Anke Burmester

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.

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.

The use of ITS DNA sequence analysis and MALDI-TOF mass spectrometry in diagnosing an infection with Fusarium proliferatum

Abstract:  Although mycoses are among the most common diseases worldwide, infections with Fusarium spp. occur only rarely. Mostly patients suffering from underlying immune deficiency are infected with this mould, resulting in a considerably decreasing prognosis. In immunocompromised patients, cutaneous manifestations are more often associated with Fusarium sp. than with Candida sp. or Aspergillus sp. We describe one patient with acute lymphoblastic leukaemia, who was first treated with chemotherapy after GMALL protocol 07/03. After relapse, the patient was successfully transplanted in second remission with a human leukocyte antigen (HLA)‐matched unrelated peripheral blood stem cell graft. Ten months later, the patient died from respiratory insufficiency and recurrence of leukaemia. Previously, Aspergillus antigen was detected in blood. In the latter course, disseminated papules appeared. One of these was examined histologically and mycologically. Conventional cultural diagnostics led to the diagnosis of a fusariosis, further supported by internal transcribed spacer (ITS) sequencing and matrix assisted laser desorption/ionisation–time‐of‐flight mass spectrometry (MALDI‐TOF) mass spectrometry, both determining the isolated strain as Fusarium proliferatum, which is a very infrequent pathogen within this genus. Our investigations underline the potential of MALDI‐TOF MS based identification of Fusarium species as an innovative, time and cost efficient alternative to ITS sequencing.

Integrative transformation of a zygomycete, Absidia glauca, with vectors containing repetitive DNA

SummaryExperimental evidence for integration of transformed DNA into the genome of Absidia glauca, a member of the fungal class of zygomycetes is presented. According to the limited knowledge on the molecular biology of these fungi, autonomous replication of transformed plasmids seems to be the preferential mode of DNA propagation. By inserting fragments of highly repetitive DNA elements into an autonomously replicating vector conferring neomycin resistance, we were able to obtain integrative transformation events. With such plasmids we observed stable mitotic propagation of a selective marker gene (NPT II under the control of a homologous actin promoter). Analysis of DNA from transformants in Southern type experiments, as well as restriction analysis of retransformants into Escherichia coli, provide evidence that integration of foreign DNA into the genome of Absidia glauca is possible. These transformation events are often associated with the appearance of mutant phenotypes.

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.

4-Dihydrotrisporin-Dehydrogenase, an Enzyme of the Sex Hormone Pathway of Mucor mucedo: Purification, Cloning of the Corresponding Gene, and Developmental Expression

ABSTRACT The NADP-dependent 4-dihydrotrisporin-dehydrogenase is a (−) mating-type-specific enzyme in the pathway from β-carotene to trisporic acid. This substance and its isomers and derivatives represent the general system of sexual communication in zygomycetes. The (−) mating type of Mucor mucedo was stimulated by trisporic acid and the enzyme was purified by ion exchange and affinity chromatography. Several peptides of the 26-kDa protein, digested with trypsin, were sequenced by mass spectrometry. Oligonucleotides based on protein sequence data were used for PCR amplification of genomic DNA. The primary PCR fragment was sequenced and the complete gene, TSP2, was isolated. A labeled TSP2 hybridization probe detects a single-copy gene in the genome of M. mucedo. Northern blot analysis with RNAs from different growth stages reveals that the expression of the gene depends on the developmental stage of the mycelium in both mating types of M. mucedo. At the enzyme level, activity is found exclusively in the (−) mating type. However, renaturation of proteins in sodium dodecyl sulfate-containing gels revealed the TSP2 gene product in both mating types. Analyzing the protein sequence places the enzyme in the short chain dehydrogenase superfamily. Thus, it has an evolutionary origin distinct from that of the previously isolated 4-dihydromethyltrisporate dehydrogenase, which belongs to the aldo/keto reductase superfamily. Apart from the TSP2 genes in the three sequenced zygomycetous genomes (Phycomyces blakesleeanus, Rhizopus oryzae, and Mucor circinelloides), the closest relative is the Myxococcus xanthus CsgA gene product, which is also a short chain dehydrogenase, involved in C signaling and fruiting body formation.

Green fluorescent protein as a reporter for gene expression in the mucoralean fungus Absidia glauca

Abstract. Mucoralean fungi (Zygomycota) are used for many industrial processes and also as important model organisms for investigating basic biological problems. Their genetic analysis is severely hampered by low transformation frequencies, by their strong tendency towards autonomous replication of plasmids instead of stable integration, and by the lack of reliable genetic reporter systems. We constructed plasmids for transforming the model zygomycete Absidia glauca that carry the versatile reporter gene coding for green fluorescent protein (GFP). gfp expression is controlled either by the homologous actin promoter or the promoter for the elongation factor of translation, EF1α. These plasmids also confer neomycin resistance and carry one of two genetic elements (rag1, seg1) that improve mitotic stability of the plasmid. The gfp constructs were replicated extrachromosomally and could be recovered from retransformed Escherichia coli cells. gfp expression was monitored by epifluorescence microscopy. The gfp reporter gene plasmids presented here for the model zygomycete A. glauca constitute the first reliable system that allows the monitoring of gene expression in this important group of fungi.

The SEG1 element: a new DNA region promoting stable mitotic segregation of plasmids in the zygomycete Absidia glauca

SummaryA series of new vectors for the model zygomycete Absidia glauca was constructed on the basis of the structural neomycin resistance (Neor) gene controlled by the promoter of the gene for elongation factor 1 (TEF). In order to select for transformed colonies with a stable Neor phenotype, spores from primary transformants were pooled and grown for two sporulation cycles under non-selective conditions. Southern blot analysis of DNA from single spore isolates originating from independent transformant pools allowed the identification of two autonomously replicating plasmids. Retransformation of Escherichia coli and restriction analysis of the two plasmids provided evidence for spontaneous in vivo insertion of a new DNA element (SEG1) from the A. glauca genome. The inserted regions in both plasmids are essentially identical and do not represent repetitive DNA. Compared with other autonomously replicating vectors, these SEG1-containing plasmids are mitotically extremely stable and are passed on to the vegetative spore progeny of a retransformed A. glauca strain. We assume that SEG1 contains structural elements involved in partitioning and stable segregation of plasmids. For the construction of stable transformants of A. glauca, the SEG1 element may be regarded as a major breakthrough, because stabilization of transformed genetic traits by integration is difficult to achieve in all mucoraceous fungi and all known replicating plasmids are mitotically unstable.

The mating-related loci sexM and sexP of the zygomycetous fungus Mucor mucedo and their transcriptional regulation by trisporoid pheromones.

The putative mating type locus of mucoralean fungi consists of a single high mobility group (HMG)-domain transcription factor gene, sexM or sexP, flanked by genes for an RNA helicase and a triosephosphate transporter. We used degenerate primers derived from the amino acid sequence of the RNA helicase to sequence a fragment of this gene from Mucor mucedo. This fragment was extended by inverse PCR to obtain the complete sequences of the sex loci from both mating types of M. mucedo. The sex loci in M. mucedo reflect the general picture obtained previously for Phycomyces blakesleeanus, presenting a single HMG-domain transcription factor gene, sexM and sexP in the minus and plus mating types, respectively. These are located next to a gene for RNA helicase. Transcriptional analysis by quantitative real-time PCR showed that only transcription of sexM is considerably stimulated by adding trisporoid pheromones, thus mimicking sexual stimulation, whereas sexP is only slightly affected. These differences in regulation between sexM and sexP are supported by the observation that the promoter sequences controlling these genes show no similarities. The protein structures themselves are considerably different. The SexM, but not the SexP protein harbours a nuclear localization sequence. The SexM protein is indeed transported to nuclei. This was shown by means of a GFP fusion construct that was used to study the localization of SexM in the yeast Saccharomyces cerevisiae. The fusion protein is highly enriched in nuclei.

Transformation of the mycoparasite Parasitella simplex to neomycin resistance

SummaryThe facultatively parasitic zygomycete Parasitella simplex was transformed to neomycin resistance by a vector, which had been developed primarily for transformation of its host Absidia glauca. This plasmid, pAmNEF21, contained the bacterial resistance gene for neomycin (NPTII) under the control of the promoter region from the gene for elongation factor 1 (tef) isolated from A. glauca. Both flanking regions of the marker gene contain parts of the structural tef gene. DNA isolated from two Parasitella transformants was re-transformed in E. coli and the resulting plasmids, pAt21 and pAt35, were analyzed. The restriction map and Southern blot analysis show that both plasmids are rearranged. They had lost the structural tef information and were found to contain new DNA fragments, which were identical in both cases. Southern blot analysis of the transformants indicates that the rearranged plasmids are present in the fungal transformants and that the changes are not the result of re-transformation in E. coli. Plasmids were only recovered after growth under selective conditions. Southern blot analysis and re-transformation with undigested transformant DNA shows that the plasmids are replicated autonomously.