Faculty Opinions recommendation of Efficient implementation of RNA interference in the pathogenic yeast Cryptococcus neoformans.

Cryptococcus neoformans is a major pathogen of humans throughout the world. Using commercial monoclonal antibodies to capsular epitopes, strains of C. neoformans manifest five serotypes: A, B, C, D and AD. Previous studies demonstrated significant divergence among serotypes A, B, C and D, which are typically haploid. In contrast, most strains of serotype AD are diploid or aneuploid and result from recent hybridization between strains of serotypes A and D. Whether serotypes A, B, C and D represent strictly asexual lineages is not known. Using comparative genealogical analyses of two genes, the authors investigated whether recombination occurred among strains within serotypes A and D. For each of 14 serotype AD strains, a portion (642 bp) of the orotidine monophosphate pyrophosphorylase (URA5) gene was cloned and sequenced. Each of these 14 strains contained two different alleles and sequences for both alleles were obtained. The URA5 gene genealogy was compared to that derived from the laccase (LAC) gene, which was reported recently for the same 14 strains. For both genes, each of the 14 serotype AD strains contained two phylogenetically distinct alleles: one allele was highly similar to those from serotype A strains and the other to alleles from serotype D strains. However, within both the serotype A allelic group and the serotype D allelic group, there was significant incongruence between genealogies derived from URA5 and LAC. The results suggest recombination in natural populations of both serotypes A and D.

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The spindle pole body of the pathogenic yeast Cryptococcus neoformans: variation in morphology and positional relationship with the nucleolus and the bud in interphase cells

Journal of Electron Microscopy ◽

10.1093/jmicro/dfp054 ◽

2009 ◽

Vol 59 (2) ◽

pp. 165-172 ◽

Cited By ~ 7

Author(s):

M. Yamaguchi ◽

S. K. Biswas ◽

Y. Kuwabara ◽

M. Ohkusu ◽

M. Shimizu ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Spindle Pole Body ◽

Spindle Pole ◽

Pathogenic Yeast ◽

Pole Body ◽

Interphase Cells

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Superoxide Dismutase Influences the Virulence of Cryptococcus neoformans by Affecting Growth within Macrophages

Infection and Immunity ◽

10.1128/iai.71.1.173-180.2003 ◽

2003 ◽

Vol 71 (1) ◽

pp. 173-180 ◽

Cited By ~ 179

Author(s):

Gary M. Cox ◽

Thomas S. Harrison ◽

Henry C. McDade ◽

Carlos P. Taborda ◽

Garrett Heinrich ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Superoxide Dismutase ◽

Cryptococcus Neoformans ◽

Fungal Infections ◽

Reactive Oxygen ◽

Oxygen Species ◽

Wild Type ◽

Pathogenic Yeast ◽

Antioxidant Defense Systems

ABSTRACT Superoxide dismutase (SOD) is an enzyme that converts superoxide radicals into hydrogen peroxide and molecular oxygen and has been shown to contribute to the virulence of many human-pathogenic bacteria through its ability to neutralize toxic levels of reactive oxygen species generated by the host. SOD has also been speculated to be important in the pathogenesis of fungal infections, but the role of this enzyme has not been rigorously investigated. To examine the contribution of SOD to the pathogenesis of fungal infections, we cloned the Cu,Zn SOD-encoding gene (SOD1) from the human-pathogenic yeast Cryptococcus neoformans and made mutants via targeted disruption. The sod1 mutant strains had marked decreases in SOD activity and were strikingly more susceptible to reactive oxygen species in vitro. A sod1 mutant was significantly less virulent than the wild-type strain and two independent reconstituted strains, as measured by cumulative survival in the mouse inhalational model. In vitro studies established that the sod1 strain had attenuated growth compared to the growth of the wild type and a reconstituted strain inside macrophages producing reduced amounts of nitric oxide. These findings demonstrate that (i) the Cu,Zn SOD contributes to virulence but is not required for pathogenicity in C. neoformans; (ii) the decreased virulence of the sod1 strain may be due to increased susceptibility to oxygen radicals within macrophages; and (iii) other antioxidant defense systems in C. neoformans can compensate for the loss of the Cu,Zn SOD in vivo.

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Role of Alternative Oxidase Gene in Pathogenesis of Cryptococcus neoformans

Infection and Immunity ◽

10.1128/iai.71.10.5794-5802.2003 ◽

2003 ◽

Vol 71 (10) ◽

pp. 5794-5802 ◽

Cited By ~ 79

Author(s):

Shamima Akhter ◽

Henry C. McDade ◽

Jenifer M. Gorlach ◽

Garrett Heinrich ◽

Gary M. Cox ◽

...

Keyword(s):

Oxidative Stress ◽

Cryptococcus Neoformans ◽

Mutant Strain ◽

Alternative Oxidase ◽

Temperature Sensitive ◽

Wild Type ◽

Phagocytic Cells ◽

Pathogenic Yeast ◽

Oxidase Gene ◽

Oxidative Pathway

ABSTRACT We identified a homologue of the alternative oxidase gene in a screen to identify genes that are preferentially transcribed in response to a shift to 37°C in the human-pathogenic yeast Cryptococcus neoformans. Alternative oxidases are nucleus-encoded mitochondrial proteins that have two putative roles: they can function in parallel with the classic cytochrome oxidative pathway to produce ATP, and they may counter oxidative stress within the mitochondria. The C. neoformans alternative oxidase gene (AOX1) was found to exist as a single copy in the genome, and it encodes a putative protein of 401 amino acids. An aox1 mutant strain was created using targeted gene disruption, and the mutant strain was reconstituted to wild type using a full-length AOX1. Compared to both the wild-type and reconstituted strains, the aox1 mutant strain was not temperature sensitive but did have significant impairment of both respiration and growth when treated with inhibitors of the classic cytochrome oxidative pathway. The aox1 mutant strain was also found to be more sensitive to the oxidative stressor tert-butyl hydroperoxide. The aox1 mutant strain was significantly less virulent than both the wild type and the reconstituted strain in the murine inhalational model, and it also had significantly impaired growth within a macrophage-like cell line. These data demonstrate that the alternative oxidase of C. neoformans can make a significant contribution to metabolism, has a role in the yeast's defense against exogenous oxidative stress, and contributes to the virulence composite of this organism, possibly by improving survival within phagocytic cells.

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Capsule Growth in Cryptococcus neoformans Is Coordinated with Cell Cycle Progression

mBio ◽

10.1128/mbio.00945-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 42

Author(s):

Rocío García-Rodas ◽

Radames J. B. Cordero ◽

Nuria Trevijano-Contador ◽

Guilhem Janbon ◽

Frédérique Moyrand ◽

...

Keyword(s):

Cell Cycle ◽

Cryptococcus Neoformans ◽

Mutant Strain ◽

Virulence Factor ◽

Cell Cycle Progression ◽

Regulatory Elements ◽

Pathogenic Yeast ◽

Cycle Progression ◽

Content Type ◽

Polysaccharide Capsule

ABSTRACT The fungal pathogen Cryptococcus neoformans has several virulence factors, among which the most important is a polysaccharide capsule. The size of the capsule is variable and can increase significantly during infection. In this work, we investigated the relationship between capsular enlargement and the cell cycle. Capsule growth occurred primarily during the G1 phase. Real-time visualization of capsule growth demonstrated that this process occurred before the appearance of the bud and that capsule growth arrested during budding. Benomyl, which arrests the cells in G2/M, inhibited capsule growth, while sirolimus (rapamycin) addition, which induces G1 arrest, resulted in cells with larger capsule. Furthermore, we have characterized a mutant strain that lacks a putative G1/S cyclin. This mutant showed an increased capacity to enlarge the capsule, both in vivo (using Galleria mellonella as the host model) and in vitro. In the absence of Cln1, there was a significant increase in the production of extracellular vesicles. Proteomic assays suggest that in the cln1 mutant strain, there is an upregulation of the glyoxylate acid cycle. Besides, this cyclin mutant is avirulent at 37°C, which correlates with growth defects at this temperature in rich medium. In addition, the cln1 mutant showed lower intracellular replication rates in murine macrophages. We conclude that cell cycle regulatory elements are involved in the modulation of the expression of the main virulence factor in C. neoformans. IMPORTANCE Cryptococcus neoformans is a pathogenic fungus that has significant incidence worldwide. Its main virulence factor is a polysaccharide capsule that can increase in size during infection. In this work, we demonstrate that this process occurs in a specific phase of the cell cycle, in particular, in G1. In agreement, mutants that have an abnormal longer G1 phase show larger capsule sizes. We believe that our findings are relevant because they provide a link between capsule growth, cell cycle progression, and virulence in C. neoformans that reveals new aspects about the pathogenicity of this fungus. Moreover, our findings indicate that cell cycle elements could be used as antifungal targets in C. neoformans by affecting both the growth of the cells and the expression of the main virulence factor of this pathogenic yeast.

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

Genome ◽

10.1139/g08-004 ◽

2008 ◽

Vol 51 (4) ◽

pp. 272-281 ◽

Cited By ~ 20

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.

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Isolates of Cryptococcus neoformans from Infected Animals Reveal Genetic Exchange in Unisexual, α Mating Type Populations

Eukaryotic Cell ◽

10.1128/ec.00097-08 ◽

2008 ◽

Vol 7 (10) ◽

pp. 1771-1780 ◽

Cited By ~ 68

Author(s):

Tien Bui ◽

Xiaorong Lin ◽

Richard Malik ◽

Joseph Heitman ◽

Dee Carter

Keyword(s):

Cryptococcus Neoformans ◽

Mating Type ◽

Fungal Pathogens ◽

Genetic Recombination ◽

Genetic Exchange ◽

Pathogenic Yeast ◽

Asexual Propagation ◽

Sexual Recombination ◽

Single Mating ◽

Almost All

ABSTRACT Sexual reproduction and genetic exchange are important for the evolution of fungal pathogens and for producing potentially infective spores. Studies to determine whether sex occurs in the pathogenic yeast Cryptococcus neoformans var. grubii have produced enigmatic results, however: basidiospores are the most likely infective propagules, and clinical isolates are fertile and genetically diverse, consistent with a sexual species, but almost all populations examined consist of a single mating type and have little evidence for genetic recombination. The choice of population is critical when looking for recombination, particularly when significant asexual propagation is likely and when latency may complicate assessing the origin of an isolate. We therefore selected isolates from infected animals living in the region of Sydney, Australia, with the assumption that the relatively short life spans and limited travels of the animal hosts would provide a very defined population. All isolates were mating type α and were of molecular genotype VNI or VNII. A lack of linkage disequilibrium among loci suggested that genetic exchange occurred within both genotype groups. Four diploid VNII isolates that produced filaments and basidium-like structures when cultured in proximity to an a mating type strain were found. Recent studies suggest that compatible α-α unions can occur in C. neoformans var. neoformans populations and in populations of the sibling species Cryptococcus gattii. As a mating type strains of C. neoformans var. grubii have never been found in Australia, or in the VNII molecular type globally, the potential for α-α unions is evidence that α-α unisexual mating maintains sexual recombination and diversity in this pathogen and may produce infectious propagules.

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