curved. It is diagnosed by the following uniquely fixed nucleotide characters: mating type idiomorph positions 760 (G), 893 (G), 918 (T), 1073 (C), 1878 (C), 2309 (T), 2498 (C), 2561 (C), 2874 (T), 3251 (T), 3379 (T), 3545 (A), 3564 (C), 3582 (A), 4808 (C), 5981 (T), 6092 (C); reductase positions 30 (G), 501 (T), 591 (G), 706 (C), 794 (C), 919 (T); Translation elongation factor position 616 (C); histone H3 positions 148 (C), 252 (C), 300 (T), 404 (T); Tri01 positions 57 (G), 150 (G), 419 (T), 440 (T), 566 (A), 878 (C), 895 (T), 932 (T), 974 (T), 1032 (T), 1224 (A), 1292 (C), 1305 (A); phosphate permease 1 position 81 (A); ammonia ligase 1 positions 197 (T), 560 (T), 662 (T), 674 (T), 693 (T), 863 (G), 938 (G); beta tubulin 1 position 445 (A). O'Donnell et al., Fungal Gen. Biol. 41: SNPs used for molecular diagnostics: Luminex 30 10

11 F. graminearum phylogenetic species Data sets for different genes Gene Haplotypes # species recognized ITS/28S 20 0 β-tubulin 37 2 Histone H EF1-α 35 4 Reductase 42 6 α tubulin 31 8 MAT URA-Tri101-PHO Combined Starkey et al FGB 44: The multigene arms race is on 35 genes for phylogenetics 31 Molecular taxonomy as it is practiced Much more emphasis on phylogenetic (evolutionary) relationships See Assembling the Fungal Tree of Life aftol.org Mycologia 98 (6) Dec Little emphasis by taxonomists of development of molecular diagnostics Some species specific PCR primers Some real time PCR kits Definition of species boundaries using molecular methods is just as problematic as with other methods 32 Overview presentation Introduction Material and methods (DNA extraction, PCR, Sequencing) Sequence based identification The ribosomal operon Protein coding genes Molecular taxonomy Phylogenetic species concept Detection of fungi Real time PCR assays Hybridization based assays 11

12 Detection methods Detection of colonies on plates (CFU) Flow cytometry Microscopy Nucleic acid probes Immunological methods Amplification (PCR) based methods Quantification level Detection limit, time Real time vs. traditional PCR amplification curve Real time PCR assays Based on specific PCR primers and a reporter probe Several detection systems TaqMan SyberGreen Molecular Beacons Currently many assays are developed in various lab Quantification against a standard curve 15,000 30,000 euro for the machine Usually 96 well format ~ 4 probes per well 36 12

13 38 Hybridization based techniques Dot blot, Colony, Southern, Northern, Western, In situ Detection of the label: Colorimetry Radioactivity Fluorescence Luminescence Magnetometry Etc 13

14 LSU rdna based oligonucleotide probes for common airborne fungi Zeng et al. 2004; FEMS Microb Lett 237: 79 DNA Microarrays DNA Chip DNA microarrays 14

15 Detection of Candida and Aspergillus sp. by microarray analysis Leinberger et al. JCM 2005; 43: 4943 Microarray with oligonucleotides for known biosynthetic genes Aflatoxin (A. flavus: 29 genes) Ochratoxin (P. nordicum: 25 genes) Patulin (P. expansum: 2 genes) Trichothecenes (F. graminearum: 25 genes, F. sporotrichioides: 18 genes, F. poae: 1 gene) Fumonisin (F. verticillioides: 23 genes) Control (actin: 7 genes various fungi) Used to examine the expression of biosynthetic genes under different growth conditions (mrna quantification) Promising tool for identification of mycotoxin producing fungi Next generation sequencing 454 sequencing (pyrosequencing) Ghannoum et al, Characterization of the oral fungal microbiome (mycobiome) in healthy individuals, PLOS pathogens,

16 Next generation sequencing 454 sequencing (pyrosequencing) The most cosmopolitan OTUs, occurring in more than half of the samples (>50%), or in nine or ten of the countries sampled (9 or more Countries), were dominated by taxa in the Class Dothideomycetes. Interpretation of Molecular Assays Some assays can be used directly from environmental samples Arrays and Real time PCR are overkill for pure cultures much cheaper to sequence Some assays can only detect where the assay is designed for Distinguishing dead and living material is technically difficult Most methods are not properly validated Comparison of molecular detection results with classical microbiological results is a challenge 47 Conclusions Real time PCR assays and hybridization assays now being developed in many labs Comparison of classical results with molecular results is a problem There is progression from ribosomal gene sequencing (basic studies), through single protein gene sequencing (more resolution) to multigene phylogenies (for determining species boundaries) Molecular diagnostics requires reliable sampling of variation within a species A well stocked library and a good microscope are still necessary for fungal identification Barcoding : identification of species of all Fungi; ITS is the prime barcode 16

17 Aren t they cool the new barcodes they ve given us? As if that s something Look, daddy, look at the big AACACTGTATCTAATTATT!!!! 17