Lecture Abstracts

Hyphal Growth and Development

J. G. H. Wessels Dept. of Plant Biology, University of Groningen, Kerklaan 30, 9 751 NN Haren, The Netherlands

Growth by means of apically extending hyphae provides fungi with a certain mobility to locate organic resources, permits penetration of solid substrata, and enables formation of multihyphal structures. Plants are totally dependent on these hypha-based fungal activities, both for their degradation and nutrition, although pathological interactions have also developed.

Fundamental to hyphal growth is extension of the wall at the growing apex (cellular morphogenesis). Apart from the apical cytoskeleton, plastic deformation and subsequent hardening of the wall play important roles (steady-state growth theory). The inside to outside growth of the wall also leads to efficient translocation of proteins over the nascent wall at the apex (bulk-flow hypothesis).

Among secreted proteins are extracellular enzymes and wall proteins, such as hydrophobins. Hydrophobins display self-assembly into amphipathic films when confronted with a hydrophilic-hydrophobic interface (molecular morphogenesis). In this way, hydrophobins are thought to be involved in, for instance, formation of aerial hyphae, dissemination of spores, formation of fruit bodies, and numerous interactions between fungi and plants. Hydrophobins may therefore be central to evolution of both fungi and plants. Hydrophobin-based activities of fungi also inspire industrial applications.

Fungal Genetics: From Fundamental Research to Biotechnology

Karl Esser, Allgemeine Botanik, Ruhr-Universitat, D-44780 Bochum

Fungal genetics is a rather young discipline in science as compared to biotechnology which is, albeit unconciously, correlated with begin of human civilization. It was at first almost exclusively devoted to fundamental research and came only during the last two decades in close relation to biotechnology, when it appeared meaningful to apply chromosomal genetics in a concerted manner to improve the production or transformation capacities of industrial fungi.

A landmark in the young relation between fungal genetics and biotechnology was the discovery of fungal plasmids and its relationship to mitochondrial DNA. Thus it became possible to incooperate also the fungi in the concept of genetic engineering, previously established for prokaryotes. After a short historical survey I will present trends in fundamental research of chromosomal and extrachromosomal genetics, involving breeding systems, recombination, gene expression, extrachromsomal genetic elements and genetic engineering. This will be followed by trends in biotechnology and in conclusion with the future development of fungal genetics.

Molecular Genetics of Vegetative Incompatibility in the Filamentous Fungus Podospora anserina

Begueret Joel, IBGC,- CNRS-UPR9026,- Laboratoire de Genetique Moleculaire des Champignons Filamenteux,- 33077 Bordeaux France

Vegetative incompatibility is common to filamentous fungi. This process prevents the formation of heterokaryons between strains which are non-isogenic for het loci. In most cases the filaments of incompatible strains can fuse but a necrotic reaction rapidly destroys the heterokaryotic cells. Cloning genes involved in vegetative incompatibility has now been initiated in several species and some genes have been characterized providing the basis to understand their function and the mechanisms that are induced after the coexpression of incompatible het genes.

In Podospora anserina we have cloned genes from several het loci. Comparison of differents alleles from the same locus provide evidence that incompatibility might be the consequence of the evolutionary divergence in natural populations, of genes that have a basic function in cell physiology.

From sequence data and mutant phenotypes, it appears that some het genes, but also mod genes, the mutations of which suppress or attenuate incompatibility, encode proteins that are involved in signal transduction essential for some differentiation stages.

These results will be discussed with reference to the role of vegetative incompatibility in natural populations of fungi.

Sexual Development in Podospora anserina: Reproductive Structures, Mating Types and Peroxisomes
Adoutte A., Arnaise S., Berteaux-Lecellier V., Coppin E. Debuchy R., Graia F., Picard M., Zickler D.
IGM, Batiment 400, Universite de Paris-Sud, 91405 Orsay Cedex, France

Two interesting developmental problems were brought to light during the study of sexual reproduction in P. anserina.

The mat+ and mat- mating types were shown to control a recognition process not only during fertilization, but also at a later stage, when reproductive nuclei which have devided in a common cytoplasm form a mat+/mat- pair within the ascogenous hyphae (1). We found that mutations in the single mat+ gene, FPR1, and in two of the three mat- genes, FMR1 and SMR2, inpaired the formation of mat+lmat- dicaryons and led to production of uniparental progeny. These arose from homocaryotic ascogenous hyphae with one or a pair of mat mutant nuclei. In contrast, mutations in SMR1 caused sterility. Complementation studies clarified the role played by each mat gene. The absence of internuclear complementation in SMR2 mutants indicated that the SMR2 protein returns to the nucleus in which the gene has been transcribed. Thus, SMR2 may control mat- identity. Opposite data suggest that FMR1 is not the main determinant of identity, but acts as an enhancer of SMR2. In agreement with that, SMR2 and FMR 1 proteins were shown to interact in the yeast two-hybrid system. The sterility of the SMR1 mutants and their internuclear complementation suggest a different function for SMR1, which might act either prior to or after FMR1 and SMR2. As it can be present in either mat+ or mat- nuclei, or in both, it is not a bona fide mating type gene. Identification of the mat target genes will help to elucidate how mat genes ensure recognition between nuclei.

A link between sexual development and peroxisomes was suggested by the demonstration that the car1 gene, involved in caryogamy, encodes a peroxisomal protein (2). The analysis of revertants has identified new genes involved in sexual development and fatty acid metabolism. These should clarify the role of peroxisomes in the sexual cycle.

In an attempt to discover new developmental pathways, we have cloned and are sequencing three genes involved in the formation of the male gametes and/or the female organs.

1- Zickler et al. (1995) Genetics 140, 493-503. 2- Berteaux-Lecellier et al. (1995) Cell 81, 1043-1051

Control of Sexual Development in Schizophyllum commune by the B Mating Type Locus

Erika Kothe, Jorg Hegner, Klaus Lengeler, Angela Mankel, Carola Siebert-Bartholmei and Jurgen Wendland
Marburg, Germany

The multiallelic B mating type locus that controls nuclear migration and pseudoclamp fusion encodes a pheromone receptor system. Analysis of different alleles of the pheromone receptor gene revealed putative binding sites for intracellular factors involved in signal transduction and adaptation. They are conserved between receptors of different specificity. Putative extracellular pheromone binding sites display sequence diversity between different specificities.

The pheromones encoded by three genes of the locus B l show no amino acid sequence similarity. However, all three genes were active in transformation of a B 2 recipient strain. Transformation with one of the pheromone genes, bap1(l), induced B-regulated development in strains with different specificities indicating promiscuous binding of pheromones. This makes the multiallelic pheromone receptor system of S. commune an ideal system to investigate specificity of ligand-receptor interaction.

Activation of the pheromone receptor by binding of a compatible pheromone triggers nuclear migration. The genes transcriptionally regulated by the pheromone response are investigated using the differential display technique.

Aspergillus Conidiation

John Clutterbuck. Molecular Genetics, University of Glasgow, Glasgow G11 6NU UK

The first genetic investigations of conidiation concentrated on its most conspicuous stages, for the obvious reason that the resulting mutants were easy to identify and interpret. However, this proved to be a fruitful strategy, since mutants with the most conspicuous phenotypes did indeed turn out to be in genes with key roles in development. Only a small number of genes were identified by this route: what proportion of conidiation genes (whatever they may be) have been discovered? Biochemistry suggest only a minority. Subsequent genetic studies have picked on a limited number of new mutants with lesser developmental effects, while the biochemical route has led to further relevant genes, some with identifiable phenotypic effects, but some with none.

Extensions of the original studies in two directions ask, on the one hand, what leads to initiation of conidiation, and on the other, what determines the character of the resulting conidia? The nature of conidia is problematic because their properties are mainly negative: the function of a spore is prolonged inactivity. So far, mutation studies have yielded spore-defective mutants which are difficult to study because of their indistinct phenotypes, while biochemistry has revealed clusters of genes, the only one of which to be dissected in detail remains of unknown function.

However the initiation of conidiation is a much more open field, the main block to its study in the past being definition of a narrow enough area to allow any sense of progress. For the fungus, the alternatives to conidiation are either the even more complex (and interesting) process of sexual reproduction., which has been under study for some time, or the production of aerial mycelium, a field which was, for historical reasons, unpalatable to Aspergillus Geneticists. Now, this nettle has also been gasped, and is bearing interesting fruit.

Signal Transduction Pathways Controlling Multicellular Development in Dictyostelium

Richard A. Firtel. Department of Biology, Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0634, USA

Dictyostelium discoideum grows as single-celled vegetative amoebae. Multicellular development is initiated by starvation. Up to 105 cells chemotactically agregate to form a mound structure that then undergoes cell type differention and morphogenesis to produce a mature fruiting body. Aggregation in Dictyostelium is mediated by extracellular cAMP that interacts with G protein-coupled serpentine receptors. This initiates a variety of intracellular signaling pathways that include the activation of adenylyl cyclase and the relay of the signal, activation of guanylyl, cyclase that regulates chemotactic movement, and the activation of aggregation-stage gene expression. Upon formation of the mound, there is a developmental switch in which the cAMP signal changes from nanomolar oscillatory pulses to a high micromolar, more continuous, signal. This results in the adaptation of the aggregation-stage pathways and the induction of a signaling pathway regulated by the same receptors but is G protein independent. The rise in extracellular cAMP activates the transcription factor GBF that induces a developmental transcription cascade that induces the expression of a variety of genes, including additional G protein subunits, receptors, other transcription factors, and a ras/ras-GAP complex. These gene products are then required for both morphogenesis and subsequent cell-type differentiation. Molecular and genetic analysis has identified nine distinct developmentally regulated heterotrimeric G protein subunits, four cAMP receptors, components of multiple MAP kinase cascades, a variety of other kinases controlled through signal transduction pathways including cAMP-dependent protein kinase, and pathways differentially controlled by protein tyrosine phosphorylation that play central roles in regulating development. How these diverse signal transduction pathways are integrated to control various aspects of multicellular development will be described. The discusion will concentrate on how cAMP receptors differentially mediate G protein-dependent and independent pathways to control development.

N-glycosylation in Aspergillus: Consequences for Heterologous Gene Expression

William Hintz, Dean Goodman, Joshua Eades. Biology Department, University of Victoria, Victoria, B.C., Canada

The emerging field of glycobiology has had a significant impact on the production of recombinant proteins. The post-translational modification of proteins by the addition of sugar residues can significantly affect protein stability, conformation and functional activity. Glycosylation also plays an important role in cell-cell and intracellular protein targeting, These factors can have important effects on the commercial development of recombinant products, particularly in the health care and pharmaceutical industries. During the recombinant production of therapeutic proteins, the composition and types of oligosaccharides can be very sensitive to host cells and culture conditions. Heterogeneity in chain composition, chain structure and the utilization of alternate glycosylation sites is common during the recombinant production of therapeutic proteins and may, as a result, affect such factors as serum half-life, antigenicity, resistance to proteases and biological activity. To obtain stable and functional recombinant proteins, methods must be developed which yield glycoproteins having highly uniform carbohydrate moieties. This could be achieved by post-purification processing of recombinant proteins with enzymes which modify the carbohydrate moieties in a highly specific and predictable manner. Alternately the custom production of recombinant glycoproteins could also be achieved in vivo through the genetic alteration of the glycosylation pathway in the host expression system. This method, while more difficult to develop, would provide a more effective and lasting means to produce glycoproteins with specific glycosylation patterns. Our objective is to characterize the complete set of carbohydrate processing enzymes produced by the filamentous fungus Aspergillus in order to tailor the glycosylation pattern of recombinant therapeutic proteins produced in this fungus. The downstream products of such N-glycan remodeling research will include highly uniform glycoforms for pharmaceutical drugs. In this paper, we report the isolation and cloning of a novel cytosolic mannosidase from Aspergillus nidulans. Sequence analysis revealed a coding region of 3383bp containing three short introns. The deduced amino acid sequence of the A. nidulans mannosidase gene showed considerable homology to both the rat cytosolic/ER and yeast vacuolar mannosidases and, in common with these enzymes, did not contain a recognizable transmembrane domain. Phylogenetic analysis indicated that these cytosolic enzymes form a closely related group (Group III) which is distinct from both the mannosidase I enzymes (Group I) and the mannosidase II/ lysosomal mannosidase (Group II) enzymes.

Genetic Regulation of Nitrogen Metabolism

George A. Marzluf, Hongoo Pan, and Bo Feng. Department of Biochemistry, Ohio State University, Columbus, Ohio 43210 USA

In Neurospora crassa, expression of many structural genes for nitrogen catabolic enzymes and permeases is regulated by nitrogen repression and by pathway-specific induction. Transcription of the nit-3 gene is highly regulated by NIT2, a global, positive-acting regulatory protein and by NIT4, a minor control protein that mediates nitrate induction. NIT2 is a DNA-binding protein which recognizes sites containing GATA core sequences; NIT4 has a GAL4-like Cys6/Zn2 DNA binding motif and binds to an 8 bp palindromic sequence TCCGCGGA and closely related sequences. Three NIT2 and two NIT4 binding sites occur in the nit-3 promoter, most as a cluster of sites at approximately -1,000 bp. All of the binding sites contribute to nit-3 gene expression, but one NIT2 and one NIT4 site which lie immediately adjacent are of primary importance.

NMR is a negative-acting regulatory protein which functions in nitrogen repression; nmr mutants are largely insensitive to nitrogen repression. A specific NMR-NIT2 protein-protein interaction has been identified with the yeast 2-hybrid system and by direct in vitro protein binding studies. NMR binds to two distinct regions of NIT2, one an -helical segment in the DNA.binding domain, the second approximately 12 amino acids at the carboxyl terminus which also appear to form an -helix. Amino acid substitutions in either of these regions result in nitrogen derepression and in the loss of the ability to bind to NMR.

Molecular Regulation of the Penicillin Biosynthesis in Aspergillus nidulans

Axel A. Brakhage, Katharina Then Bergh and Olivier Litzka. Lehrstuhl fur Mikrobiologie, Universitat Munchen, Maria-Ward-Str. la, D80638 Munchen, F.R.G.

The secondary metabolite penicillin is produced by some filamentous fungi only, most notably by Aspergillus nidulans and Penicillium chrysogenum. Starting from the three amino acid precursors, the penicillin biosynthesis is catalysed by three enzymes which are encoded by the following three genes: acvA (pcbAB), ipnA (pcbC) and aat (penDE). The genes are organised into a gene cluster. In A. nidulans, acvA and ipnA are bidirectionally transcribed. The aat gene lies 825 bps downstream of ipnA. Several regulatory circuits affecting the regulation of these penicillin biosynthesis genes have been identified. A moving window analysis of the 872-bp intergenic region between acvA and ipnA using reporter gene fusions indicated that the divergently orientated promoters are, at least in part, physically overlapping and share common regulatory elements. Removal of nucleotides -353 to -432 upstream of the acvA gene led to a 10-fold increase of acvA-uidA expression and simultaneously, to a reduction of ipnA-lacZ expression to about 30 %. Band shift assays and methyl interference analysis using partially purified protein extracts revealed that a short DNA element within this region was specifically bound by a protein (complex) which we designated PENR, for penicillin regulator. Deletion of 4 bps within the identified protein binding site caused the same contrary effects on acvA and ipnA expression, as observed for all of the deletion clones lacking nts -353 to -432. In addition, band shift assays and mutagenesis experiments led to the identification of a functional DNA element in the aat promoter region which was specifically bound by the same PENR protein (complex) as the intergenic region between acvA and ipnA. Hence, PENR seems to be involved in the regulation of all penicillin biosynthesis genes. Furthermore, the promoter regions of the corresponding genes of the -lactam producing fungi Penicillium ch sogenum and Acremonium chrysogenum also diluted the complex formed of the A. nidulans probes with PENR in vitro, suggesting that these -lactam biosynthesis genes are regulated by analogous DNA elements and proteins.

Efficient Production of Secreted Proteins by Aspergillus: Progress, Limitations and Prospects

Cees A.M.J.J. van den Hondel, Robin J. Gouka, Johanna (H.) G. M. Hessing, Jan C. Verdoes, Robert F.M. van Gorcom and Peter J. Punt. TNO Nutrition and Food Research Institute, Department of Molecular Genetics and Gene Technology, PO Box 5815, 2280 HV Rijswijk, the Netherlands

Aspergilli, such as A. niger and A. oryzae, are widely used for the commercial production of a variety of extracelluar enzymes due to their ability to secrete large amounts of proteins and their status as "GRAS organisims". To achieve high levels of fungal-enzyme production long-lasting classical strain-improvenient programs have been used.

The application of modern molecular-geiietic techniques, however, has greatly accelerated fungal strain improvement and has created new opportunities for the production of fungal enzymes, but also of non-fungal enzymes and phamraceutical proteins. In the last years all the "basic tools" have been developed, which are required for (molecular)-genetic analysis of A. niger and construction of high production strains. To prevent degradation of nonfungal proteins, several protease-deficient niutants of A. niger have also been isolated. In addition, strategies have been developed by which efficient production of fungal and non-fungal proteins can be achieved. These strategies are based on the use of well characterised strong, constitutive or regulated gene expression signals and the generation of multicopy strains. Furthermore a gene-fusion strategy has been developed which improves the production of mature non-fungal proteins. Although substantial progress has been made, systematic studies on protein production have identified clear limitations that restrict the production yields of fungal and especially of some non-fungal proteins by Aspergillus.

In the lecture the progress, limitations and prospects for efficient production of secreted proteins bv Aspergillus will be discussed and illustrated with some examples of our systematic study on the production of fungal and non-fungal proteins in Aspergillus, such as A. niger glucoamylase, plant Cyamopsis tetragonoloba -galactosidase, human Interleukin 6 and antibody fragments.

Mating and the Requirement for Allelic Pairing

Aramayo, R., Peleg, Y., Addison, R., and Metzenberg, R.L. Department of Biomolecular Chemistry, University of Wisconsin, Madison VW 53706, U.S.A.

A puzzling phenomenon seen in ascomycetous fungi is that of "ascus dominant" mutations. A classic example of this is the mutation Round spore in Neurospora crassa. In crosses heterozygous for the mutant allele, all of the ascospores, including the four that contain the wild type allele, are “round” (spherical) rather than having the spindle-shaped spores typical of the species. The simple ad hoc explanation of ascus dominance is that the mutant makes a toxic or inhibitory product that interferes with the formation or function of the wild type product - that is, the dominant mode of expression of the mutant allele is due to negative complementation in the zygote. The effects on the zygote are somehow carried into the ascospores.

Our study of an ascus-dominant mutation called Asm-1 suggests a different mechanism. Asm-1 (Ascospore maturation) has both a vegetative phenotype and a sexual phase phenotype. When crossed to wild type or its equivalent, Asm-1 causes all spores in almost all asci to fail to mature; they remain very small, white, and inviable. The significant point is that Asm-1 is a deletion mutant, and cannot be making a toxic product. We have found that when a normal allele, Asm-l+, reintegrated into the genome in a defined but ectopic location, corrects the vegetative phenotype of the deletion mutant, but completely fails to correct the ascus-dominant failure of spore maturation. However, when two Asm-1 strains of opposite mating type are both subjected to re-insertion of Asm-l+ at the same "wrong" location, mature black ascospores are produced. Our interpretation of this and related experiments is that, not only is Asm1+ function needed for ascospore maturation, but the wild type allele must be paired with its homolog in the zygote for it to be correctly expressed during later development. A phenomenon analogous to this occurs in the diploid somatic cells of certain Drosophila mutants, and is known as transvection. We suggest that Asm-1 acts in a way formally similar or identical to transvection, and speculate that other ascus-dominant mutations like Round spore may exert their effects by transvection.

Fungal Avirulence Genes, Major Players in the Gene-for-gene Game

Pierre J.G.M. De Wit1, Paul JM.J Vossen1, Miriam Kooman-Gersmann1, Ralph Vogelsang1, Matthieu H.A.J Joosten1, Guy Honee1, and Jacques JM Vervoort2. 1 Department of Phytopathology, Wageningen Agricultural University, Wageningen, 6709 PD, The Netherlands. 2Department of Biochemistry, Wageningen Agricultural University, Wageningen, 6703 HA, The Netherlands.

The interaction between the biotrophic fungal pathogen Cladosporium fulvum and tomato complies with the gene-for-gene model. Resistance, expressed as a hypersensitive response (HR) followed by other defence responses, is based on recognition of products of avirulence genes from C. fulvum (race-specific elicitors) by receptors (putative products of resistance genes) in the host plant tomato. The AVR9 elicitor is a 28 arnino acid (aa) peptide and the AVR4 elicitor a 106 aa peptide which induces HR in tomato plants carrying the complementary resistance genes CJ9 and Cf4, respectively. The 3-D structure of the AVR9 peptide, as determined by 1H NMR, revealed that AVR9 belongs to a family of peptides with a cystine knot motif. This motif occurs in channel blockers, peptidase inhibitors and growth factors. The CJ9 resistance gene encodes a membrane-anchored extracellular glycoprotein which contains leucine-rich repeats (LRRs). 125I labeled AVR9 peptide shows the same affinity for plasma membranes of Cf9+ and Cf9- tomato leaves. Membranes of solanaceous plants tested so far all contain homologs of the Cf9 gene and show similar affinities for AVR9. It is assumed that for induction of HR, at least two plant proteins (presumably CF9 and one of his homologs) interact directly or indirectly with the AVR9 peptide which possibly initiates modulation and dimerisation of the receptor, and activation of various other proteins involved in downstream events eventually leading to HR. We have created several mutants of the Avr9 gene, expressed them in the potato virus X (PVX) expression system and tested their biological activity on Cf9 genotypes of tomato. A positive correlation was obseverd between the biological activity of the mutant AVR9 peptides and their affinity for tomato plasma membranes. Recent results on structure and biological activity of AVR4 peptides encoded by aviruient and virulent alleles of the Avr4 gene (based on expression studies in PVX) will also be presented.

Engineering Mycoviruses to Understand and Alter Fungushost Pathogenic Interactions

Donald L. Nuss, Center for Agricultural Biotechnology, University of Maryland ,Biotechnology Institute, College Park, MD, 20742, USA.

Interactions between the fungal pathogen Cryphonectria parasitica and its plant host are significantly altered by virulence-attenuating mycoviruses of the genus hypovirus, thus providing a unique paradigm for examining mechanisms that drive fungal pathogenic processes. The utility of this system has been validated by recent observations that hypovirus infection attenuates fungal virulence by disrupting a key fungal G-protein (CPG-1) linked signal transduction pathway. We now report that hypovirus infection and CPG-1 transgenic suppression elevate cAMP levels three to five fold. This result is consistent with the prediction that C. parasitica CPG-1, like mammalian Gi subunits, function to negatively regulate adenylyl cyclase. Fungal genes that are specifically regulated through the CPG-1 pathway were recently identified with the aid of mRNA differential display. It was also possible to mimic the effect of both hypovirus infection and CPG-1 transgenic suppression on the expression of these genes by drug-induced elevation of cAMP levels. These results identify G-protein-regulated cAMP accumulation as a determinant of hypovirus-mediated alteration of fungal gene expression and virulence.

The recent development of an infectious cDNA copy of a hypovirus RNA has provided the ability to genetically modify hypoviruses that confer specific phenotypic traits on their fungal host and to generate "engineered" hypovirulent fungal strains with enhanced biocontrol potential. The development of a hypovirus transfection system has allowed the introduction of infectious hypovirus synthetic RNA transcripts into fungal pathogens other than C. parasitica. Moreover, these infections produced profound phenotypic changes, including hypovirulence. Combined, these basic and technical advances have provided new opportunities for broadening the potential application of hypoviruses for purposes of understanding and controlling fungal pathogenesis.

Increasing Fungal Pathogenicity Towards Insects

R.J. St. Leger, Boyce Thompson Institute for Plant Research at Cornell University Ithaca. NY 14853-1801 USA

We have developed molecular biology methods to elucidate pathogenic processes in the deuteromycete entomopathogen Metarhizium anisopliae and have cloned 53 cDNAs which are specifically expressed when the fungus is induced by physical and/or chemical stimuli to alter its saprobic growth habit, develop a specialized infection structure (the appressorium) and attack its insect host. We are testing the involvement of selected genes in pre-penetration growth and development (e.g. genes encoding protein kinases), cuticle penetration (protease) and post-penetration subduing of the host (toxins). In addition to gene disruption studies (for those activities which exists as only one or two gene copies) we are constitutively overexpressing genes in Metarhizium as possible means of speeding infection. We are also using Metarhizium genes to transform a weaker entomopathogen, Aschersonia aleyrodis and determine the effects on pathogenicity.

These experiments are allowing us to assess the genetic and biochemical factors which regulate/limit the degree of fungal pathogenicity to insects and provide new and important resources for manipulating microbial-plant pest interactions and for engineering advanced biopesticides.

Mechanisms of Resistance to Azole Antifungal Agents in the Human Fungal Pathogen Candida albicans

D. Sanglard, F. Ischer, M. Monod, J. -L. Pagani and J. Bille. Institut de Microbiologie, Centre Hospitalier Universitaire Vaudois (CHUV), 1011 Lausanne (SWITZERLAND)

Oropharyngeal candidiasis (OPC) caused by the human pathogen Candida albicans is a common infection in patients with the acquired immunodefficiency syndrome (AIDS). Azole antifungal agents, and especially fluconazole, have been used widely to treat OPC in hospitals. An increasing number of cases of clinical resistance against this antifungal correlating with in vitro resistance have been reported recently. Our goal is to investigate the mechanisms of resistance to azole antifungal agents at the molecular level in C. albicans clinical isolates. We have shown in a recent study [1] that sequential fluconazole resistant yeast isolates from patients failed to accumulate [3 H]-fluconazole. This phenomenon was linked to an increase of mRNA for a recently cloned (ATP Binding Cassette) ABC-transporter gene called CDR1 and of mRNA of a gene conferring benomyl resistance (BENr, the product of which belongs to the class of Major Facilitator multidrug efflux transporters. Therefore, in C. albicans clinical isolates, different multidrug transporters can be involved in the appearance of resistance to fluconazole and potentially to other azole derivatives such as itraconazole and ketoconazole. In fact, a mutant of the model yeast Saccharomyces cerevisiae lacking the ABC-transporter Stsl or C. albicans mutants lacking the ABC-transporter Cdr1 were hypersusceptible to the azole derivatives fluconazole, itraconazole and ketoconazole, thus showing that indeed these ABC-transporters can use these compounds as substrates. Surprisingly, the disruption of the BENr gene in C. albicans did not result in hypersusceptibility to azole antifungal agents. Other factors that could render C. albicans clinical isolates resistant to azole derivatives have been also characterized more recently in our laboratory: i) New multidrug transporters genes have been isolated, but their involvement in the resistance of clinical isolates to azole derivatives is still not clear. ii) Changes in the affinity of azole derivatives to their cellular target, i.e. a cytochrome P450 (14DM), have been also observed using a system enabling an expression screening of different 14DM genes. Specific mutations in the 14DM gene coding region altering the affinity of 14DM to azole derivatives have been mapped by this method.

[1] Sanglard, D., Kuchler, K., Ischer, F., Pagani, J.-L., Monod, M. and J. Bille (1995). Antimicrob. Agents Chemother. 39: 2378-2386.

Mechanisms of Fungal Pathogenesis

O. C. Yoder. Department of Plant Pathology, Comell University, Ithaca, NY 14853 USA

It has long been hypothesized that fungal pathogens produce molecules which condition host tissues for colonization, and that other fungal molecules function to elicit resistance responses in cells of potential hosts. In recent years direct evidence in support of both types of molecule has been obtained. Thus, it can be predicted that the occurrence of disease is determined by the balance between susceptibility-producing vs. resistance-producing molecules in any given fungus/host interaction. The goal of this lecture is to briefly survey fungal factors known with reasonable certainty to be involved in fungal pathogenesis, then focus on a particular case study from our own laboratory, and finally indulge in the conjecture that future investigations will reveal common mechanisms of pathogenesis for fungal pathogens of both plants and animals.

A CASE STUDY: The Tox1 locus of the haploid, filamentous ascomycete Cochliobolus heterostrophus determines the essential difference between the two forms of the fungus found in nature. Race T and race 0 are pathogenic to maize, but race T is distinguished by its extreme virulence to maize with Texas (T) male sterile cytoplasm, and by its ability to produce T-toxin, a family of linear polyketides which specifically affect T-cytoplasm. When the two races are crossed, only parental type progeny segregate, in a 1:1 ratio, thereby defining the single locus Tox1 which controls T-toxin production and high virulence. We have investigated the molecular nature of Tox1 to evaluate the role of T-toxin in pathogenesis and to determine the genetic mechanism underlying the evolution of a new pathogenic race. Insertional mutagenesis allowed the cloning and sequencing of three genes at the Tox1 locus: PKS1, DEC1, and RED1 encode a polyketide synthase (PKS1), a decarboxylase (DEC1), and a reductase (RED1), respectively. Site specific gene disruption revealed that PKS1 and DEC1 are required for production of T-toxin and high virulence, whereas RED1 is associated with no apparent phenotype. We propose that PKS1 is needed to construct the T-toxin polyketide carbon chain and that DEC1 activates the molecule by removing the terminal carboxyl group. Surprisingly, although PKS1 and DEC1 are linked to Tox1, genetic and physical analyses have revealed that they are not linked to each other, and indeed are on two different chromosomes! The resolution of this apparent paradox is that Tox1 maps to the breakpoints of chromosomes which are reciprocally translocated in race T with respect to race 0. Thus, PKS1 is at a locus now called Tox1A on one of the translocated chromosomes, and DEC1 and RED1 are at a locus (Tox1B) on the other translocated chromosome. These three genes are found only in race T, and not in race 0 or in any other fungus examined to date. The data suggest an explanation for how race T evolved, ie., the Tox1 locus was not inherited by race T from an ancestral strain but rather was transferred horizontally to C. heterostrophus from an alien organism; after insertion of this heterologous DNA into the genome, a reciprocal translocation occurred, resulting in "Tox1" residing on two different chromosomes.

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