XXIV Fungal Genetics Conference at Asilomar
Plenary Session Abstracts
Genome Structures and Dynamics
The Kluyveromyces polysporus genome: gene losses, convergence of genome contents, and the origin of yeast species.
Kenneth H. Wolfe. Smurfit Institute of Genetics, Trinity College, Dublin 2, Ireland. firstname.lastname@example.org
We report an 8x draft genome sequence of Kluyveromyces polysporus, a yeast species that underwent whole-genome duplication (WGD) but represents the clade of post-WGD species that is most distantly related to Saccharomyces cerevisiae. We find that the subsequent process of loss of superfluous copies of genes in K. polysporus proceeded almost independently of that in S. cerevisiae. At the time the two lineages diverged, their common ancestor still retained about 9000 genes and 80% of them were members of duplicated pairs resulting from the WGD. The genomes of the two lineages then shrank independently and converged onto their current size of around 5600 genes each. The functional categories of genes that survived in duplicate in the two genomes are similar, confirming the role of natural selection in this process, but at loci where each species retained only one copy they show almost random choice of which copy to retain. Thus, K. polysporus contains pairs of duplicated chromosomal regions that are superficially similar to those in S. cerevisiae, but whose actual makeup is very different. About 45% of the single-copy genes in these two species are paralogs, not orthologs. These observations support the hypothesis that two polyploid lineages can become reproductively isolated from one another if they lose alternative copies of duplicated genes (Dobzhansky-Muller incompatibility).
Evolution of gene clusters in fungi: lessons from isoprenoid gene clusters.
Christiane Bömke and Bettina Tudzynski, Universität Münster, Institut für Botanik, Schlossgarten 3, D-48149 Münster, Germany
With the availability of fungal genome sequences, we are now able to gain insights into the evolution of gene clusters for biosynthesis of secondary metabolites. Horizontal as well as vertical transmission of gene clusters, followed by selective loss during evolution have been proposed to explain the existence of similar clusters in distantly, and theír missing in closely related species.
We work on identification of gibberellin (GA)- and other isoprenoid gene clusters inside and outside the genus Fusarium. After disproving the hypothesis of horizontal gene transfer between higher plants and fungi, we suggest that both groups of organisms have evolved the GA biosynthesis pathways independently. Analyzing about 50 Fusarium species, we showed that only the closely related members of the Gibberella fujikuroi species complex, but not other Fusarium species, contain the GA gene cluster. On the other hand, fungi not closely related to the genus Fusarium, such as Sphaceloma and Phaeosphaeria, produce GAs. Magnaporthe grisea contains an active GA-specific diterpene cyclase in a rudimentary cluster. In Phoma betae, a similar gene cluster have been identified which is responsible for the production of the GA-like compound aphidicolin. The conserved intron positions in the diterpene cyclase genes and the physical linkage to a pathway-specific GGPP synthase gene let us assume that fungal diterpenoid gene clusters have one phylogenetic origin.
Genome defense by repeat-induced point mutation: an evolutionary dead end?
Michael Freitag. Dept. of Biochemistry and Biophysics, Center for Genome Research and Biocomputing, Oregon State University, USA
In 1987, Eric Selker and colleagues uncovered the first eukaryotic “genome defense” system, a hypermutation phenomenon later dubbed “repeat-induced point mutation” (RIP). This premeiotic process detects and inactivates duplicated DNA segments of varying length, i.e. gene-sized to large chromosome segments. RIP takes place after fertilization, during a stage characterized by pre-meiotic nuclear divisions, but before karyogamy occurs. The RIP machinery peppers both copies of duplicated sequences with C:G to T:A transition mutations. While the mechanisms involved in homologous DNA pairing and the subsequent mutagenesis remain obscure, RIP has recently enjoyed much attention because of its potential role in shaping fungal genomes. RIP appears evolutionary conserved within the true ascomycetes because at least one essential component of the RIP machinery, the putative DNA methyltransferase RID, is retained in many taxa. Moreover, active RIP has by now been demonstrated in several ascomycetes and putative relics of RIP have been found in many additional species. Evolutionary implications and our work with mutants defective in RIP will be discussed.
Evolution of Silencing Proteins in Hemiascomycetes.
Laura Rusche and Meleah Hickman, Duke University, Durham, North Carolina
In S. cerevisiae, the mating type of haploid yeast is determined by the MAT locus, which can express a or alpha genes, encoding master transcriptional regulators. To enable the cells to switch mating type, the a and alpha genes are also encoded at the silent mating-type loci, HMRa and HMLα. These two loci are constitutively silenced by the Sir proteins. Two of the four Sir proteins, Sir2p and Sir3p, have paralogs that arose in a whole-genome duplication that occurred in the ancestry of Saccharomyces. Hence, this duplication event may have enabled the Sir proteins to become more specialized for silencing. We are exploring the evolution of the Sir proteins by investigating the functions of Sir homologues in two species, Kluyveromyces lactis and Ashbya gossypii, which diverged from S. cerevisiae prior to the whole-genome duplication.
DNA Repair and Recombination in Ustilago maydis.
M. Kojic, Q. Zhou, N. Mazloum, N. Mao, and W. K. Holloman, Department of Microbiology and Immunology, Cornell University Weill Medical College, New York, NY 10021
Genome stability relies on a network of interacting systems that detect and repair disturbances in the integrity of DNA. Inactivation or impairment of these systems can lead to chromosome aberrations, mutation, and death. In many cases elements of these systems are highly conserved, indicating universality of the mechanisms and by inference the importance of these systems in preserving the genome of the hosts. A central pathway that processes potentially lethal types of DNA damage employs a mechanism that enables repair by homologous recombination. Components of this pathway in eukaryotes such as Homo sapiens include Rad51 whose function is to search for DNA sequence homology and promote strand exchange, plus factors that enable and enhance Rad51’s activity. These latter include the product of the BRCA2 breast cancer susceptibility gene, that serves as the key regulator of Rad51, and also the Rad51 paralogs, proteins structurally related to Rad51 that serve to promote its function. In the emerging view, a DNA molecule sustaining a double-strand break is resected from the exposed duplex ends to reveal protruding single-stranded stretches. These pair with a homologous DNA sequence, after which DNA synthesis proceeds with the undamaged homologous sequence serving as a template to fill in the missing nucleotides. Following resolution of the joint molecule intermediates or dissociation and reannealing of the invading strand the repair process is completed.
We are interested in recombinational repair processes in Ustilago maydis, a basidiomycete phytopathogenic fungus that has served as a model organism for the elucidation of the central mechanism of recombinational repair for many years. The recombinational repair system in U. maydis relies on orthologs of the human Rad51 protein, BRCA2 (termed Brh2 in U. maydis), and Dss1. Brh2 enables recombinational repair of DNA by controlling Rad51 and is in turn regulated by Dss1, a small polypeptide. Interplay with Rad51 is conducted via the BRC element located in the N-terminal region of the protein and through an unrelated domain, CRE, at the C-terminus. Mutation in either BRC or CRE severely reduces functional activity, but repair deficiency of the brh2 mutant can be complemented by expressing BRC and CRE on different molecules. This intermolecular complementation is dependent upon the presence of Dss1. Brh2 molecules associate through the region overlapping with the Dss1-interacting domain to form at least dimer size complexes, which in turn, can be dissociated by Dss1 to monomer. The model that emerges from this work proposes that cooperation between BRC and CRE domains and the Dss1-provoked dissociation of Brh2 complexes are requisite features of Brh2’s molecular mechanism.
Host-pathogen and symbiotic interactions
The Remarkably Diverse Effector Proteins of Hyaloperonospora parasitica.
Jim Beynon, Rebecca Allen, Laura Baxter, Rachel Baumber, Peter Bittner-Eddy, Mary Coates, Kate Fisher, Sharon Hall, Linda Hughes, Sarah Lee, Julia Meitz, Anne Rehmany and Laura Rose1. Warwick HRI, Warwick University, Wellesbourne, Warwick, CV35 9EF, UK. 1Department of Evolutionary Biology, University of Munich, Großhadernerstr. 2, 82152 Planegg-Martinsried, Germany
Hyaloperonospora parasitica is an obligate oomycete and the causal agent of downy mildew on Arabidopsis. Recently we have cloned two pathogenicity effector genes, ATR1 and ATR13, from the pathogen and shown them to code for unique proteins that are under amazing levels of diversifying selection. This implies that they are locked in an “arms race” with the plant’s pathogen detection system and consequently these levels of diversity are mirrored in the host resistance genes, RPP1 and RPP13, associated with recognition of ATR1 and ATR13, respecitively. We have used this natural variation to identify key amino acids involved in determining specificity in the ATR13/RPP13 interaction. Using bioinformatic analyses we have identified a further 140 candidate effectors and they reveal a fascinating picture of the effects of diversifying selection presumably resulting from their interactions with Arabidopsis defence mechanisms. It is difficult to ascribe all the diversity seen between ATR13 alleles to the presence of a single host resistance gene and we will describe the complexity of the ATR13/RPP13 interaction.
Molecular insights into mutualism in a fungal-plant interaction.
Barry Scott, Daigo Takemoto, Aiko Tanaka, and Carla Eaton. Massey University, New Zealand.
Key requirements for microbes to initiate and establish mutualistic symbiotic interactions with plants are evasion of potential host defense responses and strict control of microbial growth. We have recently shown that reactive oxygen species (ROS) produced by a specific fungal NADPH oxidase isoform NoxA, have a critical role in regulating hyphal growth in the mutualistic interaction between Epichloë festucae and perennial ryegrass (Tanaka et al. 2006). In professional phagocytes, activation of the membrane associated NADPH oxidase (gp91phox) involves cytosolic recruitment of a heterotrimeric phagocyte oxidase complex (phox) and the small GTP binding protein Rac2 in response to microbial or inflammation signals. E. festucae homologues of p67phox and Rac2, designated noxR and racA, were cloned and deletions of each generated (Takemoto et al. 2006; Tanaka et al. unpublished). Symbiota containing these mutants had a similar stunting phenotype to noxA. Hyphae in these symbiota were hyper-banched and the biomass dramatically increased. Over-expression of noxR results in hyper-branching of E. festucae in culture, as does treatment of wild-type cells with the ROS inhibitor DPI. Using yeast two-hybrid and pull-down assays NoxR was shown to interact with RacA. A single amino acid substitution in the predicted RacA-binding site of NoxR (R101E) abolished this interaction. A noxR construct containing this mutation failed to complement the noxR mutation in planta. Taken together these data demonstrate that NoxR is a key regulator of NoxA in the symbiosis, where it acts together with RacA to spatially regulate ROS production and control hyphal growth and patterning.
Magnaporthe grisea infection of rice and parallel studies in wheat scab.
Jin-Rong Xu. Department of Botany and Plant Pathology, Purdue University. West Lafayette, IN 47906. Email: email@example.com
Rice blast caused by Magnaporthe grisea is one of the most severe fungal diseases of rice throughout the world. In the past decade, it has been developed as a model system to study fungal-plant interactions. Several signal transduction pathways regulating infection-related morphogenesis and fungal-plant interactions have been identified. The PMK1 MAP kinase is essential for infectious growth and the formation of the highly specialized infection structure known as appressorium. Several upstream components of the PMK1 pathway, including MST7, MST11, MST50, RAS2, and MGB1, were identified and functionally characterized. One downstream transcription factor MST12 and a few virulence factors regulated by PMK1 also have been characterized. Our results indicate that well conserved signaling pathways may have different upstream signal inputs and outputs in fungal pathogens. A MAP kinase pathway homologous to PMK1 also is essential for pathogenesis in Fusarium graminearum, the causal agent of wheat and barley scab. Microarray analysis was used to compare genes regulated by this MAP kinase pathway in these two pathogens with distinct infection mechanisms. A few genes commonly regulated by PMK1 and its homolog in F. graminearum were selected for functional analysis. Several pathogenicity factors known in M. grisea also were analyzed for their roles in F. graminearum. One of them is the TBL1 transducin-beta gene that is required for infectious growth after the initial invasion.
Joyce E. Longcore. Department of Biological Sciences, University of Maine, Orono, ME 04469-5722.
Batrachochytrium dendrobatidis (Bd), described in 1999, is an amphibian pathogen and the probable cause of declines of amphibian populations on five continents. In addition to numerous genera and species in Australia and Central America, examples of affected species are the mountain yellow-legged frog in California, the midwife toad in Spain and the Kihansi spray toad in Africa; however, infection also can be present without seeming to affect population numbers. Bd, the only chytrid pathogen of vertebrates, develops determinant, spherical thalli within keratinized skin cells of hosts; at maturity each sporangium produces a discharge papilla that extends to the skin surface. The chytrid releases motile zoospores into the exterior environment. Bd also can live without an amphibian host. In pure culture, individual thalli with thread-like rhizoids grow up to 40 µm dia on 1 % tryptone medium, and mature in ~ 4 days at 23 C. During maturation the entire nucleated cytoplasm divides into asexual zoospores, which exit the zoosporangium through 1-several discharge papillae. Sexual reproduction is unknown, and is rarely reported in the Rhizophydiales, the order in which this species is classified. Field research has contributed to knowledge of the distribution and effects of Bd but only pure cultures have made possible fulfillment of Koch’s Postulates, experimental inoculations, progress in methods to detect Bd, studies of population genetics and whole genome sequencing.
Molecular dissection of the cell wall of Cryptococcus
Lorina Baker, Charles Specht, Isaac Banks and Jennifer Lodge
The fungal cell wall provides structure and protection from the environment, and it is intimately involved with interactions with host cells during infection. Fungal walls are composed of various polysaccharides and proteins, and virulence factors are often cell wall associated. Exposure of specific wall polysaccharides on the cell surface provoke specific host responses. Chitin, a rigid, insoluble, polymer of N-acetylglucosamine, has been shown to be an essential component of the cell wall of many fungi. Chitin also can be enzymatically deacetylated to chitosan, a more flexible and soluble polymer. Cryptococcus neoformans is a fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. We have shown that both chitin and chitosan are present in the cell wall of vegetatively growing C. neoformans yeast cells, and that the levels of both rise dramatically as cells grow to higher density in liquid culture. Chitosan is also present during when the fungi is growing in a mouse model. Although C. neoformans has eight putative chitin synthases, and none are essential, one of them, Chs3, appears to produce the bulk of the chitin which is converted to chitosan. Deletion of a putative regulator of chitin synthase also substantially reduces the production of chitosan. We have also identified three chitin deacetylases that account for all of the chitosan in the wall. The data suggests a model for chitosan production in vegetatively growing C. neoformans where the three chitin deacetylases convert chitin generated by the chitin synthase, Chs3, into chitosan. In C. neoformans, chitosan helps to maintain cell integrity and aids in bud separation. Additionally, chitosan is necessary for maintaining normal capsule width, and lack of chitosan results in a “leaky melanin” phenotype. Mutants in the chitin deacetylases are less viable in the mouse model, suggesting that chitosan is critical for C. neoformans interactions with its host.
Development and metabolism
Transcriptional controls of carbon source utilization in Aspergillus nidulans.
Michael Hynes. Department of Genetics, University of Melbourne, Parkville, Australia.
In Aspergillus nidulans, acetate utilisation involves acetyl-CoA formation in the cytoplasm and metabolism via the glyoxalate cycle in peroxisomes and via the TCA cycle in mitochondria. A cytoplasmic carnitine-acetyl transferase allows acetyl-CoA to enter the organelles as acetyl-carnitine which is converted back to acetyl-CoA by a second carnitine-acetyl transferase encoded by acuJ, a gene required for growth on both acetate and fatty acids which are converted to acetyl-CoA by peroxisomal beta-oxidation. A peroxisomal targeting mutation results in loss of growth on fatty acids indicating that AcuJ is necessary for transport of acetyl-CoA from peroxisomes to the mitochondria. Growth on acetate is not affected, indicating that acetyl-CoA can be metabolised via the glyoxalate cycle in the cytoplasm. This is supported by the ability of various peroxisome mutants to grow on acetate but not on fatty acids. There are three classes of genes - those induced by acetate only; those induced by both acetate and fatty acids including acuJ and genes for the glyoxalate cycle and a very large number induced only by fatty acids including genes encoding beta-oxidation enzymes and peroxins. The transcription factor FacB, specific for acetate induction, is regulated by glucose repression and induction by acetyl-carnitine. FarA, FarB (orthologs of the highly conserved cutinase transcription factors) and ScfA, are required for fatty acid induction. FarA orthologs are also found in the hemi-ascomycetes, Candida albicans, Debaryomyces hansenii and Yarrowia lipolytica. Other carbon sources metabolised via the TCA cycle, like acetate, fatty acids and amino acids, require gluconeogenesis. Two transcription factors are required for induction of relevant enzymes in response to either malate or oxaloacetate.
Growth and developmental control in A. nidulans and A. fumigatus.
Jae-Hyuk Yu. Department of Bacteriology, University of Wisconsin, Madison WI 53706 USA
Reproduction of fungi results in the formation of enormous numbers of spores that are efficient agents for genome protection, survival and propagation. Spores are also the primary agent for infecting hosts for many animal and/or plant pathogenic fungi. Asexual sporulation is a common reproductive mode for a diverse group of fungi. In some fungi asexual spore formation is intimately related with production of toxic secondary metabolites called mycotoxins, which cause various adverse health effects.
The genetically tractable fungus Aspergillus nidulans has served as an excellent model system for studying multicellular development and secondary metabolism. Overall goal of our research program is to understand the mechanisms underlying the regulation of vegetative growth and asexual development (conidiation) in A. nidulans and Aspergillus fumigatus. Via comparative functional studies of the four key A. nidulans regulators FadA (Ga), FlbA (RGS), FluG and BrlA in A. fumigatus, we found that 1) A. fumigatus and A. nidulans share conserved G protein and RGS-mediated growth signaling; 2) A. fumigatus may have the distinct and persistent upstream (fluG-level) regulatory mechanisms for activation of conidiation; and 3) BrlA, a key transcription factor necessary for conidiophore development in A. nidulans, is also essential for conidiation in A. fumigatus.
In addition to the comparative studies, we further dissected the regulation of spore formation in A. nidulans. Two distinct genetic screens have identified two key genes called sfgA and vosA that control initiation and completion of sporulation, respectively. Characterization of these genes revealed that both the beginning and the end of asexual spore formation require balanced activities of various positive and negative regulators. The genetically programmed instruction for the regulation of spore formation may explain how a fungus governs vegetative growth and reproduction appropriately. The detailed roles of these genes in developmental control are also presented.
The necrotrophic fungus, Sclerotinia sclerotiorum subverts host pathways by inducing apoptosis for disease development.
Marty Dickman, Jeff Rollins1, Changbin Chen2, and Kyoungsu Kim. Texas A&M University, Institute for Plant Genomics and Biotechnology. Department of Plant Pathology and Microbiology,College Station, Texas 77843 USA
1University of Florida, Department of Plant Pathology, Gainesville, FL. 2 University of Calofornia, San Francisco, Department of Biochemistry & Biophysics San Francisco, CA94143-2200
Sclerotinia sclerotiorum is an extremely broad host range, economically important necrotrophic fungal plant pathogen. Effective pathogenesis by this fungus requires the secretion of oxalic acid (OA). Studies with OA mutants strongly suggest that oxalic acid is an essential pathogenicity determinant in S. sclerotiorum and is required for proper sclerotial development. Studies conducted to determine the mode of action of OA have indicated a multifunctional role for this organic acid. OA modulates ambient pH by acidification, which serves as a regulatory cue for processes linked to pathogenicity and differentiation including the activation of an OA induced mitogen activated protein kinase (SMK1) that is required for sclerotial development. Increases in cAMP levels impairs sclerotial formation and cAMP also inhibits the activation of S. sclerotiorum MAPK; thus cAMP-mediated sclerotial inhibition is modulated through MAPK. Cross-talk between these two pathways is mediated by the small G-protein, Rap-1.
Previous studies have shown that oxalate suppresses the oxidative burst from plants. Moreover, non-pathogenic OA- mutants, were unable to inhibit plant reactive oxygen species (ROS) induction. Thus a previously unrecognized function of oxalate is to suppress ROS generation and thereby compromise plant defense responses. ROS is not only involved with pathogenic (sclerotial) development in Sclerotinia, but is also critical for Sclerotinia’s ability to sucessfully colonize host plant tissue. Evidence will be presented indicating that ROS is used as a signal to trigger host plant–cell death encoded pathways, resulting in apoptosis entirely for the benefit of the fungus. Thus, there is an apparent dual ROS regulatory scheme occuring; Sclerotinia suppresses plant defense via OA, while also generating ROS and inducing plant cell death pathways via OA. Thus, Sclerotinia modulates the redox environment during disease development.
An intelligent primitive eukaryote: environmental sensing in Phycomyces.
Luis M. Corrochano. Departamento de Genetica, Universidad de Sevilla, Spain. firstname.lastname@example.org
The giant fruiting bodies, sporangiophores, of the Zygomycete Phycomyces blakesleeanus are characterized by their complex behavior. Phycomyces sporangiophores are aerial hyphae that grow out from the mycelium several centimeters into the air carrying at their top a small sphere filled with spores, the sporangium. Sporangiophore growth is governed by several environmental signals, including light, gravity, touch, wind, and the presence of nearby objects. These signals allow sporangiophore growth towards open air for efficient spore dispersal. Phototropism, sporangiophore growth towards light, has been investigated in detail. Phycomyces can react to a wide interval of light intensities, spanning ten orders of magnitude, and is able to respond to a small number of photons paralleling the sensitivity of the human eye. Two photoreceptors systems operating at different intensity ranges and a complex adaptation mechanism are used by Phycomyces to deal with the wide intensity range of light that can be perceived by the fungus. Mutants in mad genes have been isolated by their defect in phototropism. Some of the mad gene products are required for sporangiophore growth, but others are only required for phototropism and other light responses in this fungus. The discovery that the madA gene product is similar to Neurospora WC-1, a photoreceptor and transcription factor, suggests that the photoreception mechanism in Phycomyces may be related to that in Neurospora. A draft of the Phycomyces genome has been recently completed by the Joint Genome Institute (DoE, USA). The genome sequence will serve as a valuable resource to understand the molecular mechanisms regulating sporangiophore growth in Phycomyces.
Quorum sensing in bacterial-fungal interactions
Deborah Hogan. Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, NH 03755, USA
Candida albicans is responsible for a wide range of opportunistic fungal infections. Pathogenicity of C. albicans is largely dependent on its ability to undergo morphological interconversions between yeast and hyphal forms within the host. The control of these transitions is complex, and many environmental and chemical factors can influence the regulatory signaling cascades that govern C. albicans morphology. Recent studies have shown that C. albicans morphology is also influenced by bacterially-secreted products. In our studies, we have found that 3-oxo-C12 homoserine lactone, a signaling molecule produced by the Gram-negative bacterium Pseudomonas aeruginosa, is a potent inhibitor of hypha formation. Other molecules possess similar inhibitory activities, such as farnesol, a small molecule secreted by C. albicans itself. Using a combination of genetic and biochemical analyses, we have found that C12 compounds impact elements of the Ras1-cAMP signaling pathway, which positively regulates the yeast-to-hypha transition, by a mechanism separate from Ras1 activation. Furthermore, our data indicate that 3OC12HSL and farnesol impact the regulation of transcriptional responses other than those known to be involved in morphogenesis, suggesting that extracellular chemical signals may coordinately regulate multiple properties that are relevant in multicellular microbial communities.
Sex, time and evolution
Protein Folding, Environmental Contingency, and Evolution: Hsp90’s Role in Fungal Drug Resistance.
Leah E. Cowen1 and Susan L. Lindquist2. 1Department of Medical Genetics and Microbiology, University of Toronto, Toronto, ON M5S 1A8 Canada; email@example.com. 2Whitehead Institute for Biomedical Research, Cambridge, MA, 02142 USA.
Hsp90 is an essential molecular chaperone in all eukaryotes that regulates the form and function of many key signal transducers. Hsp90 has a capacity to buffer the expression of genetic variation and to reveal it in response to environmental stress. As a capacitor for the storage and release of genetic variation in plants and flies, and likely other organisms, Hsp90 may play a role in evolution. In cancer cells, Hsp90 may promote evolution in a different way. It chaperones mutated cell regulators that are prone to misfolding but have activated oncogenic potential; rather than buffering the effects of new mutations, it allows them to have immediate phenotypic effects.
We exploited the emergence of fungal drug resistance as the ideal system for directly testing Hsp90’s role in the evolution of new traits. In Saccharomyces cerevisiae and Candida albicans, we found that Hsp90 potentiated the evolution of drug resistance in a very different way. It enabled the phenotypic consequences of new mutations by chaperoning calcineurin, an unmutated regulator of cell signaling and key sensor of environmental stress. Hsp90’s role in drug resistance was to enable crucial responses to specific stresses, including changes in the composition of cell membranes and cell walls. Pharmacological inhibitors of Hsp90 function abrogated drug resistance of diverse fungal pathogens, including C. albicans and Aspergillus fumigatus. Hsp90 inhibitors are now in clinical trials as anticancer agents, suggesting new therapeutic strategies for fungal infections. In nature, Hsp90 function can be overwhelmed by global protein misfolding due to stress, including elevated temperatures. We found that febrile temperatures reached in humans challenged by infection abrogated drug resistance, suggesting a specific clinical benefit of fever. Here we further explore the therapeutic potential of Hsp90 inhibitors and the molecular mechanisms by which Hsp90 acts to couple environmental contingency to the evolution and assimilation of new traits.
Comparative genomics of Coccidioides species.
John W. Taylor1, Jason Stajich1, Thomas J. Sharpton1, Garry T. Cole2. 1University of California, Plant and Microbial Biology, Berkeley, CA, 94720-3102; 2University of Texas, Biology, San Antonio, TX, 78249
Coccidioides species are ascomycetous fungi found in hot, dry areas of the New World. They associate with endemic small mammals and have been the focus of research because they can cause life-threatening disease in otherwise healthy humans. Their virulence is reflected in their being the only fungi on the DHHS Select Agent list. Variation in gene sequences and microsatellite repeats have been used to recognize two species of Coccidioides, C. immitis and C. posadasii, and at least five populations therein. Through the efforts of the Coccidioides community, the Broad Institute and TIGR, there will be high-quality sequences available for at least two individuals from each of these five populations, and of a close relative, Uncinocarpus reesii. Already, genomes have been released for U. reesii and four Coccidioides individuals. This collection of genomes provides a rich resource for comparative genomics, conditional upon the quality of annotation. With annotated genomes for Coccidioides and Uncinocarpus, we now have searched for genes specific to Coccidioides or the outgroup, Uncinocarpus, for genes showing unusual rates of evolution, and for genes showing the effects of strong selection. Using microarrays, we have compared transcription profiling in two individuals from a single population and found that individual differences in gene transcription are significant. We will use transcription profiling of additional individuals to winnow the pool of genes showing significant changes in transcription in key points of the Coccidioides life cycle, especially the shift from saprobe to parasite. Genes that are identified as interesting from our comparative genomics, or from comparative transcription, or both, will become candidates for gene disruption or modification to test their role in pathogenicity using appropriate mammalian cell lines and a murine model of coccidioidomycosis.
Unwinding the Neurospora Circadian Clock
Deborah Bell-Pedersen, Department of Biology, Texas A&M University, firstname.lastname@example.org
Organisms from bacteria to humans use a circadian clock to control daily biochemical, physiological, and behavioral rhythms. We now have a firm understanding of the molecular oscillators that form the core of the circadian timing system. However, new data reveal that the circadian system is universally more complex than a single molecular feedback loop oscillator regulating all overt rhythmicity. Mounting evidence suggests that multiple oscillators comprise the Neurospora crassa clock. We have identified two N. crassa mutant strains that display circadian rhythms in the absence of the FRQ/WCC oscillator (FWO), considered to be the core of the fungal clock. These mutant strains uncover a novel circadian oscillator(s), which can function in cells that lack the FWO, but that is coupled to the FWO when the system is intact. Secondly, we have identified a new class of genes that cycle in mRNA accumulation in strains that lack FRQ. Characterization of one of these genes, ccg-16, has demonstrated that ccg-16 mRNA rhythms are generated by a FRQ-less oscillator (FLO) that, similar to the FWO, requires functional WC proteins for activity. These data raise the possibility that the FWO and the FLO are coupled through the shared WC proteins. Critical questions that can now be addressed in N. crassa, and that are relevant to the organization of all clocks, including the human clock, are; what are the roles of multiple circadian oscillators in cells, and how do these oscillators communicate with each other to coordinately control rhythmicity?
Sex: How Cryptococcus neoformans Controls Itself.
Brynne C. Stanton1, Mark W. Staudt1, and Christina M. Hull1,2. 1Department of Biomolecular Chemistry, 2Department of Medical Microbiology and Immunology, University of Wisconsin, 1300 University Avenue, Madison, WI 53706
Cryptococcus neoformans is an opportunistic fungal pathogen that affects primarily immunocompromised individuals. Infections with C. neoformans are thought to be caused by spores, which can result from sexual development. During sexual development haploid a and alpha cells fuse and initiate a process controlled by the homeodomain proteins Sxi1alpha and Sxi2a. Our experiments are focused on determining the molecular mechanisms by which Sxi1alpha and Sxi2a control sexual development and, ultimately, spore production. Our hypothesis is that Sxi1alpha and Sxi2a control sexual development by directly regulating the transcription of key targets to specify the dikaryotic state. To identify targets of Sxi1alpha and Sxi2a, we are taking several integrated approaches, including using C. neoformans microarrays to identify genes regulated by Sxi1alpha and Sxi2a. Targets of interest are being tested for direct regulation by Sxi1alpha and/or Sxi2a using chromatin immunoprecipitations. Promoter sequences from both bioinformatic and microarray targets are being tested using in vitro DNA binding experiments with purified Sxi1alpha and Sxi2a proteins. Preliminary DNA binding studies show that the homeodomain regions of Sxi1alpha and Sxi2a bind specifically to the promoter sequences of many microarray targets, including a putative homolog of the clampless (CLP1) gene. SXI1alpha and SXI2a are the first identified sexual cycle regulators in C. neoformans, and the characterization of the pathway they control will reveal how sexual development and spore formation occur in C. neoformans.
Origin and maintenance of virulence in human pathogenic fungi
Arturo Casadevall. Albert Einstein College of Medicine, Bronx, NY. email@example.com
Human pathogenic fungi are acquired from other humans (e.g. Candida spp.) or the environment (e.g. Cryptococcus neoformans, Histoplasma capsulatum, etc). Human fungal infections are common, yet disease is rare, implying that humans posses a natural high resistance to fungal diseases. In fact, only a very small minority of fungal species have the capacity for mammalian virulence. This raises the fundamental question of whether pathogenic fungal species are different, and if so, why. In general, fungi acquired from other humans are normally part of the commensal flora and diseases caused by these fungi usually reflect a disruption of the host-microbe equilibrium. For example, candidiasis is often associated with antibiotic use, immunosuppression, or compromise integument and mucosal membranes. In contrast, human diseases caused by fungi acquired from the environment can occur in both normal or immunocompromised hosts, and the occurrence of these diseases often involve impaired immunity or unusual exposures with large inocula. For example, the likelihood of cryptococcosis increases dramatically in individuals with advanced HIV infection and both histoplasmosis and coccidioidomycosis can occur in normal individuals following infection with large inocula. Hence, whereas the origin of virulence for commensal fungi may be understood in the context of a disrupted host-microbe interaction, the origin and maintenance of virulence for the environmental fungi is more difficult to explain. Environmentally acquired fungal pathogens are free-living microbes that are adapted to specific ecologic sites and have no obvious needs for mammalian infection to procreate or survive. Most perplexing is the finding that soil organisms such as C. neoformans and H. capsulatum manifest sophistical intracellular virulence strategies that appear almost tailor made for mammalian virulence, despite having no requirement for these qualities. In recent years, an explanation for the origin of virulence among environmentally acquired fungal pathogens was proposed based on the emergence of certain traits as part of selection by environmental predators such as small animals, amoebae, and slime mold. According to this view, the emergence of virulence among the environmentally acquired pathogenic fungi is accidental, and involves the serendipitous selection of microbial characteristics that can function in mammalian factors by biotic and physical factors in the environment. The concept of ‘accidental virulence’ provides a useful construct for evaluating the origin, function, and maintenance of virulence factors that allows great freedom in approaching fundamental questions of microbial virulence. With these conceptual tools at hand, it is possible to take a different view of fungal virulence and its conceivable role in past extinctions such as the demise of dinosaurs.
Return to the XXIV FGC main page
FGSC home page