scholarly journals Centromere deletion in Cryptococcus deuterogattii leads to neocentromere formation and chromosome fusions

2019 ◽  
Author(s):  
Klaas Schotanus ◽  
Joseph Heitman
Keyword(s):  
Growth Temperature ◽  
Fungal Pathogen ◽  
Species Complex ◽  
Wild Type ◽  
Genomic Changes ◽  
Growth Defects ◽  
Human Fungal Pathogen ◽  
Mutant Strains ◽  
Chromosome Fusions ◽  
Active Centromere

AbstractThe human fungal pathogen Cryptococcus deuterogattii is RNAi-deficient and lacks active transposons in its genome. C. deuterogattii has regional centromeres that contain only transposon relics. To investigate impact of centromere loss on the C. deuterogattii genome, either centromere 9 or 10 was deleted. Deletion of either centromere resulted in neocentromere formation and interestingly, the genes covered by these neocentromeres maintained wild-type expression levels. In contrast to cen9Δ mutants, cen10Δ mutant strains exhibited growth defects and were aneuploid for chromosome 10. At an elevated growth temperature (37°C), the cen10Δ chromosome was found to have undergone fusion with another native chromosome in some isolates and this fusion restored wild-type growth. Following chromosomal fusion, the neocentromere was inactivated, and the native centromere of the fused chromosome served as the active centromere. The neocentromere formation and chromosomal fusion events observed in this study in C. deuterogattii may be similar to events that triggered genomic changes within the Cryptococcus/Kwoniella species complex and may contribute to speciation throughout the eukaryotic domain.

scholarly journals Centromere deletion in Cryptococcus deuterogattii leads to neocentromere formation and chromosome fusions

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Klaas Schotanus ◽  
Joseph Heitman
Keyword(s):  
Fungal Pathogen ◽  
Species Complex ◽  
Wild Type ◽  
Genomic Changes ◽  
Growth Defects ◽  
Human Fungal Pathogen ◽  
Mutant Strains ◽  
Chromosome Fusions ◽  
The Impact ◽  
Active Centromere

The human fungal pathogen Cryptococcus deuterogattii is RNAi-deficient and lacks active transposons in its genome. C. deuterogattii has regional centromeres that contain only transposon relics. To investigate the impact of centromere loss on the C. deuterogattii genome, either centromere 9 or 10 was deleted. Deletion of either centromere resulted in neocentromere formation and interestingly, the genes covered by these neocentromeres maintained wild-type expression levels. In contrast to cen9∆ mutants, cen10∆ mutant strains exhibited growth defects and were aneuploid for chromosome 10. At an elevated growth temperature (37°C), the cen10∆ chromosome was found to have undergone fusion with another native chromosome in some isolates and this fusion restored wild-type growth. Following chromosomal fusion, the neocentromere was inactivated, and the native centromere of the fused chromosome served as the active centromere. The neocentromere formation and chromosomal fusion events observed in this study in C. deuterogattii may be similar to events that triggered genomic changes within the Cryptococcus/Kwoniella species complex and may contribute to speciation throughout the eukaryotic domain.

scholarly journals Epigenetic dynamics of centromeres and neocentromeres in Cryptococcus deuterogattii

PLoS Genetics ◽  
2021 ◽  
Vol 17 (8) ◽  
pp. e1009743
Author(s):  
Klaas Schotanus ◽  
Vikas Yadav ◽  
Joseph Heitman
Keyword(s):  
Dna Methylation ◽  
Transposable Element ◽  
Fungal Pathogen ◽  
Fusion Partner ◽  
Epigenetic Memory ◽  
Chromosome Fusion ◽  
Human Fungal Pathogen ◽  
Chromosome Fusions ◽  
The Impact ◽  
Active Centromere

Deletion of native centromeres in the human fungal pathogen Cryptococcus deuterogattii leads to neocentromere formation. Native centromeres span truncated transposable elements, while neocentromeres do not and instead span actively expressed genes. To explore the epigenetic organization of neocentromeres, we analyzed the distribution of the heterochromatic histone modification H3K9me2, 5mC DNA methylation and the euchromatin mark H3K4me2. Native centromeres are enriched for both H3K9me2 and 5mC DNA methylation marks and are devoid of H3K4me2, while neocentromeres do not exhibit any of these features. Neocentromeres in cen10Δ mutants are unstable and chromosome-chromosome fusions occur. After chromosome fusion, the neocentromere is inactivated and the native centromere of the chromosome fusion partner remains as the sole, active centromere. In the present study, the active centromere of a fused chromosome was deleted to investigate if epigenetic memory promoted the re-activation of the inactive neocentromere. Our results show that the inactive neocentromere is not re-activated and instead a novel neocentromere forms directly adjacent to the deleted centromere of the fused chromosome. To study the impact of transcription on centromere stability, the actively expressed URA5 gene was introduced into the CENP-A bound regions of a native centromere. The introduction of the URA5 gene led to a loss of CENP-A from the native centromere, and a neocentromere formed adjacent to the native centromere location. Remarkably, the inactive, native centromere remained enriched for heterochromatin, yet the integrated gene was expressed and devoid of H3K9me2. A cumulative analysis of multiple CENP-A distribution profiles revealed centromere drift in C. deuterogattii, a previously unreported phenomenon in fungi. The CENP-A-binding shifted within the ORF-free regions and showed a possible association with a truncated transposable element. Taken together, our findings reveal that neocentromeres in C. deuterogattii are highly unstable and are not marked with an epigenetic memory, distinguishing them from native centromeres.

scholarly journals Neocentromeres lack epigenetic memory and hallmarks of native centromeres in Cryptococcus deuterogattii

2020 ◽  
Author(s):  
Klaas Schotanus ◽  
Vikas Yadav ◽  
Joseph Heitman
Keyword(s):  
Dna Methylation ◽  
Transposable Element ◽  
Fungal Pathogen ◽  
Fusion Partner ◽  
Epigenetic Memory ◽  
Chromosome Fusion ◽  
Human Fungal Pathogen ◽  
Dna Modifications ◽  
Chromosome Fusions ◽  
Active Centromere

AbstractDeletion of native centromeres in the human fungal pathogen Cryptococcus deuterogattii leads to neocentromere formation. Native centromeres span truncated transposable elements, while neocentromeres span actively expressed genes. Neocentromeres in cen10Δ mutants are unstable and chromosome-chromosome fusions occur. After chromosome fusion, the neocentromere is silenced and the native centromere of the chromosome fusion partner remains as the sole active centromere. In the present study, the active centromere of a fused chromosome was deleted to investigate if epigenetic memory promoted re-activation of a silenced neocentromere. Our results show that the silenced neocentromere is not re-activated and instead a novel neocentromere forms directly adjacent to the deleted centromere of the fused chromosome. To explore the epigenetic organization of neocentromeres, we characterized the distribution of the heterochromatic histone modification H3K9me2 and 5mC DNA methylation. Native centromeres were enriched for both H3K9me2 and 5mC DNA methylation marks, while neocentromeres lacked these specific histone and DNA modifications. To study centromere dynamics, the actively expressed URA5 gene was introduced into a native centromere. Introduction of the URA5 gene led to loss of CENP-A from the native centromere, and a neocentromere formed directly adjacent to the native centromere location. Remarkably, the silenced native centromere remained enriched for heterochromatin, yet the integrated gene was expressed and devoid of H3K9me2. Analysis of multiple CENP-A distribution profiles revealed centromere drift in C. deuterogattii, a previously unknown phenomenon in fungi. The CENP-A-enriched region shifted within the pericentric regions, and a truncated transposable element in centromere 5 acted as a barrier between the CENP-A-associated regions of chromatin. Interestingly, this truncated transposable element was devoid of CENP-A binding or H3K9me2 modification and was instead marked by 5mC DNA methylation. Taken together, our findings reveal novel aspects about the epigenetic mechanisms that distinguish native centromeres and neocentromeres.

scholarly journals Pmt-Mediated O Mannosylation Stabilizes an Essential Component of the Secretory Apparatus, Sec20p, in Candida albicans

2004 ◽  
Vol 3 (5) ◽  
pp. 1164-1168 ◽  
Author(s):  
Yvonne Weber ◽  
Stephan K.-H. Prill ◽  
Joachim F. Ernst
Keyword(s):  
Endoplasmic Reticulum ◽  
Candida Albicans ◽  
Fungal Pathogen ◽  
Wild Type ◽  
Rapid Degradation ◽  
Vesicle Traffic ◽  
Human Fungal Pathogen ◽  
Low Levels ◽  
Lumenal Domain ◽  
Secretory Apparatus

ABSTRACT Sec20p is an essential endoplasmic reticulum (ER) membrane protein in yeasts, functioning as a tSNARE component in retrograde vesicle traffic. We show that Sec20p in the human fungal pathogen Candida albicans is extensively O mannosylated by protein mannosyltransferases (Pmt proteins). Surprisingly, Sec20p occurs at wild-type levels in a pmt6 mutant but at very low levels in pmt1 and pmt4 mutants and also after replacement of specific Ser/Thr residues in the lumenal domain of Sec20p. Pulse-chase experiments revealed rapid degradation of unmodified Sec20p (38.6 kDa) following its biosynthesis, while the stable O-glycosylated form (50 kDa) was not formed in a pmt1 mutant. These results suggest a novel function of O mannosylation in eukaryotes, in that modification by specific Pmt proteins will prevent degradation of ER-resident membrane proteins via ER-associated degradation or a proteasome-independent pathway.

scholarly journals Cryptococcus neoformans Gene Involved in Mammalian Pathogenesis Identified by a Caenorhabditis elegans Progeny-Based Approach

2005 ◽  
Vol 73 (12) ◽  
pp. 8219-8225 ◽  
Author(s):  
Robin J. Tang ◽  
Julia Breger ◽  
Alexander Idnurm ◽  
Kimberly J. Gerik ◽  
Jennifer K. Lodge ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Caenorhabditis Elegans ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Infection Model ◽  
Microbial Pathogenesis ◽  
Wild Type ◽  
C Elegans ◽  
Growth Defects ◽  
High Throughput Method

ABSTRACT Caenorhabditis elegans can serve as a substitute host for the study of microbial pathogenesis. We found that mutations in genes of the fungal pathogen Cryptococcus neoformans involved in mammalian virulence allow C. elegans to produce greater numbers of progeny than when exposed to wild-type fungus. We used this property to screen a library of C. neoformans mutants for strains that permit larger C. elegans brood sizes. In this screen, we identified a gene homologous to Saccharomyces cerevisiae ROM2. C. neoformans rom2 mutation resulted in a defect in mating and growth defects at elevated temperature or in the presence of cell wall or hyperosmolar stresses. An effect of the C. neoformans rom2 mutation in virulence was confirmed in a murine inhalation infection model. We propose that a screen for progeny-permissive mutants of microorganisms can serve as a high-throughput method for identifying novel loci related to mammalian pathogenesis.

scholarly journals Strains and Strategies for Large-Scale Gene Deletion Studies of the Diploid Human Fungal Pathogen Candida albicans

2005 ◽  
Vol 4 (2) ◽  
pp. 298-309 ◽  
Author(s):  
Suzanne M. Noble ◽  
Alexander D. Johnson
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Large Scale ◽  
Gene Deletion ◽  
Selectable Marker ◽  
Wild Type ◽  
Position Effects ◽  
Human Fungal Pathogen ◽  
Chromosome Position ◽  
Knockout Mutants

ABSTRACT Candida albicans is the most common human fungal pathogen and causes significant morbidity and mortality worldwide. Nevertheless, the basic principles of C. albicans pathogenesis remain poorly understood. Of central importance to the study of this organism is the ability to generate homozygous knockout mutants and to analyze them in a mammalian model of pathogenesis. C. albicans is diploid, and current strategies for gene deletion typically involve repeated use of the URA3 selectable marker. These procedures are often time-consuming and inefficient. Moreover, URA3 expression levels—which are susceptible to chromosome position effects—can themselves affect virulence, thereby complicating analysis of strains constructed with URA3 as a selectable marker. Here, we describe a set of newly developed reference strains (leu2Δ/leu2Δ, his1Δ/his1Δ; arg4Δ/arg4Δ, his1Δ/his1Δ; and arg4Δ/arg4Δ, leu2Δ/leu2Δ, his1Δ/his1Δ) that exhibit wild-type or nearly wild-type virulence in a mouse model. We also describe new disruption marker cassettes and a fusion PCR protocol that permit rapid and highly efficient generation of homozygous knockout mutations in the new C. albicans strains. We demonstrate these procedures for two well-studied genes, TUP1 and EFG1, as well as a novel gene, RBD1. These tools should permit large-scale genetic analysis of this important human pathogen.

scholarly journals SpRY Cas9 Can Utilize a Variety of Protospacer Adjacent Motif Site Sequences To Edit the Candida albicans Genome

mSphere ◽  
2021 ◽  
Vol 6 (3) ◽  
Author(s):  
Ben A. Evans ◽  
Douglas A. Bernstein
Keyword(s):  
Candida Albicans ◽  
Genome Editing ◽  
Fungal Pathogen ◽  
Wild Type ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Protospacer Adjacent Motif ◽  
Motif Site ◽  
Life Threatening ◽  
Genomic Locations

ABSTRACT Candida albicans is a human fungal pathogen capable of causing life-threatening infections. The ability to edit the C. albicans genome using CRISPR/Cas9 is an important tool investigators can leverage in their search for novel therapeutic targets. However, wild-type Cas9 requires an NGG protospacer adjacent motif (PAM), leaving many AT-rich regions of DNA inaccessible. A recently described near-PAMless CRISPR system that utilizes the SpRY Cas9 variant can target non-NGG PAM sequences. Using this system as a model, we developed C. albicans CRISPR/SpRY. We tested our system by mutating C. albicans ADE2 and show that CRISPR/SpRY can utilize non-NGG PAM sequences in C. albicans. Our CRISPR/SpRY system will allow researchers to efficiently modify C. albicans DNA that lacks NGG PAM sequences. IMPORTANCE Genetic modification of the human fungal pathogen Candida albicans allows us to better understand how fungi differ from humans at the molecular level and play essential roles in the development of therapeutics. CRISPR/Cas9-mediated genome editing systems can be used to introduce site-specific mutations to C. albicans. However, wild-type Cas9 is limited by the requirement of an NGG PAM site. CRISPR/SpRY targets a variety of different PAM sequences. We modified the C. albicans CRISPR/Cas9 system using the CRISPR/SpRY as a guide. We tested CRISPR/SpRY on C. albicans ADE2 and show that our SpRY system can facilitate genome editing independent of an NGG PAM sequence, thus allowing the investigator to target AT-rich sequences. Our system will potentially enable mutation of the 125 C. albicans genes which have been previously untargetable with CRISPR/Cas9. Additionally, our system will allow for precise targeting of many genomic locations that lack NGG PAM sites.

scholarly journals Adenylyl Cyclase Functions Downstream of the Gα Protein Gpa1 and Controls Mating and Pathogenicity of Cryptococcus neoformans

2002 ◽  
Vol 1 (1) ◽  
pp. 75-84 ◽  
Author(s):  
J. Andrew Alspaugh ◽  
Read Pukkila-Worley ◽  
Toshiaki Harashima ◽  
Lora M. Cavallo ◽  
Deanna Funnell ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Signal Transduction Pathway ◽  
Growth Defect ◽  
Wild Type ◽  
Camp Production ◽  
Mutant Strains ◽  
Opportunistic Fungal Pathogen ◽  
Mutant Phenotypes

ABSTRACT The signaling molecule cyclic AMP (cAMP) is a ubiquitous second messenger that enables cells to detect and respond to extracellular signals. cAMP is generated by the enzyme adenylyl cyclase, which is activated or inhibited by the Gα subunits of heterotrimeric G proteins in response to ligand-activated G-protein-coupled receptors. Here we identified the unique gene (CAC1) encoding adenylyl cyclase in the opportunistic fungal pathogen Cryptococcus neoformans. The CAC1 gene was disrupted by transformation and homologous recombination. In stark contrast to the situation for Saccharomyces cerevisiae, in which adenylyl cyclase is essential, C. neoformans cac1 mutant strains were viable and had no vegetative growth defect. Furthermore, cac1 mutants maintained the yeast-like morphology of wild-type cells, in contrast to the constitutively filamentous phenotype found upon the loss of adenylyl cyclase in another basidiomycete pathogen, Ustilago maydis. Like C. neoformans mutants lacking the Gα protein Gpa1, cac1 mutants were mating defective and failed to produce two inducible virulence factors: capsule and melanin. As a consequence, cac1 mutant strains were avirulent in animal models of cryptococcal meningitis. Reintroduction of the wild-type CAC1 gene or the addition of exogenous cAMP suppressed cac1 mutant phenotypes. Moreover, the overexpression of adenylyl cyclase restored mating and virulence factor production in gpa1 mutant strains. Physiological studies revealed that the Gα protein Gpa1 and adenylyl cyclase controlled cAMP production in response to glucose, and no cAMP was detectable in extracts from cac1 or gpa1 mutant strains. These findings provide direct evidence that Gpa1 and adenylyl cyclase function in a conserved signal transduction pathway controlling cAMP production, hyphal differentiation, and virulence of this human fungal pathogen.

scholarly journals The Siderophore Iron Transporter of Candida albicans (Sit1p/Arn1p) Mediates Uptake of Ferrichrome-Type Siderophores and Is Required for Epithelial Invasion

2002 ◽  
Vol 70 (9) ◽  
pp. 5246-5255 ◽  
Author(s):  
Petra Heymann ◽  
Michaela Gerads ◽  
Martin Schaller ◽  
Francoise Dromer ◽  
Günther Winkelmann ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Fungal Pathogen ◽  
Systemic Infection ◽  
Specific Process ◽  
Human Fungal Pathogen ◽  
Iron Salts ◽  
Mutant Strains ◽  
Epithelial Invasion ◽  
Triacetylfusarinine C

ABSTRACT The human fungal pathogen Candida albicans contains a close homologue of yeast siderophore transporters, designated Sit1p/Arn1p. We have characterized the function of SIT1 in C. albicans by constructing sit1 deletion strains and testing their virulence and ability to utilize a range of siderophores and other iron complexes. sit1 mutant strains are defective in the uptake of ferrichrome-type siderophores including ferricrocin, ferrichrysin, ferrirubin, coprogen, and triacetylfusarinine C. A mutation of FTR1 did not impair the use of these siderophores but did affect the uptake of ferrioxamines E and B, as well as of ferric citrate, indicating that their utilization was independent of Sit1p. Hemin was a source of iron for both sit1 and ftr1 mutants, suggesting a pathway of hemin uptake distinct from that of siderophores and iron salts. Heterologous expression of SIT1 in the yeast Saccharomyces cerevisiae confirmed the function of Sit1p as a transporter for ferrichrome-type siderophores. The sit1 mutant was defective in infection of a reconstituted human epithelium as a model for human oral mucosa, while the SIT1 strain was invasive. In contrast, both sit1 and SIT1 strains were equally virulent in the mouse model of systemic infection. These results suggest that siderophore uptake by Sit1p/Arn1p is required in a specific process of C. albicans infection, namely epithelial invasion and penetration, while in the blood or within organs other sources of iron, including heme, may be used.

scholarly journals Genetic analysis of sirtuin deacetylases in hyphal growth of Candida albicans

2021 ◽  
Vol 3 (12) ◽  
Author(s):  
Guolei Zhao ◽  
Laura Rusche
Keyword(s):  
Candida Albicans ◽  
Nuclear Localization ◽  
Fungal Pathogen ◽  
Environmental Changes ◽  
Hyphal Growth ◽  
Morphological Plasticity ◽  
Environmental Cues ◽  
Wild Type ◽  
Human Fungal Pathogen ◽  
Redundant Role

Candida albicans is a major human fungal pathogen that encounters varied host environments during infection. In response to environmental cues, C. albicans switches between ovoid yeast and elongated hyphal growth forms, and this morphological plasticity contributes to virulence. Environmental changes that alter the cell’s metabolic state could be sensed by sirtuins, which are NAD+-dependent deacetylases. Here we studied the roles of three sirtuin deacetylases, Sir2, Hst1, and Hst2, in hyphal growth of C. albicans. We made single, double, and triple sirtuin knockout strains and tested their ability to switch from yeast to hyphae. We found that true hyphae formation was significantly reduced by the deletion of SIR2 but not HST1 or HST2. Moreover, the expression of hyphal-specific genes HWP1, ALS3, and ECE1 decreased in the sir2Δ/Δ mutant compared to wild-type. This regulation of hyphae formation was dependent on the deacetylase activity of Sir2, as a point mutant lacking deacetylase activity had a similar defect in hyphae formation as the sir2Δ/Δ mutant. Finally, we found that Sir2 and Hst1 were localized to the nucleus, with Sir2 specifically focused in the nucleolus. This nuclear localization suggests a role for Sir2 and Hst1 in regulating gene expression. In contrast, Hst2 was localized to the cytoplasm. In conclusion, our results suggest that Sir2 plays a critical and non-redundant role in hyphal growth of C. albicans.

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