Past Awardees
| Name: | Ricky Festa |
| Lab: | Thiele |
| Conference: | 9th International Conference on Cryptococcus and Cryptococcosis |
| Date: | May 15 – 19, 2014 |
| Location: | Amsterdam, The Netherlands |
| Purpose: | To present poster and talk |
Abstract:
Copper is an important metal that is essential for numerous processes in eukaryotes, however, it is toxic at high levels. Broadly, my work is focused on understanding copper homeostasis in C. neoformans to help us understand what role the metal plays during infection. We have characterized the response of C. neoformans to both high and low copper, and determined that the Cuf1 transcription factor is responsible for the expression of genes dealing with both copper acquisition and detoxification. Furthermore, copper is used by the host to kill invading pathogens, making it an interesting target to perturb during infection.
The host immune system uses a variety of weapons in an attempt to eliminate infectious agents, such as production of reactive oxygen and nitrogen species as well as iron sequestration. In addition, evidence is mounting to support a role for elevated copper (Cu) during infection. In fact, under control of the Cu responsive transcription factor, Cuf1, C. neoformans mounts a strong defensive response to elevated Cu in the lung through expression of two Cu-specific metallothioneins. Without these metallothioneins, C. neoformans is unable to establish a successful infection. This response highlights Cu as an important player in the lung, and presents a possible target for anti-Cryptococcal therapies.
In collaboration with the Franz lab in the Chemistry Department here at Duke, we have developed a strategy that takes advantage of the toxic properties of copper using a small antifungal copper-binding molecule (8HQ) that is activated in the context of the oxidative burst by macrophages during infection. This protected form of the molecule (QBP) is aimed at preventing outright host copper imbalances. We have determined that when activated, QBP that is converted to 8HQ is fungicidal and the mode of action is most likely by bombarding the fungus with elevated copper levels. Furthermore, we have tested this concept in the context of in vitro infection as well as infection in the mouse lung, and have shown efficacy when QBP treatment was administered. The conditional activation of Cu ionophores by host innate immune cells manipulates and intensifies the hostile antimicrobial environment as a new approach to combat infectious disease.
| Name: | Shannon Esher |
| Lab: | Alspaugh |
| Conference: | 9th International Conference on Cryptococcus and Cryptococcosis |
| Date: | May 15 – 19, 2014 |
| Location: | Amsterdam, The Netherlands |
| Purpose: | To present poster and talk |
Abstract:
AIM-HII: A new method to rapidly identify Agrobacterium-mediated insertion sites in C. neoformans
Agrobacterium-mediated transformation (AMT) is a commonly used tool to generate mutations in a variety of plants and fungi, including Cryptococcus neoformans. A. tumefaciens is a soil bacterium which has the ability to deliver a portion of its own DNA to plants and fungi. The T-DNA integrates into the host genome, generally yielding a single, random insertion. Insertional mutagenesis libraries can therefore be generated and screened for phenotypes of interest, followed by identification of responsible mutation events. Traditionally, the locations of T-DNA insertion in C. neoformans have been identified by tedious PCR-based methods, creating a significant bottleneck and limiting the efficiency of the system.
We have developed a high-throughput genomic sequencing and analysis pipeline to facilitate the identification of insertions generated by AMT. Agrobacterium-mediated Insertional Mutagenesis High-throughput Insert Identification (AIM-HII) combines batch sampling, whole genome sequencing, and bioinformatics analysis tools to rapidly identify the locations of insertion. This method allows for the identification of 20-30 pooled mutants in the time it would take to identify 1-2 mutants using traditional methods. In brief, a library of 1500 C. neoformans AMT mutants was screened for phenotypes of interest. The genomic DNA of 28 mutants was harvested, pooled, and sequenced. Whole genome sequencing data was generated, and reads including the T-DNA insert sequence were extracted from the raw data files. Next, the T-DNA sequence was clipped off of these reads, yielding only C. neoformans genomic DNA sequence. Finally, this resulting genomic sequence was aligned to the C. neoformansgenome, and locations of insertion were identified. Of the 28 mutants identified, we have confirmed insertions in several previously identified genes, as well as a number of unannotated genes. Through our analysis we have also identified previously undescribed ATM insertion-induced events. These include possible chromosomal rearrangements, large deletions, and extensions and truncations of the integrated T-DNA. Many of these events would have been overlooked using the traditional PCR-based methods.
Our work introduces and utilizes a new tool that will greatly facilitate insertional mutagenesis screens in the future. Through this work we have identified a number of known and novel genes involved in C. neoformans virulence attributes. Work to further characterize the function of these genes is ongoing.
2013 Meritorious Research Travel Award Recipients
| Name: | Kyla Selvig |
| Lab: | Alspaugh |
| Conference: | Munro Lab |
| Date: | July 23 – August 12, 2013 |
| Location: | University of Aberdeen |
| Purpose: | To conduct experiments |
Abstract:
I am a fourth year PhD student in the Alspaugh lab. A major component of my thesis project is to analyze the role of the alkaline response transcription factor, Rim101, in altering the cell wall of Cryptococcus neoformans during infection. Without Rim101-regulated gene expression, C. neoformans becomes highly immunogenic and induces dramatic inflammation in both mouse and rabbit models of infection. Electron microscopy has shown that the C. neoformans cell wall structure is dramatically altered in a rim101Δ mutant. My goal is to identify the Rim101-mediated cell wall changed that prevent this immune recognition. To do this, I will compare the cell wall composition of WT and rim101Δ mutant cells. Specifically, I will use specialized chromatography and mass spectrometry to identify cell wall components that are altered on the rim101Δ mutant. I will be traveling to the University of Aberdeen to carry-out these experiments with Dr. Carol Munro.
| Name: | Pooja Strope |
| Lab: | Dietrich |
| Conference: | Yeast Genetics and Genomics |
| Date: | July 23 – August 12, 2013 |
| Location: | Cold Spring Harbor, NY |
| Purpose: | To present poster and talk |
Abstract:
Genetic diversity in 93 sequenced Saccharomyces cerevisiae strains
We have sequenced and assembled the genomes of 93 haploid or self-diploidized Saccharomyces cerevisiae strains with the goals of studying the sequence variation and testing genotype-phenotype associations to understand many biological processes. The sequence variation data along with high throughput quantitative phenotyping of the 93 strains (plus 8 previously sequenced strains) will be used to identify candidate quantitative trait genes by haplotype association mapping. These 93 strains were isolated from diverse geographical locations and origins, such as trees, soil, wine, and human infections. We sequenced the 93 genomes (Illumina Hiseq, 100 X 100 bp paired end reads, average insert size of 300 bp, 35x – 600x coverage), assembled the genomes de novo and used the reference genome to scaffold the contigs and annotate the sequences. We are currently depositing all the annotated chromosomes to GenBank so that the yeast community can readily access and utilize the data. We have successfully assembled and annotated the complete 2-micron plasmid and the mitochondrial sequences from all the 93 strains. Most of the mitochondrial genome sequences are highly conserved between strains but we saw extensive variation in the numbers and positions of introns in the COX1 and COBgenes. Variation was also seen in the 21S_rDNA, which in 39 strains contains the SCEI-encoding homing intron. Among the total ~5900 genes in the reference nuclear genome, ~4600 genes are highly similar (≥ 97% identity) in all strains, ~1100 genes are varied (< 97% identity, mostly introgressions) in one or more strains, and a total of 69 genes from the reference gene set are missing from one or more of the 93 strains. We have also identified ~200 genes in these strains that are not in the reference gene set. We are continuing to annotate these genomes and to identify the polymorphisms in these strains. Further, we will perform phenotypic analyses including environmental stress and drug stress experiments to then associate the phenotypes with the underlying genotypes of the strains.
| Name: | Deborah Springer |
| Lab: | Heitman |
| Conference: | 5th FEBS Advanced Lecture Course, Human Fungal Pathogens: Molecular Mechanisms of Host-Pathogen Interactions and Virulence |
| Date: | May 25 – 31, 2013 |
| Location: | La Colle sur Loup, France |
| Purpose: | To present poster and talk |
Abstract:
Cryptococcus gattii VGIII infections in HIV/AIDS patients in Southern California: Identification of local environmental source
The Cryptococcus pathogenic species complex, which includes the globally distributed human pathogens C. neoformans and C. gattii, is an important contributor to the fungal disease burden in immunocompromised and immunocompetent individuals worldwide. Cryptococcus is annually responsible for >1,000,000 infections, >620,000, deaths and 1/3 of all AIDS related deaths. The ongoing and expanding outbreaks of C. gattii in the western United States have stimulated increased clinical and environmental surveys that have advanced our understanding of the ecology and virulence of C. gattii. C. gattii is comprised of four distinct molecular types, without genetic exchange, suggesting these types represent distinct species. C. gattii molecular types VGII and VGIII are causing two distinct expanding outbreaks in the western US. VGII, with a predilection for apparently healthy hosts and high virulence is causing the outbreak on Vancouver Island that has expanded its host range to Washington, Oregon, and south into California. On the other hand, VGIII is responsible for an ongoing outbreak in HIV/AIDS patients in Southern California and the Southwestern US. Both VGII and VGIII have been isolated from clinical and veterinary samples obtained from patients and animals in the western US, suggesting the two molecular types occur in the environment. In contrast to the clinical presence and increased environmental surveys, only two environmental isolates of C. gattii have ever been reported from the Californian environment: CBS7750 (VGII, Eucalyptus) and WM161 (VGIII, Eucalyptus). The incongruence of the frequent clinical presence and uncommon environmental isolation suggest an unknown ecological reservoir for C. gattii in California. Here we report the isolation of C. gattii VGIII MATa and MATa isolates from environmental sources in Southern California. These isolates are fertile under laboratory conditions and produce abundant spores. Positive isolates were obtained from soil debris from tree species not previously recorded as hosts for C. gattii and from sites in close proximity to the residences of patients confirmed to have infections cause by C. gattii. MLST analysis provides additional evidence that these environmental isolates may be the source of human infections. Virulence studies in macrophages and animal models demonstrate that these environmental and clinical VGIII isolates are of similar virulence. Taken together, our studies reveal an environmental source and risk of C. gattii to HIV/AIDS patients with implications for the >1,000,000 infections annually for which the causative isolate is rarely assigned to a species. Thus, the global health burden caused by C. gattii could be considerably more substantial than currently appreciated.
| Name: | Lukasz Kozubowski |
| Lab: | Heitman |
| Conference: | 27th Fungal Genetics Conference |
| Date: | March 12 – 17, 2013 |
| Location: | Asilomar, Pacific Grove, CA |
| Purpose: | To present talk and poster |
Abstract:
Metazoan-like mitotic events in the basidiomycetous budding yeast Cryptococcus neoformans – a human fungal pathogen
Mitosis in ascomycetous budding yeasts is characterized by several features that are distinct from those of metazoans. In Saccharomyces cerevisiae, centromeres are always clustered in a single spot, the kinetochores are fully assembled for the majority of the cell cycle, and the nuclear envelope (NE) does not break down (closed mitosis). Currently it is not clear how these mechanisms evolved or whether these features are a universal characteristic hallmark of the budding mode of cellular division. Here we report an analysis of key mitotic events in the basidiomycetous human fungal pathogen Cryptococcus neoformans. The dynamics of microtubules, the kinetochore, NE and the nucleolus were analyzed by time-lapse microscopy using fluorescently tagged proteins. In striking contrast to ascomycetous budding yeast, centromeres in C. neoformans were not clustered in non-dividing cells. Prior to mitosis, centromeres underwent gradual clustering, eventually forming a single spot, which then migrated into the daughter cell where the chromosomal division occurred. One set of chromosomes migrated back to the mother cell and subsequent de-clustering of centromeres occurred in both cells. Analysis of individual components of the kinetochore indicated that kinetochores assemble in a step-wise manner in C. neoformans. While the inner kinetochore (Cse4, Mif2) was present throughout the entire cell cycle, the middle kinetochore (Mtw1) assembled prior to mitosis when centromeres underwent clustering, and this was then followed by assembly of the outer kinetochore (Dad1, Dad2). Formation of the outer kinetochore during mitosis, as observed in metaozoans that undergo an open mitosis, prompted us to examine the fate of the NE at various cell cycle stages. Several lines of evidence suggested that C. neoformans undergoes a semi-open mitosis. The nuclear pore marker GFP-Nup107, and a nucleolar marker GFP-Nop1 dispersed into the cytoplasm during metaphase, a nuclear membrane marker Ndc1 exhibited a localization pattern that also suggests a partial opening of the NE during mitosis. A semi-open mitosis was further confirmed by transmission electron microscopy. In summary, our data demonstrate that key mitotic events in C. neoformans are similar to that of metazoan cells. This study sheds new light on the evolution of mitosis during fungal speciation.
| Name: | Silvia Calo Varela |
| Lab: | Heitman |
| Conference: | 27th Fungal Genetics Conference |
| Date: | March 12 – 17, 2013 |
| Location: | Asilomar, Pacific Grove, CA |
| Purpose: | To present talk and poster |
Abstract:
RNAi-dependent epimutations evolve antifungal drug resistance in the zygomycete fungal pathogen Mucor
Microorganisms evolve via a panoply of mechanisms spanning aneuploidy, sexual/parasexual reproduction, mutators, Hsp90, and even prions. Mechanisms that may seem detrimental can be repurposed to generate diversity.
The pathogenic fungus Mucor circinelloides grows as a hyphae aerobically, but as a yeast in anaerobic conditions or in the presence of the immunosuppressive drug FK506. FKBP12 is a protein folding enzyme conserved throughout eukaryotes that interacts with FK506 and mediates antifungal activity of this drug. The FK506-FKBP12 complex inhibits the protein phosphatase calcineurin and thereby blocks hyphal growth of M. circinelloides. Continued exposure to FK506 yields resistant isolates, which exhibit hyphal growth emerging from the yeast colony. Some isolates harbor a variety of mutations in the fkbA gene that encodes FKBP12. However, other isolates harbor no mutations in the fkbA gene. These unusual epimutant isolates also revert frequently within several generations of vegetative growth in drug-free media and are restored to wild-type (yeast growth in the presence of FK506). Northern and Western analyses revealed a loss of fkbA mRNA and FKBP12 protein in the epimutants. High-throughput sequencing and Northern blot also detected sRNA generated from fkbA in the epimutant strains, revealing a new role for RNAi in the development of transient, reversible resistance to an antifungal drug treatment. RNAi could be triggered via dsRNA production from an overlap in the 3’ regions of the mRNA of fkbA and its neighboring gene patA, which encodes a putative polyamine transporter. Our results reveal a novel epigenetic RNAi-based epimutation mechanism controlling phenotypic plasticity in fungi.
2012 Meritorious Research Travel Award Recipients
| Name: | Ying-Lien Chen |
| Lab: | Heitman |
| Conference: | 11th ASM Conference on Candida and Candidiasis |
| Date: | March 29-April 2, 2012 |
| Location: | San Francisco, CA |
| Purpose: | To present talk and poster |
Abstract:
Convergent evolution of calcineurin pathway roles in thermotolerance and virulence in Candida glabrata
Candida glabrata is an emerging human fungal pathogen that is frequently drug- tolerant, resulting in difficulties in treatment and a higher mortality in immunocompromised patients. The calcium-activated protein phosphatase calcineurin plays critical roles in controlling drug tolerance, hyphal growth, and virulence in diverse fungal pathogens via distinct mechanisms involving survival in serum or growth at host temperature (37°C and higher). Here, we comprehensively studied the calcineurin signaling cascade in C. glabrata and found novel and uncharacterized functions of calcineurin and its downstream target Crz1 in governing thermotolerance, intracellular architecture, and pathogenesis in murine ocular, urinary tract, and systemic infections. This represents a second independent origin of a role for calcineurin in thermotolerant growth of a major human fungal pathogen, distinct from that which arose independently in Cryptococcus neoformans. Calcineurin also promotes survival of C. glabrata in serum via mechanisms distinct from C. albicans and thereby enables establishment of tissue colonization in a murine systemic infection model. To understand calcineurin signaling in detail, we performed global transcript profiling analysis and identified calcineurin- and Crz1-dependent genes in C. glabrata involved in cell wall biosynthesis, heat shock responses, and calcineurin function. Regulators of calcineurin (RCNs) are a novel family of calcineurin modifiers, and two members of this family were identified in C. glabrata: Rcn1 and Rcn2. Our studies demonstrate that Rcn2 expression is controlled by calcineurin and Crz1 to function as a feedback inhibitor of calcineurin in a circuit required for calcium tolerance in C. glabrata. In contrast, the calcineurin regulator Rcn1 activates calcineurin signaling. Interestingly, neither Rcn1 nor Rcn2 are required for virulence in a murine systemic infection model. Taken together, our findings show that calcineurin signaling plays critical roles in thermotolerance and virulence, and that Rcn1 andRcn2 have opposing functions in controlling calcineurin signaling in C. labrata.
| Name: | Makoto Inoue |
| Lab: | Shinohara |
| Conference: | FASEB Summer Research Conference – Osteopontin Biology |
| Date: | August 5-10, 2012 |
| Location: | Saxtons River, VT |
| Purpose: | To present talk and poster |
Abstract:
Inflammation triggered by acute infections must be properly controlled; otherwise, excessive inflammation is detrimental to hosts. However, the mechanism that regulates inflammation is still poorly understood. Here, we show that the CD40 signaling in macrophages suppresses TNF-mediated inflammation and protect mice from lethal effects by acute fungal infection by Cryptococcus neoformans and septic shock. Ligation of CD40 on macrophages to inhibit TNF production is achieved by T cell contact to macrophages, suggesting negative regulation of innate inflammation by adaptive immunity. To exert the inhibitory effect of CD40 signaling, intracellular osteopontin (OPN) is essential as an adaptor molecule. Such suppressive role of OPN was observed in immunocompetent mice going through acute inflammation, but OPN works to enhance inflammation and immunity in immunocompromised mice. Appropriate “switching” of OPN’s role from pro-inflammatory to anti-inflammatory, depending on the presence of adaptive immune cells, is therefore essential for hosts to cope with acute inflammation.
| Name: | Marianna Feretzaki |
| Lab: | Heitman |
| Conference: | Gordon Research Conference: Cellular and Molecular Fungal Biology |
| Date: | June 17-22, 2012 |
| Location: | Holderness, NH |
| Purpose: | To present poster |
Abstract:
Genetic Circuits that Govern Opposite- and Same-Sex Mating in Cryptococcus neoformans
Cryptococcus neoformans is a human fungal pathogen with a defined sexual cycle. Nutrient limited conditions and pheromones induce a dimorphic transition from unicellular yeast to multicellular hyphae and production of infectious spores. Sexual differentiation is initiated by fusion of cells of the same or
opposite mating type. Opposite- and same-sex mating are governed by shared components of the conserved pheromone sensing Cpk1 MAPK signaling transduction cascade and Mat2, the major transcriptional regulator of the pathway. However, the downstream targets of the pathway are largely unknown and homology-based methods have failed to yield downstream transcriptional regulators or other targets. In this study, we applied insertional mutagenesis via Agrobacterium tumefaciens trans-kingdom DNA delivery methods to identify mutants with a same-sex mating defect. In addition to factors known to be involved in sexual development (Crg1, Ste7, Mat2 and Znf2), three novel key regulators of sexual development were identified by our screen: Znf3, Spo11 and Ubc5. Znf3, a novel zinc finger transcription factor, orchestrates hyphal development during opposite- and same-sex mating and plays a key role in cell-cell fusion. Phenotypic and transcriptional analyses indicate that Znf3 governs mating by enhancing pheromone production. Virulence assays in murine models provide evidence that Znf3 is also required for virulence. Spo11 and Ubc5 are important factors of sporulation. Spo11 is a DNA topoisomerase IV, an ortholog that delivers DNA double strand breaks that provoke meiotic recombination, and Ubc5 a ubiquitin-conjugating enzyme. Both share significant identity with Saccharomyces cerevisiae Spo11 and Ubc5, key regulators of sporulation. We show that Spo11 and Ubc5 are required for sporulation during opposite- and same-sex mating in C. neoformans and are important factors for meiosis. These studies illustrate the power of unbiased genetic screens to reveal novel circuits that operate sexual reproduction.
| Name: | Kyla Selvig |
| Lab: | Alspaugh |
| Conference: | Molecular Mycology: Current Approaches to Fungal Pathogenesis |
| Date: | August 1-17, 2012 |
| Location: | Marine Biological Laboratory, Woods Hole, MA |
| Purpose: | To present talk |
Abstract:
Cryptococcus neoformans is an opportunistic fungal pathogen that causes significant disease in HIV/AIDS patients. This pathogen adapted the conserved fungal alkaline response pathway (Rim101 pathway) to sense and respond specifically to the environment within its infected host, allowing it to cause disease. The conserved function of the Rim101 pathway is to activate the Rim101 transcription factor to regulate changes in gene expression required for adaptation to alkaline conditions. C. neoformans requires this pathway to properly modify its cell wall in order to allow for the formation of its protective polysaccharide capsule. The Rim pathway normally follows a well characterized signaling cascade to activate Rim101. However in C. neoformans, this pathway has both novel connections with the cAMP/PKA pathway and adaptations to respond to host signals other than pH. Despite this unique activation, at least a portion of the classical Rim101 pathway appears to be intact. The Rim101 transcription factor is activated by a signaling complex docked on the ESCRT machinery located on endocytic vesicles. C. neoformans contains orthologs to all of the Rim101 signaling proteins that make up this endocytic signaling complex. One of these proteins, Rim20, has previously been shown to be required for Rim101 activation. I have disrupted the orthologs encoding the other two components of this complex, Rim13 and PalC. Both show phenotypes consistent with defects in Rim101 activation, such as defects in capsule formation and growth on high salt concentrations. This evidence shows that in addition to the PKA pathway, Rim101 activation requires conserved members of this pathway. C. neoformans lacks orthologs to the most upstream members of the pathway, the Rim21 receptor and the Rim8 arrestin. Therefore, elucidating these components of the C. neoformans pathway is the next step in identifying the host-specific signals that activate Rim101 in this pathogen.
| Name: | Sheng Sun |
| Lab: | Heitman |
| Conference: | Molecular Mycology: Current Approaches to Fungal Pathogenesis |
| Date: | August 1-17, 2012 |
| Location: | Marine Biological Laboratory, Woods Hole, MA |
| Purpose: | To present talk and poster |
Abstract:
Sexual reproduction in fungi is governed by a specialized genomic region called the mating-type locus (MAT). The human fungal pathogenic and basidiomycetous yeast Cryptococcus neoformans has evolved a bipolar mating system (a, α) in which the MATlocus is unusually large (>100 kb), and encodes >20 genes including homeodomain (HD) and pheromone/receptor (P/R) genes. To understand how this unique bipolar mating system evolved, we investigated MAT in the closely related species Tsuchiyaea wingfieldii and Cryptococcus amylolentus and discovered two physically unlinked loci encoding the HD and P/R genes. Interestingly, the HD (B) locus sex-specific region is restricted (~2 kb) and encodes two linked and divergently oriented homeodomain genes in contrast to the solo HD genes (SXI1α, SXI2a) of C. neoformans and C. gattii. The P/R (A) locus contains the pheromone and pheromone receptor genes but has expanded considerably compared to other outgroup species (C. heveanensis) and is linked to many of the genes also found in the MAT locus of the pathogenic Cryptococcus species. Our discovery of a heterothallic sexual cycle for C. amylolentus allowed us to establish the biological roles of the sex-determining regions. Matings between two strains of opposite mating-types (A1B1 x A2B2) produced dikaryotic hyphae with fused clamp connections, basidia, and basidiospores. Genotyping progeny using markers linked and unlinked to MAT revealed that meiosis and uniparental mitochondrial inheritance occur during the sexual cycle of C. amylolentus. The sexual cycle is tetrapolar and produces fertile progeny of four mating-types (A1B1, A1B2, A2B1, and A2B2), but a high proportion of progeny are infertile, and fertility is biased towards one parental mating-type (A1B1). Our studies reveal insights into the plasticity and transitions in both mechanisms of sex determination (bipolar vs. tetrapolar) and sexual reproduction (outcrossing vs. inbreeding) with implications for similar evolutionary transitions and processes in fungi, plants, and animals.
| Name: | Chen Ding |
| Lab: | Thiele |
| Conference: | Trace element Metabolism in biology and Medicine |
| Date: | June 10-15, 2012 |
| Location: | Steamboat Springs, CO |
| Purpose: | To present talk and poster |
Abstract:
Copper homeostasis as a virulence factor in systemic infection by the human fungal pathogen Cryptococcus neoformans
Cryptococcus neoformans is a human fungal pathogen that is the causative agent of lethal meningitis in immunocompromised hosts as a consequence of HIV/AIDS, cancer chemotherapy, diabetes, maintenance on immunosuppressants due to organ transplants or other pathologies. Copper (Cu) is an important metal to C. neoformans, as it is involved in processes that include, among others, respiration, iron acquisition, melanin formation, mating and superoxide dismutase activity. Previous reports suggest that the C. neoformans Cu-responsive transcription factor, Cuf1, contributes to virulence but the precise mechanisms for this are unclear. Cuf1 homologues in other fungi are known to activate expression of genes involved in Cu acquisition. We identified all of the genes induced either by Cu deficiency or excess in C. neoformans, including the previously known Ctr4 Cu importer, a new Cu importer, Ctr1, two metallothionein genes (MT1, MT2) and others. Surprisingly, both Cu inducible and Cu repressible genes are dependent on Cuf1 for their metalloregulation. To decipher which Cuf1 target genes contribute to virulence we generated Cuf1 target gene deletions and assayed survival to intra-nasal administration in mouse models of infection. We generated reporters of C. neoformans intracellular Cu status (high Cu or Cu deficiency) and are using live animal imaging to ascertain what Cu environment C. neoformans senses when it is first encountered by alveolar macrophages. Taken together, these studies will elucidate the contributions of the C. neoformans Cu homeostasis machinery to virulence and will provide a set of tools to investigate how C. neoformans and mammalian hosts do battle over Cu.
| Name: | Pooja Strope |
| Lab: | Dietrich |
| Conference: | Yeast Genetics & Molecular Biology Meeting |
| Date: | July 30-August 5, 2012 |
| Location: | Princeton, NJ |
| Purpose: | To present talk and poster |
Abstract:
We are undertaking the sequencing and assembly of 93 Saccharomyces cerevisiae strains to study the sequence variation among a total of 101 S. cerevisiae strains (with eight previously sequenced strains), and to test the genotype-phenotype associations to understand many biological processes. One goal of this project is to fully sequence 93 haploid genomes to identify the variation among the 101 strains. The sequence variation data along with high throughput quantitative phenotyping of the 101 strains will be used to map quantitative trait loci/genes by haplotype association mapping. These strains were isolated from diverse origins and geographical locations including trees, soil, wine, and human infections. We are assembling the genomes using a de novo assembly method. Our preliminary analysis has identified a combined 65 genes from the reference S288C genome missing from one or more of the 101 strains. We have also identified ~200 genes in these strains that are not in S288C. These genes include a putative arginase from Yersinia frederiksenii (bacteria), a methyltransferase from Tetrahymena thermophile (protozoa), and a hypothetical protein from Meyerozyma guilliermondii (fungi). We also looked at the distribution of the repetitive gene families, the five classes of Ty elements. We found the number of Ty to vary in these strains. Ty1 found in 27 copies in S288C is present in between 1 and 30 copies per strain, and Ty2 found in 13 copies in S288C is present in between 2 and 20 copies per strain. Several strains lacked Ty3, Ty4, or Ty5 entirely. No evidence of novel Ty elements was found in the initial assembly. It is well documented that Ty transposition can knock out genes and alter gene expression suggesting that some phenotypic variation in these strains may be Ty mediated. Further analysis on these sequences including deletion studies will shed light on the functions of these Ty regions. Preliminary data analysis shows that there is a significant amount of variation to be found in these strains. We are continuing to assemble and annotate these genomes to identify the polymorphisms in these strains. Further, we will perform phenotypic analyses including environmental stress and drug stress experiments to then associate the phenotypes with the underlying genotypes of the strains.
2011 Meritorious Research Travel Award Recipients
| Name: | Richard Festa |
| Lab: | Thiele |
| Conference: | Molecular Mycology: Current Approaches to Fungal Pathogenesis |
| Date: | August 3-19, 2011 |
| Location: | Marine Biological Laboratory, Woods Hole, MA |
| Purpose: | Training course |
Abstract:
The C. neoformans Cuf1 copper metalloregulatory transcription factor
Copper is crucial trace element for many cellular processes in virtually all forms of life, while excess levels of free copper can lead to toxicity through the destruction of Fe-S clusters and via oxidative stress generated through Fenton chemistry. Additionally, copper is important for C. neoformans as a cofactor for proteins implicated in virulence such as laccase and Cu/Zn SOD, supporting the importance of Cu during infection. Since copper has previously been implicated in C. neoformans virulence, it must be able to acquire copper from the host, while maintaining sub-toxic concentrations. Copper homeostasis in C. neoformans is maintained via the copper importers, Ctr1 and Ctr4, dedicated chaperones that route intracellular copper and two metallothioneins that bind and detoxify copper. Unlike other yeasts such as S. cerevisiae and S. pombe that encode two copper responsive transcription factors that activate transcription of distinct sets of genes in response to high copper or copper deficiency, C. neoformans encodes only one known copper sensing transcription factor, the cryptococcal copper-dependent transcription factor 1, Cuf1. My future work will be aimed at identifying the direct targets of Cuf1 to determine what role they play in copper homeostasis and virulence. Furthermore, I hope to uncover the mechanism by which Cuf1 regulates genes in a high- and low-copper dependent manner. The Woods Hole Molecular Mycology course will aid my progression towards these goals by gaining hands on experience with different cutting edge molecular and genetic techniques, as well as relevant animal models of infection. On a broad scale, I will further the understanding of the biology of copper in C. neoformans and how this fungal pathogen interacts with mammalian hosts over copper.
| Name: | Ana Litvintseva |
| Lab: | Mitchell |
| Conference: | 8th International Conference on Cryptococcus and Crytpococcosis |
| Date: | May 1 – 5, 2011 |
| Location: | Charleston, SC |
| Purpose: | To present talk and poster |
Abstract:
Population Structure and Virulence of VNI Strains of Cryptococcus neoformans
To determine any correlation between genotypes and phenotypes of the dominant VNI population Cryptococcus neoformans, environmental and clinical isolates of with identical MLST/AFLP genotypes were compared for virulence in mice. Regardless of genotypes, most clinical strains were highly lethal, and most environmental strains failed to cause disease, although viable yeasts were recovered from the lungs of healthy mice 60 days postinfection. Subsequently, each strain was phagocytized by J774.1A murine macrophage-like cells, and after incubation at 37ºC for 24 hours, intracellular growth was significantly higher among the virulent clinical strains than the nonlethal environmental strains. These results suggest that the initial interaction between cryptococci and alveolar macrophages is a crucial determinant of whether the infection progresses.
To investigate the genetic determinants of these differences in virulence, we developed two approaches — gene expression analysis and experimental evolution. In the first approach, genome-wide transcription patterns of clinical and environmental strains with identical genotypes were compared after exposing strains to conditions that (i) simulated the initial stage of infection, phagocytosis by alveolar murine macrophage-like cells, or (ii) terminal disease, incubation in human cerebrospinal fluid (CSF). These experiments revealed that strains with identical genotypes exhibited dramatically different patterns of gene expression following phagocytosis by macrophage-like cells, and the expression profiles correlated with murine virulence. Avirulent strains had control levels of gene expression, but lethal strains displayed significant up-regulation of transporters and genes involved in neutralizing oxidative stress and DNA damage. Conversely, after incubation in CSF, none of the strains replicated well, and gene expression patterns were similar, regardless of virulence. These results support the hypothesis that virulent strains adapt to growth within the host in the early pulmonary stage of infection. In the second approach, in vitro experiments tested whether non-lethal strains became more virulent after incubation under pathogenic conditions. Two non-lethal environmental strains with different genotypes were cultured at 37ºC in media that mimicked host-like conditions of starvation and oxidative stress. After 200 generations, both strains had adapted to low glucose, nitrogen, iron and copper as well as oxidative stress. Compared to the original strains, the in vitro growth rates of the evolved strains in the restrictive media was significantly higher. These strains also exhibited greater proliferation in macrophage-like cells. These data support the hypothesis that growth within the host may lead to the selection and proliferation of clones with enhanced virulence.
| Name: | Praveen R. Juvvadi |
| Lab: | Steinbach |
| Conference: | The 26th Fungal Genetics Conference at Asilomar |
| Date: | March 15 – 20, 2011 |
| Location: | Asilomar, CA |
| Purpose: | To present talk and poster |
Abstract:
Mutational analysis of Aspergillus fumigatus calcineurin A reveals critical domains required for its function in vivo and targeting to the hyphal septum
Calcineurin, a conserved calmodulin-dependent protein phosphatase, is a heterodimer consisting of the catalytic (CnaA) and the regulatory (CnaB) subunits. It is known to play key roles in virulence, growth and stress responses of pathogenic fungi. Critically understanding the calcineurin pathways and identifying the residues indispensible for calcineurin activity in vivo will pave way for devising new drug targets for combating Aspergillosis. We previously reported that CnaA localizes at the hyphal septum implicating its importance for septum formation and conidiophore development. By constructing the ΔcnaA ΔcnaB double mutant strain of A. fumigatus and utilizing the dual fluorescent labeling technique we provide evidence on colocalization of CnaA-GFP and mcherry-CnaB fusion proteins at the hyphal septum. Surprisingly, while the CnaB-GFP fusion protein mislocalized to the cytosol in the absence of of cnaA, cnaA still localized to the hyphal septum in the absence of cnaB. By site-directed point mutagenesis of several residues in the catalytic domain, CnaB binding helix, and the calmodulin binding domain of CnaA, we identify critical domains essential for its function in vivo apart from the absolute requirement of complexing with CnaB for its function at the hyphal septum.
| Name: | Andrii Gryganskyi |
| Lab: | Vilgalys |
| Conference: | The 26th Fungal Genetics Conference at Asilomar |
| Date: | March 15 – 20, 2011 |
| Location: | Asilomar, CA |
| Purpose: | To present talk and poster |
Abstract:
Sex gene in Rhizopus oryzae
The Rhizopus oryzae species complex comprises a group of zygomycetous fungi that are common, cosmopolitan saprotrophs. Some strains are used for production of tempeh and other Asian fermented foods but they can also act as opportunistic human pathogens. Although R. oryzae reportedly has a heterothallic (+/-) mating system, most strains are incapable of sexual reproduction, and the genetic structure of its mating locus has not been characterized. Here we report on the mating behavior and genetic structure of the mating locus from 50 isolates of the R. oryzae complex, including the recently sequenced genome of isolate RA 99-880 (=FGSC 9543). All strains from the R. oryzae complex have a mating locus that is similar in overall organization to the mucoralean fungi Phycomyces blakesleeanus and Mucor circinelloides. In all of these fungi, the minus allele features a high mobility gene (HMG) flanked by an RNA helicase gene and a triose phosphate transporter gene. Within the R. oryzae complex, the plus mating allele includes a large inserted region that codes for a BTB/POZ domain gene. Phylogenetic analysis of HMGs, ITS and 28s rDNA, RPB2, mtSSU and LDH genes in R. oryzae isolates identified two distinct groups that correspond to previously described sibling species (R. oryzae sensu stricto and R. delemar). Laboratory mating assays identified a phenotypic difference between these species. When mixed with the isolates of the opposite mating type, isolates of R. oryzae s. s. produced zygospores, whereas isolates of R. delemar remained sterile. This physiological difference confirms the genetic isolation between the two cryptic species.
| Name: | Xuying Wang |
| Lab: | Heitman |
| Conference: | The 26th Fungal Genetics Conference at Asilomar |
| Date: | March 15 – 20, 2011 |
| Location: | Asilomar, CA |
| Purpose: | To present talk and poster |
Abstract:
Sex induced silencing defends the genome of Cryptococcus neoformans via RNAi
Co-suppression is a silencing phenomenon triggered by the introduction of homologous DNA sequences into the genomes of organisms as diverse as plants, fungi, flies, and nematodes. Here we report sex induced silencing (SIS), which is triggered by tandem integration of a transgene array in the human fungal pathogen Cryptococcus neoformans. A SXI2a-URA5 transgene array was found to be post-transcriptionally silenced during sexual reproduction. More than half of the progeny that inherited the SXI2a-URA5 transgene became uracil auxotrophic due to silencing of the URA5 gene. In vegetative mitotic growth, silencing of this transgene array occurred at ~250-fold lower frequency, indicating that silencing is induced during the sexual cycle. Central components of the RNAi pathway, including genes encoding Argonaute, Dicer, and an RNA-dependent RNA polymerase, are all required for both meiotic and mitotic transgene silencing. URA5-derived ~22-nt small RNAs accumulated in the silenced isolates, suggesting that SIS is mediated by RNAi via sequence-specific small RNAs. Through deep sequencing of the small RNA population in C. neoformans, we also identified abundant small RNAs mapping to repetitive transposable elements, and these small RNAs were absent in rdp1 mutant strains. Furthermore, a group of retrotransposons was highly expressed during mating of rdp1 mutant strains and an increased transposition/mutation rate was detected in their progeny, indicating that the RNAi pathway squelches transposon activity during the sexual cycle. Interestingly, Ago1, Dcr1, Dcr2, and Rdp1 are translationally induced in mating cells, and Ago1, Dcr1, and Dcr2 localize to P-bodies whereas Rdp1 appears to be nuclear, providing mechanistic insights into the elevated silencing efficiency during sexual reproduction. We hypothesize that the SIS RNAi pathway operates to defend the genome during sexual development.
2010 Meritorious Research Travel Award Recipients
| Name: | Marianna Feretzaki |
| Lab: | Heitman |
| Conference: | Molecular Mycology: Current Approaches to Fungal Pathogenesis |
| Date: | August 4 – 20, 2010 |
| Location: | Marine Biological Laboratory, Woods Hole, MA |
| Purpose: | Training course |
Abstract:
Two heterologous model hosts reveal a link between virulence and hyphal growth in Cryptococcus neoformans
Cryptococcus neoformans is a human fungal pathogen that causes infections of the central nervous system in immunocompromised and immunocompetent hosts. C. neoformans exists in two varieties with different virulence and geographical distribution. C. neoformans var. grubii (serotype A) and var. neoformans (serotype D) are predominantly opportunistic pathogens with worldwide distribution and with var. grubii being the most clinically prevalent. Human infection is thought to be caused by inhalation of spores from the environment. The small diameter of spores facilitates alveolar penetration, and following germination the yeast disseminates hematogenously. In the laboratory, nutrient limitation and pheromones induce a dimorphic transition from yeast to hyphae via sexual reproduction. The traditional sexual cycle involves α and a opposite mating type cells. Same sex mating is a modified sexual cycle involving mating between cells of the same mating type, usually α. Both forms of sexual reproduction lead to hyphal growth and infectious spores production that affect the distribution and virulence of cryptococcal strains. However same sex mating occurs in nature but not yet in laboratory for the highly virulent strains of serotype A, yet is a common feature of attenuated serotype D isolates. In our study we found that the hyperfilamentous serotype D strain XL280 is hypervirulent compared to other serotype D strains. The murine model of intranasal instillation proved to be an excellent model host to determine the pathogenicity of our strains. Galleria mellonella larvae were also used as a heterologous model host. In both heterologous model hosts the hyperfilamentous XL280 strain exhibited a hypervirulent behavior. In current studies we focus on the interactions between fungal cells and the vertebrate host and the impact it may have on filamentation and virulence of the strain. In addition we are performing linkage analysis to determine whether a single or multiple loci contribute to the observed difference in virulence. Our studies may provide evidence linking filamentation and virulence and provide new insights on virulence and its evolution.
| Name: | Ying-Lien Chen |
| Lab: | Heitman |
| Conference: | 10th Conference on Candida and Candidiasis |
| Date: | March 22 – 26, 2010 |
| Location: | Miami, Florida |
| Purpose: | To present talk and poster |
Abstract:
Calcineurin is essential for virulence and thermal stress tolerance in pathogenic Candida species
Non-albicans Candida species are emerging pathogens in bloodstream infections of immunocompromised patients. Whole genome sequences of major Candida species recently became available, and our aim is to elucidate the roles of calcineurin in virulence in C. dubliniensis, C. glabrata, and C. parapsilosis. Calcineurin is a calcium activated phosphatase that forms a heterodimer consisting of the catalytic A (Cna1) and regulatory B subunits (Cnb1). This heterodimer is required for calcineurin enzyme activity and is highly conserved between yeasts and mammals. In response to stress, the transcription factor Crz1 is dephosphorylated by calcineurin and then migrates into the nucleus to induce expression of genes encoding cell wall biosynthetic enzymes and ion pumps in yeast. In C. dubliniensis, a sister species to C. albicans, we have disrupted the CNA1, CNB1, and CRZ1 genes and demonstrated that these mutants exhibit similar but not identical phenotypes compared to C. albicans. The cna1/cna1 and cnb1/cnb1 mutants are hypersensitive to membrane perturbation (SDS) and antifungal drugs (fluconazole and caspofungin), while the crz1/crz1 mutantdisplays modest sensitivity to SDS and resistance to caspofungin as compared to the wild-type. Although filamentous growth of the calcineurin and crz1/crz1mutants was not affected, calcineurin A and B and Crz1 were all found to be essential for virulence of C. dubliniensis in a murine model of candidiasis. We extended this study to C. glabrata and surprisingly found that the calcineurin pathway is required for C. glabrata growth at high temperature. Similar to previous studies of the divergent human fungal pathogen Cryptococcus neoformans, the C. glabrata cna1 and cnb1 mutants were inviable at 40°C while crz1 mutants exhibited a growth defect at 40°C. Exposing 19 C. glabrata clinical isolates to the calcineurin inhibitors FK506 and CsA demonstrated that three isolates (15.8%) require calcineurin for growth at 40°C. In the remaining CsA-FK506 insensitive isolates, either calcineurin is not required or drug levels are insufficient for inhibition. C. glabrata cna1 and cnb1 mutants were hypersensitive to SDS and fluconazole while crz1 mutants only modestly sensitive to SDS. Unlike C. dubliniensis, C. glabrata calcineurin and crz1 mutants were not sensitive to Ca2+ ions. In contrast to the temperature sensitive phenotype displayed by C. glabrata and C. neoformans calcineurin mutants, C. parapsilosiscna1/cna1 and cnb1/cnb1 mutants exhibited cold-sensitivity at 24°C in addition to drug susceptibility and membrane dysfunction. Taken together, our data provide evidence that calcineurin plays a general role in promoting fungal growth under thermal stress in C. glabrata and C. parapsilosis, and is required for virulence in C. dubliniensis. These advances illustrate how calcineurin inhibition could be harnessed to target emerging pathogenic Candida species.
| Name: | Wenjun Li |
| Lab: | Heitman |
| Conference: | 2nd ASM Conference on Dimorphic Fungal Pathogens |
| Date: | March 22 – 26, 2010 |
| Location: | Miami, Florida |
| Purpose: | To present talk and poster |
Abstract:
Evolution of mating type (MAT) locus in the dermatophytes and dimorphic fungi
Sexual reproduction contributes to genetic diversity and virulence of pathogens. In fungi, sexual reproduction is governed by a specialized genomic region, the mating type (MAT) locus, whose gene identity, organization, and complexity are diverse. Dimorphic fungi and dermatophytes are closely related human pathogens. While sexual reproduction has been reported in dimorphic fungi and dermatophytes, the genetic basis and evolution of the MAT locus remain to be elucidated. Herein, based on genomic searches, primer walking, comparative genomics, population genetic studies, and phylogenetic analyses, we characterized and compared the MAT locus of dimorphic fungi and dermatophytes. Both groups are heterothallic with one of two mating types: plus (+) and minus (-) isolates harbor an alpha-box gene (MAT1-1) or an HMG domain gene (MAT1-2) in the MAT locus, respectively, flanked by the APN2 and SLA2 genes. In the dimorphic fungi MAT has expanded to ~6 to 9 kb by gene acquisition compared to the dermatophyte MAT locus (~3.5 kb). In Blastomyces dermatitidis, the size of MAT may be even further increased; transposons are present around or in the MAT locus and the distance between the APN2 and SLA2 genes is ~50 kb in 3 minus (-) strains. We developed specific primers to determine the mating type of Trichophyton rubrum, the most common cause of fungal infections in humans. 80 of 87 (92%) T. rubrum isolates are plus (+) with a MAT1-1 locus; the other seven isolates are minus (-) with the MAT1-2 locus. How genetic recombination occurs in the T. rubrum population with a high prevalence of the plus (+) mating type is not known, and may involve mating between strains of opposite mating types as well as same sex mating, as occurs in Cryptococcus neoformans and Candida albicans. Population genetic studies of T. rubrum applying MLST and mating tests are in progress. Highly variable genetic markers are also being implemented to test if recombination occurs in the progeny of strains produced by opposite or same sex mating.
2009 Meritorious Research Travel Award Recipients
| Name: | Cecelia Shertz |
| Lab: | Heitman |
| Conference: | Molecular Mycology: Current Approaches to Fungal Pathogenesis |
| Date: | August 5 – 21, 2009 |
| Location: | Marine Biological Laboratory, Woods Hole, MA |
| Purpose: | Training course |
Abstract:
Genetic basis of sexual development in two species of the chytridiomycota: the model water mold Allomyces macrogynus and the amphibian pathogen Batrachochytrium dendrobatidis
Sexual reproduction likely arose in a unicellular, uniflagellate ancestor to the metazoa and fungal lineages. Studying the mechanisms of sexual reproduction in the most basal fungal species provides crucial information as to the origins, evolution and maintenance of sexual reproduction in the fungal kingdom and beyond. I am investigating the genetic basis of sexual development in two species of the chytridiomycota: the model water mold Allomyces macrogynus and the amphibian pathogen Batrachochytrium dendrobatidis, a fungus responsible for the worldwide global amphibian decline in recent years. By identifying transcription factors such as high mobility group-domain proteins, alpha-box containing proteins and homeodomain-containing proteins through genomic analysis, we are able to locate genomic regions potentially responsible for sexual reproduction to investigate further.
| Name: | Elizabeth Ballou |
| Lab: | Alspaugh |
| Conference: | Molecular Mycology: Current Approaches to Fungal Pathogenesis |
| Date: | March 17 – 22, 2009 |
| Location: | Marine Biological Laboratory, Woods Hole, MA |
| Purpose: | Training course |
Abstract:
Two CDC42 paralogs modulate thermotolerance and morphogenesis in the human fungal pathogen Cryptococcus neoformans
In the human host environment, the fungal pathogen Cryptococcus neoformans must maintain tightly regulated morphogenesis events under stress conditions, including host physiological temperature (37°C). We have previously shown that RAS1 is a major regulator of polarity and morphogenesis in C. neoformans, and that this cascade is mediated via Rho-GTPases. Cdc42 is a highly conserved Rho family GTPase, which in fungi as diverse as Saccharomyces cerevisiae and Penicillium marneffei is essential to the establishment and maintenance of polarity. Unlike other eukaryotes, the C. neoformans genome encodes two highly related CDC42 paralogs. We therefore aim to understand the individual and shared contributions of these Rho-GTPases to polarized growth, budding, and pathogenicity. Using gene disruption, we have generated knockouts for both paralogs singly and in combination and have show that both paralogs are functional and required for growth at 37°C. Morphological differences, as well as differences in organization of the actin cytoskeleton and the septin collar of budding cells, between wildtype, single, and double mutants suggest that both paralogs play distinct and overlapping roles in polarity and growth at host physiological conditions.
2008 Meritorious Research Travel Award Recipients
| Name: | Teresa O’Meara |
| Lab: | Alspaugh |
| Conference: | 25th Annual Fungal Genetics Conference |
| Date: | March 17 – 22, 2009 |
| Location: | Asilomar, CA |
| Purpose: | To present talk and poster |
Abstract:
Cryptococcus neoformans Rim101/PacC homolog is involved in capsule and iron regulation
Pathogenic microorganisms such as Cryptococcus neoformans must be able to adapt to hostile host conditions including iron starvation. The host uses iron sequestration as part of the innate immune system, and iron deprivation is a powerful host-specific signal that results in the induction of microbial virulence-associated phenotypes such as capsule. Understanding the interplay of host iron availability and microbial iron acquisition has recently been recognized as an essential component of microbial pathogenesis. When C. neoformans enters a host, it responds to specific host signals and activates capsule production using the cAMP/PKA pathway. This capsule is required for evading host defenses and surviving in the host. In this study, we have identified a specific C. neoformans transcription factor, Rim101, that regulates both iron homeostasis and capsule production likely in response to PKA stimulus. The data support the hypothesis that PKA-activated transcription factors offer a previously unknown mechanism for interpreting host-specific signals through the cAMP/PKA pathway. These transcription factors are essential in responding appropriately to host-specific signals and regulating capsule production and iron homeostasis.
| Name: | Elizabeth Pekarek |
| Lab: | Vilgalys |
| Conference: | Mycological Society of America Meeting |
| Date: | July 25 – 29, 2009 |
| Location: | Snowbird, UT |
| Purpose: | To present talk and poster |
Abstract:
Multiple-species sexual compatibility and the role of reinforcement in Pleurotus pulmonarius from New Zealand
Our understanding of the genetic mechanisms of speciation is hindered by our inability to cross good biological species and produce fertile offspring. Exceptions to this rule provide a useful tool in aiding
our understanding of how species form reproductive barriers. To this end, I have characterized a collection of Pleurotus pulmonarius from New Zealand (NZP) that shows mating compatibility with three other well-documented biological species of Pleurotus. I report here that the NZP strain appears to have completely lost the prezygotic isolating barriers reported in P. pulmonarius strains from Europe, Asia, and North America. Postzygotic isolation, in the form of inviable and infertile offspring, is incomplete, but remains a strong
factor for these interspecies hybrids. This dichotomy between pre- and postzygotic isolation along with the biogeography of the species involved, suggests a reversal of the effects of reinforcement on this isolated New Zealand strain._______________________________________________________