Johannes Wöstemeyer

Identification of dermatophyte species causing onychomycosis and tinea pedis by MALDI-TOF mass spectrometry

Abstract:  Identification of dermatophytes is currently performed based on morphological criteria and is increasingly supported by genomic sequence comparison. The present study evaluates an alternative based on the analysis of clinical fungal isolates by mass spectrometry. Samples originating from skin and nail were characterized morphologically and by sequencing the internal transcribed spacer 1 (ITS1), ITS2 and the 5.8S rDNA regions of the rDNA clusters. In a blind comparative study, samples were analyzed by matrix assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF MS). The mass spectra were compared to a database comprising of the spectral data of reference strains by applying the saramis software package. All fungal isolates belonging to the taxa Trichophyton rubrum, T. interdigitale, T. tonsurans, Arthroderma benhamiae and Microsporum canis were correctly identified, irrespective of host origin and pathology. To test the robustness of the approach, four isolates were grown on five different media and analyzed. Although the resulting mass spectra varied in detail, a sufficient number of signals were conserved resulting in data sets exploitable for unequivocal species identification. Taken together, the usually widespread dermatophytes can be identified rapidly and reliably by mass spectrometry. Starting from pure cultures, MALDI‐TOF MS analysis uses very simple sample preparation procedures, and a single analysis is performed within minutes. Costs for consumables as well as preparation time are considerably lower than for PCR analysis.

Phylogeny and origin of 82 zygomycetes from all 54 genera of the Mucorales and Mortierellales based on combined analysis of actin and translation elongation factor EF-1α genes

True fungi (Eumycota) are heterotrophic eukaryotic microorganisms encompassing ascomycetes, basidiomycetes, chytridiomycetes and zygomycetes. The natural systematics of the latter group, Zygomycota, are very poorly understood due to the lack of distinguishing morphological characters. We have determined sequences for the nuclear-encoded genes actin (act) from 82 zygomycetes representing all 54 currently recognized genera from the two zygomycetous orders Mucorales and Mortierellales. We also determined sequences for translation elongation factor EF-1alpha (tef) from 16 zygomycetes (total of 96,837 bp). Phylogenetic analysis in the context of available sequence data (total 2,062 nucleotide positions per species) revealed that current classification schemes for the mucoralean fungi are highly unnatural at the family and, to a large extent, at the genus level. The data clearly indicate a deep, ancient and distinct dichotomy of the orders Mucorales and Mortierellales, which are recognized only in some zygomycete systems. Yet at the same time the data show that two genera - Umbelopsis and Micromucor - previously placed within the Mortierellales on the basis of their weakly developed columella (a morphological structure of the sporangiophore well-developed within all Mucorales) are in fact members of the Mucorales. Phylogenetic analyses of the encoded amino acid sequences in the context of homologues from eukaryotes and archaebacterial outgroups indicate that the Eumycota studied here are a natural group but provide little or no support for the monophyly of either zygomycetes, ascomycetes or basidiomycetes. The data clearly indicate that a complete revision of zygomycete natural systematics is necessary.

Repetitive DNA elements in fungi (Mycota): impact on genomic architecture and evolution

Abstract. Repetitive DNA elements, microsatellites or simple repeats, minisatellites, mobile elements that transpose at the level of DNA, retrotransposons and various derivatives thereof are ubiquitous constituents of all fungal genomes. Many of these elements, especially the different types of transposon, have been cloned and characterised at the sequence level. Their biological role, however, has not yet been sufficiently elucidated. We are far from understanding the selection mechanisms that tend to conserve repeated DNA at defined loci. There is also little insight into the mechanisms that provide the balance between spreading repetitive elements within genomes and control of their copy number. Depending on the fungal group, this balance can be stabilised at different levels. Asco- and basidiomycetes rarely contain more than 5% repetitive DNA, whereas the phylogenetically older division Zygomycota is characterised by typically more than 30%. The effects of repetitive DNAs on the expression of adjacent genes are only rarely understood and their role for genomic plasticity on an evolutionary time scale is still especially enigmatic. This survey summarises the main characteristics of well studied experimental systems and intends to define important open questions for stimulating future research.

Transfer of genetic information from the mycoparasite Parasitella parasitica to its host Absidia glauca

The infection of the model organism Absidia glauca by P. parasitica is accompanied by the fusion of both mycelia. By two lines of evidence we were able to show that this process is associated with the transfer of genes. First, auxotrophically labelled A. glauca mutants are efficiently complemented as a consequence of transfer of the parasites genetic material. Second, for a plasmidcoded dominant marker (neomycin resistance), which is expressed in either organism, we proved the presence of plasmid DNA in recombinant recipients by molecular analysis at the DNA level. We propose the term para-recombinants for describing recombinant inter-generic chimærae, which are generated as a consequence of mycoparasitism.

Evolution of host resistance in a toxin-producing bacterial-fungal alliance

The rice seedling blight fungus Rhizopus microsporus harbors endosymbiotic Burkholderia sp. for the production of the virulence factor, the antimitotic agent rhizoxin. Since the toxin highly efficiently blocks mitosis in most eukaryotes, it remained elusive how self-resistance emerged in the fungal host. In this study, rhizoxin sensitivity was systematically correlated with the nature of β-tubulin sequences in the kingdom Fungi. A total of 49 new β-tubulin sequences were generated for representative species of Ascomycota, Basidiomycota and Zygomycota. Rhizoxin sensitivity assays revealed two further amino acids at position 100 (Ser-100 and Ala-100), in addition to the known Ile-100 and Val-100, which convey rhizoxin resistance. All sensitive strains feature Asn-100. This hot spot was verified by modeling studies, which support the finding that rhizoxin preferentially interacts with the tubulin molecule in a cavity near position 100. Ancestral character state reconstructions conducted in a Bayesian framework suggest that rhizoxin sensitivity represents the ancestral character state in fungi, and that evolution of rhizoxin resistance took place in the ancestor of extant resistant Zygomycota. These findings support a model according to which endosymbiosis became possible through a parasitism—mutualism shift in insensitive fungi.

Identification and Isolation of Two Ascomycete Fungi from Spores of the Arbuscular Mycorrhizal Fungus Scutellospora castanea

ABSTRACT Two filamentous fungi with different phenotypes were isolated from crushed healthy spores or perforated dead spores of the arbuscular mycorrhizal fungus (AMF) Scutellospora castanea. Based on comparative sequence analysis of 5.8S ribosomal DNA and internal transcribed spacer fragments, one isolate, obtained from perforated dead spores only, was assigned to the genus Nectria, and the second, obtained from both healthy and dead spores, was assigned to Leptosphaeria, a genus that also contains pathogens of plants in the Brassicaceae. PCR and randomly amplified polymorphic DNA-PCR analyses, however, did not indicate similarities between pathogens and the isolate. The presence of the two isolates in both healthy spores and perforated dead spores of S. castanea was finally confirmed by transmission electron microscopy by using distinctive characteristics of the isolates and S. castanea. The role of this fungus in S. castanea spores remains unclear, but the results serve as a strong warning that sequences obtained from apparently healthy AMF spores cannot be presumed to be of glomalean origin and that this could present problems for studies on AMF genes.

Carotene derivatives in sexual communication of zygomycete fungi

Recognition between mating partners, early sexual morphogenesis and development are regulated by a family of beta-carotene derived signal compounds, the trisporoids, in zygomycete fungi. Mating type-specific precursors are released from the hyphae and exert their physiological effects upon compatible mating partners. In a cooperative synthesis pathway, later intermediates and finally trisporic acid are formed. All trisporoids occur in a number of derivatives. Trisporic acid and some precursors directly influence the transcription of genes involved in sexual development. This has been demonstrated for TSP3, encoding the carotene oxygenase involved in sexually induced cleavage of beta-carotene. Species specificity of mating despite a common and commonly recognized signaling system is maintained by several factors. Specific distribution and recognition patterns of the trisporoid derivatives and the proposed divergence in trisporoid synthesis pathways in diverse species play a role. The derivatives elicit vastly differing, partially mating type-specific responses during early sexual development. Another specificity factor is the realization of different regulation levels for the trisporoid synthesis enzymes in different species. Enzymes in the trisporoid synthesis pathway show remarkable variations in mating type-specific activity and the exact activation time during sexual development. This allows for the observed complex network of possible interactions, but at the same time forbids successful mating between dissimilar partners because the necessary transcripts or gene products are not available at the appropriate developmental stage.

Reliable amplification of actin genes facilitates deep-level phylogeny.

The gene for actin as a highly conserved and functionally essential genetic element is developing into a major tool for phylogenetic analysis within a broad organismic range. We therefore propose a set of universally applicable primers that allow reliable amplification of actin genes. For primer construction the amino acid sequences of 57 actin genes comprising fungi, animals, plants and protists were analysed, aligned and used for the definition of six well-conserved regions which are suitable as priming sites in PCR amplification experiments. Ten primers were designed for specific in vitro amplification of actin gene fragments from a wide range of microorganisms. The corresponding gene fragments provide a strong basis to isolate nearly complete actin genes for further molecular characterization and for establishing phylogenies based on actin gene trees.

Electrophoretic karyotype of the zygomycete Absidia glauca: evidence for differences between mating types

SummaryExperimental conditions for the separation of chromosomes from the model zygomycete Absidia glauca by rotating field electrophoresis were established. The sexually compatible strains of the mating type pair A. glauca CBS 100.48 (+) and CBS 101.48 (-) showed considerable differences in their electrophoretic karyotype. By Southern hybridization with homologous probes we have mapped the chromosomal locations for rDNA, the repetitive element rAg1, and the genes for actin and the elongation factor EF1. For the mapping of ubiquitin information we used a heterologous probe from U. maydis. The combination of electrophoretic karyotyping and Southern mapping proved to be a useful tool for characterizing mutant genotypes, which were induced by integrative transformation.

4-Dihydromethyltrisporate dehydrogenase from Mucor mucedo, an enzyme of the sexual hormone pathway: purification, and cloning of the corresponding gene

We have purified the NADP-dependent 4-dihydromethyltrisporate dehydrogenase from the zygomycete Mucor mucedo. The enzyme is involved in the biosynthesis of trisporic acid, the sexual hormone of zygomycetes, which induces the first steps of zygophore development. Protein was obtained from the (-) mating type of M. mucedo after induction with trisporic acid, and purified by gel filtration and affinity chromatography steps. On SDS-PAGE a band with an apparent molecular mass of 33 kDa was ascribed to the enzyme. After transferring onto PVDF membranes the protein was digested with endoprotease Lys-C, and several peptides were sequenced. Oligonucleotides derived from protein sequence data were used for PCR amplification of genomic M. mucedo DNA. The PCR fragment was used as probe for isolation of the corresponding cDNA and complete genomic DNA clones. Comparison of protein and DNA sequence data showed that the cloned fragment corresponded to the purified protein. Search for similarity with protein sequences of the Swiss-Prot database revealed a relationship to enzymes belonging to the aldo/keto reductase superfamily. Southern-blot analysis of genomic DNA with the labelled cloned fragment detected a single-copy gene in both mating types of M. mucedo. PCR with genomic DNA from other zygomycetes gave rise to several fragments. Hybridization analysis with the cloned M. mucedo fragment showed that a fragment of similar length cross-hybridized in Blakeslea trispora (Choanephoraceae) as well as in Parasitella parasitica and Absidia glauca (Mucoraceae). The promoter region of the gene contains DNA elements with similarity to a cAMP-regulated gene of Dictyostelium discoideum.