Yeast and Basidiomycete Research

Fungi are closely related to animals, making them excellent model organisms for basic cell biological and developmental studies that are directly relevant to human biology. They have therefore become one of the most intensively studied eukaryotic groups in the rapidly expanding field of genomics, and the number of complete genome sequences available for fungal species is rapidly increasing. This unprecedented quantity of information will make an unparalleled contribution to our understanding of fungal phylogeny and evolution, as well as to our understanding of how fungal cells, in our case of some selected species of human pathogens, function. In this research programme we explore fungal genomic data from a perspective of understanding functional biodiversity related to disease potential, susceptibility, population structure, and reproduction.

Human pathogenic yeasts
These research projects aim to understand the biodiversity as well as the virulence properties of selected clinically important yeast species, namely Cryptococcus neoformans, Cryptococcus gattii, and Malassezia, Trichosporon and Candida species.

Cryptococcus neoformans:Normally, C. neoformans and C. gattii reproduce asexually, but in some cases mating can occur. The existence of hybrids between C. n. var. grubii and C. n. var. neoformans demonstrates that mating occurs in nature. Recently, we have also documented a number of unique hybrids between C. neoformans and C. gattii. The recognition of the isolates involved as hybrids with this unexpected genetic background was supported by numerous analyses and was a breakthrough in the thinking of many people dealing with Cryptococcus. The full extent is not yet known, but very recent data suggest that these gattii ´ neoformans hybrids occur much more common than previously anticipated.  Our observations also suggest that the microspecies within the complex are genetically isolated by postzygotic isolation. We have also observed mitochondrial recombination which indicates that possible hybridization events may occur even more frequent than currently known based on the presence of hybrids. This is important because it allows the possibility of introgression of “aspecific” genetic material, which may have an effect on e.g. virulence [Note: this is one of our hypotheses to explain the sudden outbreak of a subtype of C. gattii at Vancouver island that recently spread into the US Northwest]. Moreover, studies on hybridization and mitochondrial recombination may also contribute to a better understanding of species concepts in these organisms, which are topics of an ongoing debate [Note at the 2005 International Congress of Cryptococcus and cryptococcosis, Boston, we participated in a lively round table debate on the topic featured in the ISHAM newsletter]. Genomic diversification within the complex is also studied using a CBS-developed Agilent microarray, based on the genome of C. neoformans isolate JEC 21 (serotype D). Results confirmed that C. n. var. grubii and C. n. var. neoformans are relatively closely related to each other, but distinct species, and the four genotypes of C. gattii could also be distinguished.Interestingly the hybridisation patterns of the AD hybrid differed widely from that of the BD hybrid. This implies that considerable differences exist among the serotype D backgrounds found in both hybrids, thus supporting the notion that the hybrids may be highly aneuploid. We coined the term ‘Taxogenomics’ for this type of investigations.
Isolates of a genetic subgroup of C. gattii referred to as AFLP genotype 6 (= PCR-fingerprint group VGII) have been recognized as responsible for a major outbreak of cryptococcosis in Vancouver Island, Canada [see our paper in PNAS, commented on Faculty of 1000], which recently extended to the Canadian and US mainland territories, thus enlarging its area of distribution and gaining continental access. This outbreak mainly affects otherwise healthy people, but animals, including marine mammals, can be affected. Ecological sampling has indicated that the same genotype occurs on many native tree species, which collectively may represent the main environmental reservoir involved in the outbreak. Recently, a Danish tourist who travelled to the part of Canada affected by the outbreak developed cryptococcal pneumonia. Detailed genotypic analysis demonstrated that the isolate obtained from this patient was identical to those from Vancouver Island. Hence, this investigation documented the first known tourist-mediated intercontinental transmission of this disease and pathogen [see our paper in EID; topic of podcast by CDC Atlanta, USA http://www2a.cdc.gov/podcasts/player.asp?f=3927].
To trace the origin of this ongoing and expanding outbreak, we used comparative genomic approaches, including comparative AFLP to search for novel, highly variable, molecular markers useful to develop an MLST scheme. Six DNA regions were selected to be sequenced for ca. 120 genotype AFLP 6 isolates, including many from the Vancouver outbreak. A microsatellite typing system was developed for this genotype in collaboration with C. Klaassen and J. Meis (CWZ, Nijmegen). In addition, our set of strains was studied by an alternative MLST scheme developed at Duke (J. Heitman). This set will be used to analyse the geographical site of origin of the outbreak by coalescence analysis, recombination and further population studies (collaboration P. Ceresini, Zürich, Switzerland). Knowing the origin of the outbreak is not only scientifically important, but is will also provide a possible clue as to understand whether a genetic event caused the hypervirulence (e.g. an introgression, a transposition, or a mating event). Furthermore, it may give insight in those isolates that are genetically most closely related to the ancestor of the outbreak population. Finally, it may unravel whether external factors, such as climate change, have contributed to the outbreak, or if a combination of these different mechanisms caused the outbreak. We presently have identified a low virulent strain that is genetically closest to the outbreak isolates. Comparative genomics approaches such as Comparative Genome Hybridization (CGH) and resequencing of this strain of low virulence will be performed to identify genomics differences (e.g. SNPs, introgressions) between the outbreak isolates and it closest neighbour of low virulence [PhD project F. Hagen, funding OvV].
The pathogenicity of C. gattii genotype AFLP 6 was further studied using the nematode worm Caenorhabditis elegans, interaction with macrophages and a mouse model system (in collaboration with Dr. R. May, Birmingham, UK; I. Polacheck, Jerusalem, Israel, respectively). The absence of an adaptive immune system in C. elegans allows this model to be used to dissect out “basic” cryptococcal virulence factors. Interestingly, considerable differences were observed among isolates from the Vancouver Island outbreak that could not be distinguished otherwise. PhD student H. Ma (University of Birmingham) performed interaction studies between selected isolates and macrophages, using our phylogenomics data for strain selection. F. Hagen (PhD student OvV) is closely interacting with H. Ma and R. May. During a research visit to CBS, H. Ma assessed virulence attributes, such as protease activity, phospholipase activity, melanisation rates, and capsule sizes (all at both 25 and 37ºC).  Virulence of a selection of these isolates was further assessed in a mouse model by I. Polacheck (Jerusalem, Israel). In what may turn into one of the highlights of our research during the past years an astonishing correlation was observed between behaviour in macrophages, mouse and origin of the isolates. This is the very first time that in vitro tests fully predicted the virulence potential of cryptococcal cells in a cell based model. This is not only interesting from the point of view to develop alternatives for animal models, but it also clearly showed that the isolates causing the Vancouver outbreak possess some unique immunomodulating properties. These results led us to propose that an introgression event may have caused the hypervirulence of these isolates. We also hypothesize that the origin of this genetic element may belong to a different haplotype of C. gattii than that causing the outbreak. Support for the possibility for inter-haplotype matings is obtained by incongruent mitochondrial phylogenies [M. Bovers, PhD thesis 2007]. This research will be taken further as the main topic of Ferry Hagen, a PhD student [OvV funding], who is just in his second year. Further collaboration on this topic will be with Jim Kronstad (molecular mycology, University of British Colombia, Vancouver, Canada), U. Himmelreich (NMR, Leuven, Belgium) and G. Janbon (molecular biology, Paris, France).
A functional genomics analysis performed in collaboration with F. Coenjaerts (UMC Utrecht) concerned a serotype D strain of C. neoformans  var. neoformans that had a mutant form of the Skn7 gene (listed by the Saccharomyces genome database as a nuclear response regulator and transcription factor required for optimal induction of heat-shock genes in response to oxidative stress). Preliminary data suggest that only few genes were upregulated after exposure of the mutant to oxidative stress, including a putative transcription factor. Further research will include a more detailed bioinformatics analysis, as well as additional genetics and microarray experiments (K. Khayhan, PhD student from Thailand, 2008–2012).

Malassezia: The genetic diversity within the lipid-dependent species Malassezia furfur was investigatedin collaboration with Roma Batra (presently Milwaukee, USA) and F. Cabañes (Barcelona, Spain). In AFLP analysis, we found several subclusters within the species. Additional techniques have been used to analyse this complex further, e.g., pulsed field gel electrophoresis as well as sequencing of the LSU and ITS regions of the rDNA and part of the chitin synthase gene. As part of the results, one of the M. furfur subclusters was shown to have a mixture of markers suggestive of a hybrid origin, even though no sexual mechanisms are known so far for any species clustering within the Malassezia lineage. Interestingly, the genome sequence of M. globosa revealed a mating type locus (see below).
In collaboration with J. Cabañes (Barcelona, Spain) two new species were described. The landmark in Malassezia research was the publication of the genome of M. globosa and M. restricta [see our paper in PNAS, commented on Faculty of 1000]. Interesting life-style adaptations were observed. Part of the genome was less close to that of the phylogenetically related Ustilago maydis, a plant pathogen, but was found to be closer related (at least in gene composition) to the distantly related Candida albicans, another inhabitant of skin. The release of this paper received substantial press coverage all over the globe [including Dutch radio shows and newspapers]. It is estimated that this information was covered by an audience of > 77 million! This paper is also important as it allows the further development of molecular tools in this field. A challenge already undertaken by research groups in Duke (Durham, USA) and Vancouver (Canada), respectively. The detection of a mating type locus suggested that sex may be possible (already suggested previously on molecular data and the presence of hybrid genotypes, see above), and we are currently looking for diversity in mating types in M. globosa, in collaboration with P&G, and J. Kronstad. A further spin off of this project will be the study of the mitochondrial genome of M. globosa. Within the ISHAM working group on Malassezia we will participate in the set up of a MLST typing scheme. Besides, the first monograph of this genus will be edited within the frame of the ISHAM working group.

Phylogenomics, Tree of Life (TOL) and Yeast Biodiversity
Phylogenetic studies are generally based on comparing DNA or protein sequences that, though found in a wide range of organisms, all arose from the same ancestral genes that occurred millions of years ago in a hypothetical common ancestor species. Such genes that occur among many organisms, but that all have a common ancestral root, are referred to as “orthologous” (= “directly related”). Orthologous groups of proteins (KOGs, or “euKaryotic clusters of Orthologous Groups of proteins”) from complete fungal genomes species were analysed in order to resolve their phylogenetic relationships. Conflicting data were obtained on the phylogenetic position of Schizosaccharomyces pombe when using different set of cophenetically similar orthologues. In collaboration with B. Dutilh (PhD student, University of Nijmegen) a bioinformatics study compared various phylogenetic tools [see our paper in Bioinformatics]. Cophenetics as an analysis tool was developed to mine phylogenetically informative single copy orthologues [see our highly accessed paper in BMC Evol. Biol.]. M. van Passel, a postdoctoral guest worker (6 months), performed a bioinformatics study on the genome signature, a genome compositional parameter in prokaryotes and fungi (see our highly accessed paper in BMC Evol. Biol.).
A main asset in the area of Yeast Biodiversity is the co-editing of the 5th edition of ‘The Yeast, a taxonomic study’. This book will set the stage for another ten years and will cover all c. 1500 know yeast species. Descriptions will include molecular phylogeny, morphology, physiology, biochemistry, gene accession numbers for the type strains, and discussions on Systematics, Ecology, Biotechnology, Food and Agriculture, and Clinical importance. This book will appear in 2008.
During the process of editing ‘The Yeasts’ it became very clear that the present phylogenetic classification of the basidiomycetous yeasts urgently needs to be revised. In order to realize this, we are in the process of establishing a global network (a so-called wiki) in order to analyze ca. 20 genes among all ca. 500 accepted species and, revise the phylogenetic classification accordingly. Interest in this project is shown by groups in China, Japan, Malaysia, Brazil, Portugal, Germany, USA and The Netherlands. Among these are also groups with genomics facilities. When realized, this new taxonomic system will result in major conceptual changes, and, it can be expected will result in highly cited papers (as was the case with the first comparative studies using D1/D2 LSU ribosomal RNA genes of all know species at that time (Fell et al. 2000). Postdocs E.K. Kuramae [KNAW renewal fund 2003–2007] and Carlos Echavarri [CURIE GRANT CT-2006-036584, 2006–2009] have contributed to the design of this TOL analysis, by selecting candidate single copy orthologues using comparative genomics approaches.
Yeast diversity was further studied in collaboration with various researchers on an ad hoc base. Clinically important yeasts from neonates and HIV-infected persons were studied from Jakarta (Indonesia) in collaboration with Retno Wahyuningsih [SPIN Mobility grant]. Interestingly, the recently described species, Candida nivariensis, a close relative of Candida glabrata, was found [paper JCM]. Type strain of current synonyms of C. albicans were studied by MLST in collaboration with F. Odds (Aberdeen, UK). Interestingly, C. stellatoidea, long time recognized as a separate and clinically important species, but presently interpreted as a synonym under C. albicans, was found to be divergent from the remainder of C. albicans. A recently obtained grant for a three-year postdoc position will merge phylogenomics knowledge and the development of innovative detection tools for Candida yeasts [grant EuroTransBio, EU, SenterNovem, 1 postdoc vacancy, 3 yr, 2008–2011]. This project will be run in collaboration with academia, hospitals and small enterprises in Flanders (Belgium) and The Netherlands. Innovative diagnostics tools were further developed using Luminex XMap technology for Cryptococcus and Malassezia yeasts in collaboration with M. Diaz and J. Fell (Key Biscayne, USA) [M. Bovers, chapter in PhD Thesis, two publications]. A new clinically important species of Trichosporon was identified using molecular tools in collaboration with Dr Saad J. Taj-Aldeen (Doha, Qatar). With V. Passoth (Uppsala, Sweden) a new species of Cryptococcus will be described. Biological control agents on mites and powdery mildews were described as Meira and Acaromyces gen. nov. with U. Gerson and A. Sztejnberg (Rehovoth, Israel). Three new species were described and recently, two more by other researchers. A paper on dual biocontrol capabilities published in Crop Science was highly accessed. With Z. Kahn (Kuwait) a new species of Cryptococcus (C. rhandawi) will be described. Together with K. Seifert (Ottawa, Canada) a new thermophilic species of Rhodosporidium (R. concentricum) is in the process of being described. B. Turchetti, a postdoc guest (one year) from Perugia (Italy) is studying psychrophilic yeasts from (disappearing) Alpine glaciers and found a number of new species of Mrakia and Leucosporidium [funding FEMS, Synthesys, University of Perugia].

A functional study of the septal pore caps (SPC) in basidiomycetes and phylogeny of the Rhizoctonia solani complex
The septal pore cap (SPC) or parenthesome is a membranous structure associated with endoplasmic reticulum covering both sides of the dolipore septum and is restricted to Basidiomycetes. Although this structure was already described in 1958 and well studied at an ultrastructural level, no functional studies have been done so far. Therefore, the composition and the precise function of the SPC at the dolipore is still unclear. The aim of this study was to isolate the SPC and to partly characterise the proteins present, so that a start can be made in understanding the function of this organelle. We used the plant pathogen Rhizoctonia solani as a model organism, because it has relatively well-studied, large SPCs. We have been the first ever, to isolate and biochemically study the SPC of basidiomycetes. Consequently, we have opened a research field, that probably will be entered by other research groups soon.
Laser microdissection with a P.A.L.M. microscope (P.A.L.M. Microlaser Technologies GmbH, Bernried, Germany) was used successfully to isolate the SPC-dolipore region. We could identify the septal regions using lectin-gold labelling of antibodies specifically targeting the septa; this analysis was done with a scanning electron microscope. In addition, we successfully enriched SPCs from R. solani cell fractions by isopycnic (buoyant density or equilibrium) centrifugation. In electron microscopic studies, we observed that plug material at the orifice of the septal pore channel remained attached via filamentous material to the SPCs. This tight connection between SPCs and pore-occluding material implicates a key role of SPCs in the process of plugging septal pores in Basidiomycetes. Such plugging is often connected to maintaining hyphal integrity in situations where some cells are damaged or otherwise strongly stressed.
Protein electrophoresis showed that a 18 kDa glycoprotein (SPC18) was present in the SPC-enriched fraction. This protein was N-terminally sequenced and afterwards the complete gene sequence was obtained. No homologue could be identified using the available sequences in genome databases. Western blot analysis, however, suggests that the protein may be limited to the R. solani lineage. Attempts are ongoing to study the nucleotide diversity of the gene within the Rhizoctonia lineage and to compare it with standard D1/D2 and ITS variable region sequences of the ribosomal DNA. Polyclonal antibodies raised against the 18 kDa glycoprotein were labelled using the immunogold technique and then used to perform immunodetection studies. The labelled antibodies were found to be localized in the SPC, the SPC membrane and plug material. Thus suggesting that SPC18 may be involved in plugging the pore upon stress and that the PSC may have a repository function for SPC to make it possible that the process of pore plugging can start directly after the cells have encountered a serious stress (prefab model).
The observed heterogeneity of SPC morphology in some of the major lineages of the Basidiomycetes, notable the Hymenochaete and Cantharellus-Rhizoctonia clades, was confirmed by analysing the SPC of Rickenella spp. and Cantharellus sp. It appeared that the observed heterogeneity in SPC morphology is characteristic of basal lineages among the basidiomycetes, This may suggest that genes involved in both types of SPC morphology may be present in these basal lineages, and that the basal organisms may thus manifest a genetic condition that existed in the ancestors of other Basidiomycetes prior to the occurrence of lineage sorting.
When all manuscripts from this project will be published, we will investigate possibilities for new funding to continue this intriguing research line.
PhD student A. Nakatani, studied > 300 global isolates of Rhizoctonia solani by sequence analysis of the ITS 1 and 2 regions. A good correlation was observed with the anastomosis groups. Per ITS clade a smaller selection of isolates was made and studied by three genes (data analysis still in progress).

Bioprospecting of fungi
The screenings have been set up and are running smoothly. One patent has been filed (decision pending). In the first half year of the project we established an efficient collaboration between CBS, Hubrecht Institute and the Department of Pharmacy at Utrecht University.