scholarly journals A Rac Homolog Functions Downstream of Ras1 To Control Hyphal Differentiation and High-Temperature Growth in the Pathogenic Fungus Cryptococcus neoformans

2005 ◽  
Vol 4 (6) ◽  
pp. 1066-1078 ◽  
Author(s):  
Marcelo A. Vallim ◽  
Connie B. Nichols ◽  
Larissa Fernandes ◽  
Kari L. Cramer ◽  
J. Andrew Alspaugh
Keyword(s):  
High Temperature ◽  
Cryptococcus Neoformans ◽  
Pathogenic Fungus ◽  
Ras Proteins ◽  
Gene Encoding ◽  
Temperature Growth ◽  
Multicopy Suppressor ◽  
Human Fungal Pathogen ◽  
Hyphal Formation ◽  
Mutant Phenotypes

ABSTRACT The Cryptococcus neoformans Ras1 protein serves as a central regulator for several signaling pathways. Ras1 controls the induction of the mating pheromone response cascade as well as a distinct signaling pathway that allows this pathogenic fungus to grow at human physiological temperature. To characterize elements of the Ras1-dependent high-temperature growth pathway, we performed a multicopy suppressor screen, identifying genes whose overexpression allows the ras1 mutant to grow at 37°C. Using this genetic technique, we identified a C. neoformans gene encoding a Rac homolog that suppresses multiple ras1 mutant phenotypes. Deletion of the RAC1 gene does not affect high-temperature growth. However, a rac1 mutant strain demonstrates a profound defect in haploid filamentation as well as attenuated mating. In a yeast two-hybrid assay, Rac1 physically interacts with the PAK kinase Ste20, which similarly regulates hyphal formation in this fungus. Similar to Rac1, overexpression of the STE20α gene also restores high-temperature growth to the ras1 mutant. These results support a model in which the small G protein Rac1 acts downstream of Ras proteins and coordinately with Ste20 to control high-temperature growth and cellular differentiation in this human fungal pathogen.

scholarly journals Calcium- and Calcineurin-Independent Roles for Calmodulin in Cryptococcus neoformans Morphogenesis and High-Temperature Growth

2005 ◽  
Vol 4 (6) ◽  
pp. 1079-1087 ◽  
Author(s):  
Peter R. Kraus ◽  
Connie B. Nichols ◽  
Joseph Heitman
Keyword(s):  
High Temperature ◽  
Cryptococcus Neoformans ◽  
Insertional Mutagenesis ◽  
Pathogenic Fungus ◽  
Critical Factor ◽  
Growth Defect ◽  
Insertion Mutant ◽  
Gene Encoding ◽  
Temperature Growth ◽  
New Genes

ABSTRACT The function of calcium as a signaling molecule is conserved in eukaryotes from fungi to humans. Previous studies have identified the calcium-activated phosphatase calcineurin as a critical factor in governing growth of the human pathogenic fungus Cryptococcus neoformans at mammalian body temperature. Here, we employed insertional mutagenesis to identify new genes required for growth at 37°C. One insertion mutant, cam1-ts, that displayed a growth defect at 37°C and hypersensitivity to the calcineurin inhibitor FK506 at 25°C was isolated. Both phenotypes were linked to the dominant marker in genetic crosses, and molecular analysis revealed that the insertion occurred in the 3′ untranslated region of the gene encoding the calcineurin activator calmodulin (CAM1) and impairs growth at 37°C by significantly reducing calmodulin mRNA abundance. The CAM1 gene was demonstrated to be essential using genetic analysis of a CAM1/cam1Δ diploid strain. In the absence of calcineurin function, the cam1-ts mutant displayed a severe morphological defect with impaired bud formation. Expression of a calmodulin-independent calcineurin mutant did not suppress the growth defect of the cam1-ts mutant at 37°C, indicating that calmodulin promotes growth at high temperature via calcineurin-dependent and -independent pathways. In addition, a Ca2+-binding-defective allele of CAM1 complemented the 37°C growth defect, FK506 hypersensitivity, and morphogenesis defect of the cam1-ts mutant. Our findings reveal that calmodulin performs Ca2+- and calcineurin-independent and -dependent roles in controlling C. neoformans morphogenesis and high-temperature growth.

scholarly journals Identification of Cryptococcus neoformans Temperature-Regulated Genes with a Genomic-DNA Microarray

2004 ◽  
Vol 3 (5) ◽  
pp. 1249-1260 ◽  
Author(s):  
Peter R. Kraus ◽  
Marie-Josée Boily ◽  
Steven S. Giles ◽  
Jason E. Stajich ◽  
Andria Allen ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Temperature ◽  
Cryptococcus Neoformans ◽  
Stress Responses ◽  
Pathogenic Fungus ◽  
High Growth ◽  
Synthesis Inhibitor ◽  
Gene Encoding ◽  
Temperature Growth ◽  
Partial Genome

ABSTRACT The ability to survive and proliferate at 37°C is an essential virulence attribute of pathogenic microorganisms. A partial-genome microarray was used to profile gene expression in the human-pathogenic fungus Cryptococcus neoformans during growth at 37°C. Genes with orthologs involved in stress responses were induced during growth at 37°C, suggesting that a conserved transcriptional program is used by C. neoformans to alter gene expression during stressful conditions. A gene encoding the transcription factor homolog Mga2 was induced at 37°C and found to be important for high-temperature growth. Genes encoding fatty acid biosynthetic enzymes were identified as potential targets of Mga2, suggesting that membrane remodeling is an important component of adaptation to high growth temperatures. mga2Δ mutants were extremely sensitive to the ergosterol synthesis inhibitor fluconazole, indicating a coordination of the synthesis of membrane component precursors. Unexpectedly, genes involved in amino acid and pyrimidine biosynthesis were repressed at 37°C, but components of these pathways were found to be required for high-temperature growth. Our findings demonstrate the utility of even partial-genome microarrays for delineating regulatory cascades that contribute to microbial pathogenesis.

scholarly journals Impact of Protein Palmitoylation on the Virulence Potential of Cryptococcus neoformans

2015 ◽  
Vol 14 (7) ◽  
pp. 626-635 ◽  
Author(s):  
Connie B. Nichols ◽  
Kyla S. Ost ◽  
Dayton P. Grogan ◽  
Kaila Pianalto ◽  
Shirin Hasan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
High Temperature ◽  
Cryptococcus Neoformans ◽  
Genome Database ◽  
Temperature Growth ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Virulence Potential ◽  
Protein Palmitoylation ◽  
And Function

ABSTRACT The localization and specialized function of Ras-like proteins are largely determined by posttranslational processing events. In a highly regulated process, palmitoyl groups may be added to C-terminal cysteine residues, targeting these proteins to specific membranes. In the human fungal pathogen Cryptococcus neoformans , Ras1 protein palmitoylation is essential for growth at high temperature but is dispensable for sexual differentiation. Ras1 palmitoylation is also required for localization of this protein on the plasma membrane. Together, these results support a model in which specific Ras functions are mediated from different subcellular locations. We therefore hypothesize that proteins that activate Ras1 or mediate Ras1 localization to the plasma membrane will be important for C. neoformans pathogenesis. To further characterize the Ras1 signaling cascade mediating high-temperature growth, we have identified a family of protein S -acyltransferases (PATs), enzymes that mediate palmitoylation, in the C. neoformans genome database. Deletion strains for each candidate gene were generated by homogenous recombination, and each mutant strain was assessed for Ras1-mediated phenotypes, including high-temperature growth, morphogenesis, and sexual development. We found that full Ras1 palmitoylation and function required one particular PAT, Pfa4, and deletion of the PFA4 gene in C. neoformans resulted in altered Ras1 localization to membranes, impaired growth at 37°C, and reduced virulence.

scholarly journals Structural analysis of fungal pathogenicity-related casein kinase α subunit, Cka1, in the human fungal pathogen Cryptococcus neoformans

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Belinda X. Ong ◽  
Youngki Yoo ◽  
Myeong Gil Han ◽  
Jun Bae Park ◽  
Myung Kyung Choi ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Pathogenic Fungus ◽  
Kinase Assay ◽  
Α Subunit ◽  
Dynamic Architecture ◽  
Human Fungal Pathogen ◽  
Life Threatening ◽  
The Difference ◽  
Anti Fungal

Abstract CK2α is a constitutively active and highly conserved serine/threonine protein kinase that is involved in the regulation of key cellular metabolic pathways and associated with a variety of tumours and cancers. The most well-known CK2α inhibitor is the human clinical trial candidate CX-4945, which has recently shown to exhibit not only anti-cancer, but also anti-fungal properties. This prompted us to work on the CK2α orthologue, Cka1, from the pathogenic fungus Cryptococcus neoformans, which causes life-threatening systemic cryptococcosis and meningoencephalitis mainly in immunocompromised individuals. At present, treatment of cryptococcosis remains a challenge due to limited anti-cryptococcal therapeutic strategies. Hence, expanding therapeutic options for the treatment of the disease is highly clinically relevant. Herein, we report the structures of Cka1-AMPPNP-Mg2+ (2.40 Å) and Cka1-CX-4945 (2.09 Å). Structural comparisons of Cka1-AMPPNP-Mg2+ with other orthologues revealed the dynamic architecture of the N-lobe across species. This may explain for the difference in binding affinities and deviations in protein-inhibitor interactions between Cka1-CX-4945 and human CK2α-CX-4945. Supporting it, in vitro kinase assay demonstrated that CX-4945 inhibited human CK2α much more efficiently than Cka1. Our results provide structural insights into the design of more selective inhibitors against Cka1.

scholarly journals Septin Function inCandida albicansMorphogenesis

2002 ◽  
Vol 13 (8) ◽  
pp. 2732-2746 ◽  
Author(s):  
Amy J. Warenda ◽  
James B. Konopka
Keyword(s):  
Fluorescent Protein ◽  
Pathogenic Fungus ◽  
Wild Type ◽  
Animal Cells ◽  
Hyphal Morphogenesis ◽  
Hyphal Formation ◽  
Green Fluorescent ◽  
Mutant Phenotypes ◽  
Germ Tubes ◽  
Selection Of

The septin proteins function in the formation of septa, mating projections, and spores in Saccharomyces cerevisiae, as well as in cell division and other processes in animal cells. Candida albicans septins were examined in this study for their roles in morphogenesis of this multimorphic, opportunistically pathogenic fungus, which can range from round budding yeast to elongated hyphae. C. albicans green fluorescent protein labeled septin proteins localized to a tight ring at the bud and pseudohyphae necks and as a more diffuse array in emerging germ tubes of hyphae. Deletion analysis demonstrated that the C. albicans homologs of the S. cerevisiae CDC3 andCDC12 septins are essential for viability. In contrast, the C. albicans cdc10Δ and cdc11Δ mutants were viable but displayed conditional defects in cytokinesis, localization of cell wall chitin, and bud morphology. The mutant phenotypes were not identical, however, indicating that these septins carry out distinct functions. The viable septin mutants could be stimulated to undergo hyphal morphogenesis but formed hyphae with abnormal curvature, and they differed from wild type in the selection of sites for subsequent rounds of hyphal formation. Thecdc11Δ mutants were also defective for invasive growth when embedded in agar. These results further extend the known roles of the septins by demonstrating that they are essential for the proper morphogenesis of C. albicans during both budding and filamentous growth.

scholarly journals Recapitulation of the Sexual Cycle of the Primary Fungal Pathogen Cryptococcus neoformans var. gattii: Implications for an Outbreak on Vancouver Island, Canada

2003 ◽  
Vol 2 (5) ◽  
pp. 1036-1045 ◽  
Author(s):  
James A. Fraser ◽  
Ryan L. Subaran ◽  
Connie B. Nichols ◽  
Joseph Heitman
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Large Scale ◽  
Vancouver Island ◽  
Sexual Cycle ◽  
Gene Encoding ◽  
Tropical Regions ◽  
Human Fungal Pathogen ◽  
Large Scale Analysis ◽  
Serotype B

ABSTRACT Cryptococcus neoformans is a human fungal pathogen that exists as three distinct varieties or sibling species: the predominantly opportunistic pathogens C. neoformans var. neoformans (serotype D) and C. neoformans var. grubii (serotype A) and the primary pathogen C. neoformans var. gattii (serotypes B and C). While serotypes A and D are cosmopolitan, serotypes B and C are typically restricted to tropical regions. However, serotype B isolates of C. neoformans var. gattii have recently caused an outbreak on Vancouver Island in Canada, highlighting the threat of this fungus and its capacity to infect immunocompetent individuals. Here we report a large-scale analysis of the mating abilities of serotype B and C isolates from diverse sources and identify unusual strains that mate robustly and are suitable for further genetic analysis. Unlike most isolates, which are of both the a and α mating types but are predominantly sterile, the majority of the Vancouver outbreak strains are exclusively of the α mating type and the majority are fertile. In an effort to enhance mating of these isolates, we identified and disrupted the CRG1 gene encoding the GTPase-activating protein involved in attenuating pheromone response. crg1 mutations dramatically increased mating efficiency and enabled mating with otherwise sterile isolates. Our studies provide a genetic and molecular foundation for further studies of this primary pathogen and reveal that the Vancouver Island outbreak may be attributable to a recent recombination event.

Fitness distribution and transgressive segregation across 40 environments in a hybrid progeny population of the human-pathogenic yeast Cryptococcus neoformans

Genome ◽  
2008 ◽  
Vol 51 (4) ◽  
pp. 272-281 ◽  
Author(s):  
Morvarid Shahid ◽  
Susan Han ◽  
Heather Yoell ◽  
Jianping Xu
Keyword(s):  
Cryptococcus Neoformans ◽  
Biological Effects ◽  
Pathogenic Fungus ◽  
Transgressive Segregation ◽  
Variable Number ◽  
Hybrid Progeny ◽  
Pathogenic Yeast ◽  
Human Fungal Pathogen ◽  
Different Temperatures ◽  
Human Pathogenic Fungus

The opportunistic human fungal pathogen Cryptococcus neoformans includes two varieties, C. neoformans var. grubii and C. neoformans var. neoformans, which correspond to serotypes A and D, respectively. Recent population genetic studies revealed that multiple natural hybridizations have occurred recently between these two divergent lineages. However, the biological effects of such hybridizations are little understood. In this study, we used colony size as a proxy for vegetative fitness to examine the phenotypic effects of hybridization between these two lineages in a laboratory cross. Two genetically diverged parental strains that differed in their growth at different temperatures and on different media as well as in their susceptibility to the common antifungal drug fluconazole were chosen. A total of 269 progeny were obtained and their vegetative growth was determined in 40 environments that differed in nutrients, temperature, and fluconazole concentration. Our analyses indicated little evidence for outbreeding depression or heterosis in the average vegetative fitness of the hybrid progeny population. The progeny, each of the three environmental variables, and their two-way, three-way, and four-way interactions all contributed significantly to the overall vegetative fitness variation. Interestingly, a variable number of progeny displayed evidence of transgressive segregation in vegetative fitness among the tested environments. Our study suggests that hybridization could play a significant role in the phenotypic evolution of this important human-pathogenic fungus.

scholarly journals Transcriptional Network of Multiple Capsule and Melanin Genes Governed by the Cryptococcus neoformans Cyclic AMP Cascade

2005 ◽  
Vol 4 (1) ◽  
pp. 190-201 ◽  
Author(s):  
Read Pukkila-Worley ◽  
Quincy D. Gerrald ◽  
Peter R. Kraus ◽  
Marie-Josée Boily ◽  
Matthew J. Davis ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Cryptococcus Neoformans ◽  
Glucose Deprivation ◽  
Melanin Production ◽  
Gene Encoding ◽  
Human Fungal Pathogen ◽  
Camp Pathway ◽  
Capsule Production ◽  
Capsule Assembly ◽  
Laccase Enzyme

ABSTRACT Cryptococcus neoformans is an opportunistic human fungal pathogen that elaborates several virulence attributes, including a polysaccharide capsule and melanin pigments. A conserved Gα protein/cyclic AMP (cAMP) pathway controls melanin and capsule production. To identify targets of this pathway, we used an expression profiling approach to define genes that are transcriptionally regulated by the Gα protein Gpa1. This approach revealed that Gpa1 transcriptionally regulates multiple genes involved in capsule assembly and identified two additional genes with a marked dependence on Gpa1 for transcription. The first is the LAC1 gene, encoding the laccase enzyme that catalyzes a rate-limiting step in diphenol oxidation and melanin production. The second gene identified (LAC2) is adjacent to the LAC1 gene and encodes a second laccase that shares 75% nucleotide identity with LAC1. Similar to the LAC1 gene, LAC2 is induced in response to glucose deprivation. However, LAC2 basal transcript levels are much lower than those for LAC1. Accordingly, a lac2 mutation results in only a modest delay in melanin formation. LAC2 overexpression suppresses the melanin defects of gpa1 and lac1 mutants and partially restores virulence of these strains. These studies provide mechanistic insights into the regulation of capsule and melanin production by the C. neoformans cAMP pathway and demonstrate that multiple laccases contribute to C. neoformans melanin production and pathogenesis.

scholarly journals The monothiol glutaredoxin Grx4 interacts with the Cryptococcus iron regulator Cirl and regulates iron homeostasis and virulence in the Cryptococcus neoformans

2018 ◽  
Author(s):  
Rodgoun Attarian ◽  
Guanggan Hu ◽  
Melissa Caza ◽  
Eddy Sanchez-Leon ◽  
Daniel Croll ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Iron Homeostasis ◽  
Pathogenic Fungus ◽  
Virulence Determinants ◽  
Rna Seq ◽  
Nuclear Retention ◽  
Poor Growth ◽  
Binding Partner ◽  
Iron Deprivation ◽  
Mutant Phenotypes

AbstractThe acquisition of iron and the maintenance of iron homeostasis are important aspects of the virulence in the pathogenic fungus Cryptococcus neoformans. In this study, we identified the monothiol glutaredoxin Grx4 as a binding partner of Cir1, a master regulator of iron-responsive genes and virulence factor elaboration in C. neoformans. Monothiol glutaredoxins are important regulators of iron homeostasis because of their conserved roles in [2Fe-2S] cluster sensing and trafficking. We confirmed that Grx4 binds Cir1 and demonstrated that iron repletion promotes the relocalization of Grx4 from the nucleus to the cytoplasm. Nuclear retention is partially dependent on Cir1 and also influenced by treatment with the proteasome inhibitor bortezomib. Cir1 remains in the nucleus in both iron replete and iron limiting conditions. We also found that a grx4Δ mutant displayed iron-related phenotypes similar to those of a cir1Δ mutant, including poor growth upon iron deprivation. Importantly, a grx4Δ mutant was avirulent in mice, a phenotype consistent with observed defects in the key virulence determinants, capsule and melanin, and poor growth at 37°C. A comparative transcriptome analysis of a grx4Δ mutant and the WT strain in low iron and iron-replete conditions confirmed a central role for Grx4 in iron homeostasis. Dysregulation of iron-related metabolism was consistent with grx4Δ mutant phenotypes related to oxidative stress, mitochondrial function, and DNA repair. Overall, the phenotypes of the grx4Δ mutant and the RNA-Seq analysis support the hypothesis that Grx4 functions as a sensor of iron levels, in part through an interaction with Cir1, to extensively regulate iron homeostasis and contribute to virulence.

scholarly journals The Cryptococcus neoformans Rho-GDP Dissociation Inhibitor Mediates Intracellular Survival and Virulence

2008 ◽  
Vol 76 (12) ◽  
pp. 5729-5737 ◽  
Author(s):  
Michael S. Price ◽  
Connie B. Nichols ◽  
J. Andrew Alspaugh
Keyword(s):  
Cryptococcus Neoformans ◽  
Cell Morphology ◽  
Culture Medium ◽  
Intracellular Survival ◽  
Murine Models ◽  
Gene Encoding ◽  
Cellular Processes ◽  
Growth Defects ◽  
Human Fungal Pathogen ◽  
Gdp Dissociation Inhibitor

ABSTRACT Rho-GDP dissociation inhibitors (Rho-GDI) are repressors of Rho-type monomeric GTPases that control fundamental cellular processes, such as cytoskeletal arrangement, vesicle trafficking, and polarized growth. We identified and altered the expression of the gene encoding a Rho-GDI homolog in the human fungal pathogen Cryptococcus neoformans and investigated its impact on pathogenicity in animal models of cryptococcosis. Consistent with its predicted function to inhibit and sequester Rho-type GTPases, overexpression of RDI1 results in cytosolic localization of Cdc42. Likely as a result of this finding, RDI1-overexpressing strains exhibited altered morphology compared to that of the wild type, with apparent defects in maintaining proper cell polarity and cytokinesis. RDI1 deletion resulted in increased vacuole size in tissue culture medium and aberrant cell morphology at neutral pH. Maintenance of normal cell morphology is vital for C. neoformans pathogenicity. Accordingly, the rdi1Δ mutant strain also showed reduced intracellular survival in macrophages and severe attenuation of virulence in two murine models of cryptococcosis. This reduction in virulence of the rdi1Δ mutant occurs in the absence of major growth defects in rich medium and with classical virulence-associated phenotypes.

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