15 april 2005

Het programma van de mycologie-sessie ziet er als volgt uit:

Deel 1. Voorzitter: Jacques Meis

14.00 - 14.15 J. Dijksterhuis: Confocal microscopy of Spitzenkörper dynamics during growth and differentiation of rust fungi.

14.15 - 14.30 F.B.J.M. Thunnissen, G.S. de Hoog, J.F.G.M. Meis & H.E. Viëtor: The Systemic Mycosis ARray Test (SMART).

14.30 - 14.45 G. Haase & H. Stender: Labeled peptide nucleic acids (PNA) as species-specific probes for fluorescence in situ hybridization (FISH) enabling an easy-to-perform identification of fungi in blood cultures.

14.45 - 15.15 P.M. Ellerbroek, A.M. Hoepelman, F. Wolbers & F.E.J. Coenjaerts:
Cryptococcal glucuronoxylomannan (GXM) inhibits adhesion of polymorphonuclear leukocytes (PMN) to stimulated endothelium in vitro by affecting both PMN and endothelial cells in both static and dynamic adhesion models.

15.15 - 15.45 Break

Deel 2. Voorzitter: Sybren de Hoog

15.45 - 16.00 T. Boekhout, V. Robert, J. Stalpers, G. Gijswit, C.P. Kurtzman, J.W. Fell & I. Roberts: Yeasts of the world, an interactive CD-ROM.

16.00 - 16.15 H.J. Deelstra, R. Bohlmann, J. Dijksterhuis, R. Kahman & H.A.B. Wösten:
Repellents of the phytopathogenic fungus Ustilago maydis.

16.15 - 16.45 M.C. Fisher: Population genetics of the AIDS-associated fungus, Penicillium marneffei in South East Asia

Om 17.15 begint de plenaire lezing van John Taylor: Co-evolution of fungi with other (micro)organisms.

Abstracts

Confocal microscopy of Spitzenkörper dynamics during growth and differentiation of rust fungi
Jan Dijksterhuis*1,2
1Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; 2Fungal Cell Biology Group, Institute of Cell and Molecular Biology, University of Edinburgh, Daniel Rutherford Building, Edinburgh, UK
The membrane-selective fluorescent dye FM4-64 was used to stain the apical vesicle cluster within the specialized Spitzenkörper (Spk) of germ tubes of Uromyces vignae and Puccinia graminis f. sp. tritici grown on glass surfaces. Spk stained within 15 min following addition of the dye. Optical sectioning by confocal microscopy of stained hyphal tips showed that the Spk was asymmetrically positioned close to the cell-substratum interface during germ tube growth. The Spk showed variations in shape and positioning during short (5 sec) time intervals. The movement to a new location in the hyphal dome was followed by new growth in that region, consistent with the view that the Spk supplies secretory vesicles for germ tube growth. A pronounced Spk disappeared at the onset of appressorium differentiation during swelling of the germ tube. However, a stained structure, similar in appearance to a Spk, was again observed during the formation of the highly polarized penetration peg.

The Systemic Mycosis ARray Test (SMART)
F.B.J.M. Thunnissen1, G.S. de Hoog2, J.F.G.M. Meis1, H.E. Viëtor3
1Canisius-Wilhelmina Ziekenhuis, Weg door Jonker bos 100, 3562 SZ Nijmegen, The Netherlands; 2Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; 3Multigen Diagnostics, Abcoude, The Netherlands

Introduction: One of the main health problems in the hospitalized population of immunocompromised patients, now that bacterial infections are largely under control, may be systemic infections by fungi. This may cause an increased morbidity and mortality in patients who might have overcome their primary disorder. Recognition of a fungal infection, as well as identification of the agent requires specialized histopathological and time consuming culturing skills. It is our central hypothesis that (1) systemic fungal infections are much more common than currently established, and that (2) the diversity of the agents concerned differs significantly from the species-spectrum published in most handbooks. In order to gain insight into this problem, first-line fungal diagnostics should be simplified to such an extent that tests can routinely be applied. To this aim we will develop an 'easy-do' diagnostic micro-array using staining and microscopic techniques available in any clinical lab and which provides specific information within a single day. Material and Methods: The project aims to develop a DNA micro-array for the identification of all potential agents of systemic filamentous fungal infection in humans, either primary pathogens or opportunists, as recently listed in the “Atlas of Clinical Fungi” (2000); a total of about 150 species will be covered. The array will initially be based on selected ribosomal and intron sequences, which have been proven to deviate sufficiently among the relevant species. The microarray procedure for genotyping will be similar to the one used for human papilloma viruses in the Canisius Wilhelmina Hospital. The procedure after PCR can be applied in any microbiology or pathology laboratory as this follows the basics of routine immunohistochemistry for visualization. Results: The setup of the HPV system will be presented. Over 36 different HPV types are present in triplicate on a microscope object slide. If a type is present in the PCR amplification this is easily visible in the light microscope. Conclusion: An 'easy-do' diagnostic micro-array is developed using staining and microscopic techniques available in any clinical lab and for diagnosis and examination of pathogenicity of systemic fungi.

Labeled peptide nucleic acids (PNA) as species-specific probes for fluorescence in situ hybridization (FISH) enabling an easy-to-perform identification of fungi in blood cultures
Gerhard Haase1, Henrik Stender2
1Institute of Medical Microbiology, University Hospital RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany; 2AdvanDx, Inc., 222 Partridge Lane, Concord, MA, U.S.A.
In PNAs, the negatively charged phosphate linkages in DNA are replaced with peptomimetic neutral amide linkages. PNA-DNA/RNA complexes form more quickly and are tighter and more specific than analogous DNA-DNA/RNA complexes thereby allowing more robust hybridization applications. Especially relative insensitivity to ionic strength and pH and resistance to nucleases and proteases during hybridization provides a wider platform for specific DNA/RNA detection thereby simplifying diagnostic procedures, such as FISH. A novel FISH method is reported that uses PNA probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining. The test is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55?C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are then examined by fluorescence microscopy. The specificity of the method was confirmed with 15 reference strains and 75 clinical isolates representing clinical important yeast species including C. albicans (n = 35), C. dubliniensis (n = 30), C. glabrata (n = 10), C. krusei (n = 3), C. parapsilosis (n = 5), and C. tropicalis (n = 7). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 13 routine and 20 simulated yeast-positive blood culture bottles (BacAlert®) and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management. Application of the described method holds a great potential for identification of other fungi causing systemic infection, e.g. in case of biopsies containing fungal elements and is therefore well suited as a reliable, cost-effective and easy-to-perform procedure in the diagnostic laboratory.

Cryptococcal glucuronoxylomannan (GXM) inhibits adhesion of polymorphonuclear leukocytes (PMN) to stimulated endothelium in vitro by affecting both PMN and endothelial cells in both static and dynamic adhesion models.
P.M. Ellerbroek, A.M. Hoepelman, F. Wolbers, F.E.J. Coenjaerts
UMCU, AGI, UTRECHT, Netherlands
Introduction. Cryptococcal infections are often characterized by a paucity of PMN in infected tissues. Previous research has shown that the capsular polysaccharide GXM inhibits PMN migration by interfering with chemotactic pathways. In this study we investigated whether GXM can affect the migration of PMN through the endothelium by interfering with adhesion. For this purpose, we employed both static and dynamic adhesion models. Methods, Results. In the static model, pre-treatment of PMN with GXM inhibited PMN adhesion to tumor necrosis factor-(TNF )-stimulated endothelium up to 44%. Treatment of TNF-stimulated endothelium with GXM led to 27% decrease in PMN adhesion. GXM treatment of both PMN and endothelium did not have an additive inhibitory effect. We demonstrated that GXM-induced L-selectin shedding does not play an important role in the detected inhibition of adhesion. L-selectin was still present on PMN after GXM treatment, since it could be further inhibited by blocking antibodies. Furthermore, blocking of GXM-related L-selectin shedding did not abolish the GXM-related inhibition of adhesion. GXM most likely exerts its effect on PMN by interfering with E-selectin-mediated binding. The use of blocking monoclonal antibodies against E-selectin - shown to decrease adhesion in the absence of GXM - did not cause additive inhibition of PMN adhesion after GXM pre-treatment. By using blocking antibodies we demonstrated that the inhibiting effect found after GXM treatment of endothelium probably involves interference with both intercellular adhesion molecule-1 and E-selectin binding. These findings were confirmed in the ‘dynamic’ rolling model. Here, pre-treatment of PMN with GXM caused inhibition of PMN rolling both on endothelium and on E-selectin transfectants.

Yeasts of the world, an interactive CD-ROM
T. Boekhout1, V. Robert1, J. Stalpers1, G. Gijswit2, C.P. Kurtzman3, J.W. Fell4, I. Roberts5
1Centraalbureau voor Schimmelcultures, P.O. Box 85167, 3508 AD Utrecht, The Netherlands, 2Expertisecenter Taxonomic Identification, University of Amsterdam, The Netherlands, 3NRRL-USDA, Peoria, USA, 4RSMAS, University of Miami, Key Biscayne, USA, 5NCYC, Norwich, UK
This CD-ROM presents a complete taxonomic data set of all currently accepted yeast species, including morphological and physiological data, and ribosomal DNA sequences. The interactive software contains modules for the comparison and integrated use of physiological, sequence and morphological information, facilitating the identification of yeasts using complementary data sets. Many species are illustrated by microscopic and macroscopic images. This product will be useful in a wide range of yeast studies throughout the agro-industrial and medical sciences.

Repellents of the phytopathogenic fungus Ustilago maydis
H.J. Deelstra1, R. Bohlmann2, J. Dijksterhuis3, R. Kahmann4, H.A.B. Wösten1
1University of Utrecht, Microbiology, Utrecht, Netherlands, 2Ludwig Maximiliaans University, Genetics, Munich, Germany, 3Fungal Biodiversity Center, Utrecht, Netherlands, 4Max Plank Institute for Terrestric Microbiology, Organismic Interactions, Marburg, Germany
Repellents of U. maydis are polypeptides of 35-53 amino acids cleaved off the larger precursor Rep1 and located in the cell wall of the dikaryon. Crosses of knockout strains do no longer project dikaryotic filaments of the phytoparasitic stage into the air when grown in vitro. Repellents seem to be analogous to the hydrophobins of filamentous fungi in this respect. A hydrophobin gene (Hum2) has also been found to be expressed in the dikaryotic phase of U. maydis. The location and function of the hydrophobin and the functions of Rep1 are the subject of the described research. Dikaryons of Hum2 and Rep1 strains were compared to the wildtype with respect to aerial hyphae formation, cell wall constitution, hydrophobicity and pathogenicity. A fusion of Hum2 with a LA epitope (Low affinity Haemagglutin Epitope) was used to study the location of Hum2 in cell fractions by immunoblotting. Immunoblotting showed the presence of Hum2-LA in the cell wall fraction of celextracts, but in such low quantities that it could not be visualized on a proteingel using standard hydrophobinextraction procedures. Scanning Electron Microscopy (SEM) showed that ÄHum2 and wildtype dikaryons produce the same amount of aerial hyphae. Surprisingly, SEM revealed that ÄRep1 strains do produce aerial hyphae under dry conditions, but up to 80% of these hyphae cluster together in bundles of 2-6 hyphae. Wetting of the ÄRep1 aerial hyphae results in their collapse, whereas wetting of ÄHum2 and wildtype aerial hyphae results in a waterdroplet with a high contact angle on the colony surface. Pathogenicity of ÄHum2 dikaryons and ÄRep1 dikaryons is comparable to the wildtype; about 50% of the exposed plants develop disease. Cell walls of ÄRep1 dikaryons contain more alkaline-soluble sugars than wildtype and ÄHum2 dikaryons.These results indicate that repellents have an effect on cell wall architecture, analogous to the effect of the hydrophobin Sc3 on the cell walls of Schizophyllum commune. Repellents, although completely different in primary sequence, share more than one function with hydrophobins.

Population genetics of the AIDS - associated fungus, Penicillium marneffei in South East Asia
M.C. Fisher
Institute of Zoology, London, United Kingdom
Globalisation of the HIV virus has resulted in a number of diseases emerging as humans become susceptible to environmental cycles of infection. The fungus Penicillium marneffei has appeared in Southeast Asia as the third most common opportunistic infection in AIDS patients. In endemic areas, epidemiological studies suggest that wild rodents may act as zoonotic reservoirs of infection, and that environmental cycles enhance infection rates. However, these hypotheses remain untested. An MLST typing scheme has been established for P. marneffei to investigate these questions.Genetic diversity within 6 genes (both intronic and exonic sequence) has been used to establish phylogenetic relationships between spatially and ecologically separated populations of the fungus. These same isolates have then been genotyped using a panel of 25 di, tri and tetra nucleotide microsatellites. The relative utility of these two classes of genetic markers (i) for describing the population structure of P. marneffei and (ii) for use as epidemiological tools, will be shown.

Co-evolution of fungi with other (micro)organisms
John W. Taylor
University of California, Department of Plant and Microbial Biology, Berkeley, California, 94720-3102, USA
Co-evolution can be defined broadly or narrowly. The advent of phylogenetic theory and methods of assessing nucleic acid variation made it possible to narrow the definition by rigorously investigating claims of co-evolution. Some of the earliest such studies now are classics, including studies of gophers and their parasitic lice1. With microbes, similarly rigorous studies of co evolution have been conducted with aphids and the bacterial genus Buchneria2. Fungi, members of a very large kingdom of complex and widely distributed eukaryotic microbes, certainly co-evolve in the broad sense with other microbes as well as with macroscopic plants and animals. However, few of the many possible instances of co-evolution have been rigorously investigated. I will touch on the possibilities for fungal co evolution with virus, bacteria, algae, animals and plants, and spend more time on examples where careful tests for co-evolution could be, or have been, applied. For virus, the interaction of double stranded RNA virus with fungi in the plant pathogen, Cryphonectria parasitica, stands out3. With bacteria, the presence in arbuscular mycorrhizal fungi of intracellular bacteria in the genus Burkholderia is remarkable.4 With microscopic plants, i.e., algae, studies aimed at recognizing species in the two partners of species of the lichen Letharia show cases of co-evolution, as well as host jumps5. With plants, studies of the co-evolution of mycorrhizae fungi and their vascular plant partners show the same pattern of co-evolution and host jumping6. One of the best-understood systems of fungal co-evolution involves the fungi farmed by attine ants7, and recently these studies have grown to include weedy fungi and bacteria used to control the “weeds”. Finally, there are cases where fungi are likely to be co-evolving with humans. Here, commensal fungi, such as Candida or Malassezia8 species should be very interesting, as well as the fungi causing superficial skin infections9. Recent studies of systemic fungi causing deep mycoses would seem to rule out long-standing co-eveolution, however there appear to be events in the recent evolution of Histoplasma species10 and Coccidioides species11 that may be best explained by the actions of migrating humans.

Presentation 15 April, 10.15 - 11.00:
Selection of potential fungal agents of bioterrorism using evolutionary criteria
Sybren de Hoog
Centraalbureau voor Schimmelcultures, P.O. Box 85167, 3508 AD Utrecht, The Netherlands

The list of potential agents of bioterrorism issued by the US Department of Occupational Safety and Environmental Health (OSEH) presently contains a single fungus, Coccidioides immitis. This is the agent of Valley Fever, a disseminating disease which commonly infects humans but exceptionally takes a fatal course, then mainly in patients with impaired acquired immunity. C. immitis is not the only fungal species known to cause fatal disease; the question then arises whether other fungi should be put on the list. Agents should combine host-specific pathogenicity with a high degree of virulence. The probability that species display these criteria optimally in terms of bioterrorism is determined by the evolutionary history of the group at hand. The fungal kingdom is reviewed in search of clades with (1) shared virulence factors including species with (2) mammal host-dependence with dual life cycles and (3) production of zoodemes while (4) fitness is increased. In addition (5) the degree of adaptation is discussed. About 400 fungal species have been reported from humans; this is less than 0.5% of the fungi known to date. Suitable criteria are encountered in only a small fraction, the remaining species being occasional opportunists or superficial pathogens. Required properties are combined in only two species. Among these is not C. immitis.

Cursussen
De jaarlijkse CBS cursus ‘Medische Mycologie’ is van 7-25 April, 2003, maar is geheel volgeboekt met 50 deelnemers. Het wordt in het algemeen aangeraden tijdig in te schrijven voor deze cursus, want er is vaak een wachtlijst. De volgende (Engelstalige) aflevering is in het voorjaar van 2004. Voor informatie, bezoek de CBS website, of mail naar info@cbs.knaw.nl.

Congressen
Geachte mycologen,

Wij willen jullie gaarne wijzen op het congres "Trends in Medical Mycology" dat dit jaar van 28 september tot 1 oktober in Amsterdam wordt gehouden.
Het is niet alleen een uniek congres omdat het in nauwe samenwerking tussen twee mycologische verenigingen (ECMM en EORTC-IFIG) wordt georganiseerd, maar ook omdat het wetenschappelijk programma een gebalanceerde mix is van fundamentele en klinisch mycologische onderwerpen. Mocht het zo zijn dat iemand zich nog niet inschreven heeft kan ik u meedelen dat speciaal voor de leden van de NVMy de inschrijvingstermijn voor het laagste bedrag nog tot 15 april is verlengd. Voor kosten en verdere informatie zie de website www.ecmm-tifi2003.org; opgave is mogelijk via een inschrijfformulier dat is te downloaden vanaf deze site. Het is wel nodig dat op het inschrijfformulier wordt vermeld dat u lid bent van de NVMy. Overigens geldt voor analisten het lage bedrag van physicians in training.
Ik hoop dat jullie allemaal overwegen om, actief danwel passief, deel te nemen aan dit belangrijke in Nederland georganiseerde congres.

Samenstelling Bestuur: e-mail-adressen
Voorzitter: Dr. J.F.G.M. Meis j.meis@cwz.nl
Secretaris: E.P.F. Yzerman e.yzerman@streeklabhaarlem.nl
Penningmeester: Mw. M.H. Dammer mennie.dammer@lvf.nl
Lid: Prof. Dr. G.S. de Hoog de.hoog@cbs.knaw.nl

Secretariaat: Streeklaboratorium voor de Volksgezondheid Kennemerland
Boerhaavelaan 26 2035 RC Haarlem
Tel. 023 5307800 Fax. 023 5307805
NB. Heeft u een e-mail adres en dit nog niet doorgegeven? Ik wil u vriendelijk verzoeken dit dan alsnog door te geven op: e.yzerman@streeklabhaarlem.nl