Damage to yeast cells of Cryptococcus neoformans by voriconazole and fluconazole: a culture and microscopic study

The opportunistic fungal pathogen Cryptococcus neoformans has two mating types, MATa and MAT alpha. The MAT alpha strains are more virulent. Mating of opposite mating type haploid yeast cells results in the production of a filamentous hyphal phase. The MAT alpha locus has been isolated in this study in order to identify the genetic differences between mating types and their contribution to virulence. A 138-bp fragment of MAT alpha-specific DNA which cosegregates with alpha-mating type was isolated by using a difference cloning method. Overlapping phage and cosmid clones spanning the entire MAT alpha locus were isolated by using this MAT alpha-specific fragment as a probe. Mapping of these clones physically defined the MAT alpha locus to a 35- to 45-kb region which is present only in MAT alpha strains. Transformation studies with fragments of the MAT alpha locus identified a 2.1-kb XbaI-HindIII fragment that directs starvation-induced filament formation in MATa cells but not in MAT alpha cells. This 2.1-kb fragment contains a gene, MF alpha, with a small open reading frame encoding a pheromone precursor similar to the lipoprotein mating factors found in Saccharomyces cerevisiae, Ustilago maydis, and Schizosaccharomyces pombe. The ability of the MATa cells to express, process, and secrete the MAT alpha pheromone in response to starvation suggests similar mechanisms for these processes in both cell types. These results also suggest that the production of pheromone is under a type of nutritional control shared by the two cell types.

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Adaptation of Cryptococcus neoformans to Mammalian Hosts: Integrated Regulation of Metabolism and Virulence

Eukaryotic Cell ◽

10.1128/ec.05273-11 ◽

2011 ◽

Vol 11 (2) ◽

pp. 109-118 ◽

Cited By ~ 63

Author(s):

Jim Kronstad ◽

Sanjay Saikia ◽

Erik David Nielson ◽

Matthias Kretschmer ◽

Wonhee Jung ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Cryptococcus Neoformans ◽

Central Carbon Metabolism ◽

Yeast Cells ◽

Phagocytic Cells ◽

Content Type ◽

The Central Nervous System ◽

Mechanisms Of Adaptation ◽

Complex Integration

ABSTRACTThe basidiomycete fungusCryptococcus neoformansinfects humans via inhalation of desiccated yeast cells or spores from the environment. In the absence of effective immune containment, the initial pulmonary infection often spreads to the central nervous system to result in meningoencephalitis. The fungus must therefore make the transition from the environment to different mammalian niches that include the intracellular locale of phagocytic cells and extracellular sites in the lung, bloodstream, and central nervous system. Recent studies provide insights into mechanisms of adaptation during this transition that include the expression of antiphagocytic functions, the remodeling of central carbon metabolism, the expression of specific nutrient acquisition systems, and the response to hypoxia. Specific transcription factors regulate these functions as well as the expression of one or more of the major known virulence factors ofC. neoformans. Therefore, virulence factor expression is to a large extent embedded in the regulation of a variety of functions needed for growth in mammalian hosts. In this regard, the complex integration of these processes is reminiscent of the master regulators of virulence in bacterial pathogens.

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Fatty Acid Synthesis Is Essential for Survival of Cryptococcus neoformans and a Potential Fungicidal Target

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00442-07 ◽

2007 ◽

Vol 51 (10) ◽

pp. 3537-3545 ◽

Cited By ~ 28

Author(s):

Methee Chayakulkeeree ◽

Thomas H. Rude ◽

Dena L. Toffaletti ◽

John R. Perfect

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Cryptococcus Neoformans ◽

Fatty Acid Synthase ◽

Acid Synthesis ◽

Yeast Cells ◽

Synthase Inhibitor ◽

The Brain

ABSTRACT Fatty acid synthase in the yeast Cryptococcus neoformans is composed of two subunits encoded by FAS1 and FAS2 genes. We inserted a copper-regulated promoter (P CTR4-2 ) to regulate FAS1 and FAS2 expression in Cryptococcus neoformans (strains P CTR4-2 /FAS1 and P CTR4-2 /FAS2, respectively). Both mutants showed growth rates similar to those of the wild type in a low-copper medium in which FAS1 and FAS2 were expressed, but even in the presence of exogenous fatty acids, strains were suppressed in growth under high-copper conditions. The treatment of C. neoformans with fluconazole was shown to have an increased inhibitory activity and even became fungicidal when either FAS1 or FAS2 expression was suppressed. Furthermore, a subinhibitory dose of fluconazole showed anticryptococcal activity in vitro in the presence of cerulenin, a fatty acid synthase inhibitor. In a murine model of pulmonary cryptococcosis, a tissue census of yeast cells in P CTR4-2 /FAS2 strain at day 7 of infection was significantly lower than that in mice treated with tetrathiomolybdate, a copper chelator (P < 0.05), and a yeast census of P CTR4-2 /FAS1 strain at day 14 of infection in the brain was lower in the presence of more copper. In fact, no positive cultures from the brain were detected in mice (with or without tetrathiomolybdate treatment) infected with the P CTR4-2 /FAS2 strain, which implies that this mutant did not reach the brain in mice. We conclude that both FAS1 and FAS2 in C. neoformans are essential for in vitro and in vivo growth in conditions with and without exogenous fatty acids and that FAS1 and FAS2 can potentially be fungicidal targets for C. neoformans with a potential for synergistic behavior with azoles.

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Isolation and Characterization of Cryptococcus neoformans Spores Reveal a Critical Role for Capsule Biosynthesis Genes in Spore Biogenesis

Eukaryotic Cell ◽

10.1128/ec.00352-08 ◽

2009 ◽

Vol 8 (4) ◽

pp. 595-605 ◽

Cited By ~ 60

Author(s):

Michael R. Botts ◽

Steven S. Giles ◽

Marcellene A. Gates ◽

Thomas R. Kozel ◽

Christina M. Hull

Keyword(s):

Cryptococcus Neoformans ◽

Fungal Pathogens ◽

Critical Role ◽

Spore Formation ◽

Yeast Cells ◽

Isolation And Characterization ◽

Specific Configuration ◽

First Time

ABSTRACT Spores are essential particles for the survival of many organisms, both prokaryotic and eukaryotic. Among the eukaryotes, fungi have developed spores with superior resistance and dispersal properties. For the human fungal pathogens, however, relatively little is known about the role that spores play in dispersal and infection. Here we present the purification and characterization of spores from the environmental fungus Cryptococcus neoformans. For the first time, we purified spores to homogeneity and assessed their morphological, stress resistance, and surface properties. We found that spores are morphologically distinct from yeast cells and are covered with a thick spore coat. Spores are also more resistant to environmental stresses than yeast cells and display a spore-specific configuration of polysaccharides on their surfaces. Surprisingly, we found that the surface of the spore reacts with antibodies to the polysaccharide glucuronoxylomannan, the most abundant component of the polysaccharide capsule required for C. neoformans virulence. We explored the role of capsule polysaccharide in spore development by assessing spore formation in a series of acapsular strains and determined that capsule biosynthesis genes are required for proper sexual development and normal spore formation. Our findings suggest that C. neoformans spores may have an adapted cell surface that facilitates persistence in harsh environments and ultimately allows them to infect mammalian hosts.

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Cryptococcus neoformans STE12α Regulates Virulence but Is Not Essential for Mating

Journal of Experimental Medicine ◽

10.1084/jem.191.5.871 ◽

2000 ◽

Vol 191 (5) ◽

pp. 871-882 ◽

Cited By ~ 117

Author(s):

Y.C. Chang ◽

B.L. Wickes ◽

G.F. Miller ◽

L.A. Penoyer ◽

K.J. Kwon-Chung

Keyword(s):

Immune Responses ◽

Cryptococcus Neoformans ◽

Mating Type ◽

Yeast Cells ◽

Mating Frequency ◽

Histopathological Analysis ◽

Cystic Lesions ◽

Wild Type Allele ◽

Wild Type ◽

Melanin Production

The Cryptococcus neoformans STE12α gene, a homologue of Saccharomyces cerevisiae STE12, exists only in mating type (MAT)α cells. In S. cerevisiae, STE12 was required for mating and filament formation. In C. neoformans, haploid fruiting on filament agar required STE12α. The ability to form hyphae, however, was not affected by deletion of STE12α when convergently growing MATa strains were present. Furthermore, ste12α disruptants were fertile when mated with MATa strains, albeit with reduced mating frequency. Most importantly, the virulence of a ste12α disruptant of serotype D strain was significantly reduced in a mouse model. When the ste12α locus was reconstituted with the wild-type allele by cotransformation, virulence was restored. Histopathological analysis demonstrated a reduction in capsular size of yeast cells, less severe cystic lesions, and stronger immune responses in meninges of mice infected with ste12α cells than those of mice infected with STE12α cells. Using reporter gene constructs, we found that STE12α controls the expression of several phenotypes known to be involved in virulence, such as capsule and melanin production. These results demonstrate a clear molecular link between mating type and virulence in C. neoformans.

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Role of Actin-Bundling Protein Sac6 in Growth of Cryptococcus neoformans at Low Oxygen Concentration

Eukaryotic Cell ◽

10.1128/ec.00120-12 ◽

2012 ◽

Vol 11 (7) ◽

pp. 943-951 ◽

Cited By ~ 7

Author(s):

Yun C. Chang ◽

Ami Khanal Lamichhane ◽

Kyung J. Kwon-Chung

Keyword(s):

Cryptococcus Neoformans ◽

Ambient Air ◽

Etiologic Agent ◽

Yeast Cells ◽

Actin Binding ◽

Environmental Niche ◽

Low Oxygen ◽

Cortical Actin ◽

Content Type ◽

Oxygen Conditions

ABSTRACTCryptococcus neoformans, the etiologic agent of cryptococcosis, is an obligately aerobic yeast that inhabits an environmental niche exposed to ambient air. The cell doubling time was significantly prolonged under 1% O2relative to that under normoxic conditions. No apparent cell cycle arrest occurred following a shift from ambient air to 1% O2. However, yeast cells became hypersensitive to the actin monomer-sequestering agent latrunculin A at 1% O2, indicating that proper actin function is critical for growth at low oxygen concentrations. We showed that Sac6, an actin-binding protein, played an important role in cell growth under low oxygen conditions. Sac6 colocalized with cortical actin patches and with the ring structures between mother cells and buds. Under low oxygen conditions, thesac6deletion mutant grew poorly, and accumulation of the actin capping protein Cap1 was observed in the vacuole of thesac6Δstrain. Furthermore, endocytic processes were hampered in thesac6Δmutant, but cell polarity and cytokinesis were not visibly disturbed. The deficiency of endocytosis in thesac6Δstrain could be rescued by 1 M sorbitol under 1% O2, but growth remained retarded. These results suggest an absence of a direct link inC. neoformansbetween endocytosis and coping with the stress of low oxygen conditions. This interpretation is further supported by the observation that deletion of three conserved genes,ABP1,CRN1, andSLA2, which play important roles in endocytosis, had no effect on growth under 1% O2. Interestingly, deletion ofSAC6inC. neoformanshad no effect on virulence in mice.

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Host environmental conditions induce small fungal cell size and alter population heterogeneity in Cryptococcus neoformans

10.1101/2020.01.03.894709 ◽

2020 ◽

Cited By ~ 1

Author(s):

Xin Zhou ◽

Hanna Zafar ◽

Poppy Sephton-Clark ◽

Sally H. Mohamed ◽

Ambre Chapuis ◽

...

Keyword(s):

Drug Resistance ◽

Cryptococcus Neoformans ◽

Lipid Membrane ◽

Population Heterogeneity ◽

Clinical Isolates ◽

Yeast Cells ◽

Small Cells ◽

Fungal Cell

AbstractFungal morphology significantly impacts the host response. Filamentation and tissue penetration by Candida and Aspergillus species are essential for virulence, while growth as a yeast allows the thermal dimorphic fungi Coccidiodes, Histoplasma, and Talaromyces to reside inside phagocytes and disseminate. The basidiomycete Cryptococcus neoformans exhibits an unusual yeast-to-titan transition thought to enhance pathogenicity by increasing fungal survival in the host lung and dissemination to the central nervous system. In a common laboratory strain (H99), in vitro and in vivo titan induction yields a heterogenous population including >10 μm titan cells, 5-7 μm yeast cells and 2-4 μm titanides. Previous reports have shown that titan cells are associated with enhanced virulence and the generation of aneuploid cells that facilitate stress adaptation and drug resistance, while small (>10 μm) cells are associated with increased dissemination. However, the relationship between titan cells, small cells, and titanides remains unclear. Here, we characterize titanides and small cells in H99 and three clinical isolates and show that titanides share the lipid membrane order of their titan mothers and the G0 quiescent-like DNA staining of mating spores. In addition, we show that both titanizing and non-titanizing isolates exhibit altered capsule structure and PAMP exposure over time during in vitro culture, and generate aneuploidy in vivo.Author summaryThe human fungal pathogen Cryptococcus neoformans causes 200,000 HIV-associated deaths each year. In the lung, Cryptococcus makes an unusual yeast-to-titan morphological switch that contributes to disease development by altering immune polarization and introducing aneuploidy underlying host stress and drug resistance. Specifically, a proportion of 5 um haploid yeast endoreduplicate and swell, converting to large (> 10 um) polyploid titan cells that can then produce genetically distinct daughter cells. We recently developed an in vitro protocol for inducing large titan cells and additionally observed a novel small “titanide” cell type. Here we investigate the nature and origin of these small cells, demonstrating that they emerge during both in vitro and in vivo mouse-passaged titan induction in the well characterised lab strain H99 and are also apparent in a titanizing clinical isolate, Zc8. We show that these titanide cells share features with titan mothers (lipid order) and with spores produced during heterothalic mating. Finally, we show that the capacity of clinical isolates to produce both titan and titanide cells impacts aneuploidy and the emergence of drug resistance in vivo.

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Comparison of Fungizone, Amphotec, AmBisome, and Abelcet for Treatment of Systemic Murine Cryptococcosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.4.899 ◽

1998 ◽

Vol 42 (4) ◽

pp. 899-902 ◽

Cited By ~ 62

Author(s):

Karl V. Clemons ◽

David A. Stevens

Keyword(s):

Body Weight ◽

Cryptococcus Neoformans ◽

Amphotericin B ◽

Rank Order ◽

Yeast Cells ◽

Treatment Regimens ◽

Equal Dose ◽

The Brain ◽

All Treatment ◽

Better Than

ABSTRACT Three lipid-based formulations of amphotericin B have been approved for use in various countries. The aim of this study was to compare Amphotec (ABCD; Sequus), AmBisome (AmBi; Nexstar), Abelcet (ABLC; The Liposome Co.), and conventional deoxycholate amphotericin B (Fungizone; Bristol Meyers Squibb) for the treatment of experimental systemic cryptococcosis. A model was established in 10-week-old female CD-1 mice by intravenous (i.v.) injection of 6.25 × 105 viableCryptococcus neoformans yeast cells. Therapy began 4 days later, with i.v. administration three times per week for 2 weeks. Mice received either no treatment, 1 mg of Fungizone per kg of body weight, or 1, 5, or 10 mg of ABCD, AmBi, or ABLC per kg. Ninety percent of control mice died between days 15 and 34. All treatment regimens except ABLC at 1 mg/kg prolonged survival compared with no treatment (P < 0.01 to 0.001). All mice receiving 5 or 10 mg of ABCD or AmBi per kg and 90% of mice given 10 mg of ABLC per kg survived, whereas ≤50% of those given other treatment regimens survived. Fungizone was the least effective of the four formulations, with 5 or 10 mg of ABCD, AmBi, or ABLC per kg resulting in a significantly better outcome than Fungizone (P < 0.001). Among the three formulations, ABCD and AmBi were equally effective, both being better than ABLC at equal 5- or 10-mg/kg doses (P < 0.001). Comparison of residual infectious burdens in various organs showed that each drug had some dose-responsive efficacy in three or more organs at escalating doses. In the brain, ABCD or AmBi at 5 or 10 mg/kg or ABLC at 10 mg/kg was more effective than Fungizone at 1 mg/kg or no treatment, while ABCD or AmBi at 1 mg/kg was as effective as ABLC at 10 mg/kg. Similar results were obtained for the kidneys and lungs. In the spleen, ABCD at 10 mg/kg cured all mice of infection and was superior to all other regimens. In the liver, AmBi at 5 mg/kg was superior to an equal dose of ABCD or ABLC. Overall, the efficacies of ABCD and AmBi were equal to that of Fungizone at 1 mg/kg and were about 10-fold better than that of ABLC, particularly in the brain; a comparative rank order of efficacies was ABCD ≅ AmBi > ABLC ≫ Fungizone. This is the first study that compared all four amphotericin B formulations.

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Role of an Expanded Inositol Transporter Repertoire in Cryptococcus neoformans Sexual Reproduction and Virulence

mBio ◽

10.1128/mbio.00084-10 ◽

2010 ◽

Vol 1 (1) ◽

Cited By ~ 37

Author(s):

Chaoyang Xue ◽

Tongbao Liu ◽

Lydia Chen ◽

Wenjun Li ◽

Iris Liu ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Gene Family ◽

Sexual Reproduction ◽

Cryptococcus Neoformans ◽

Yeast Cells ◽

Sexual Cycle ◽

Fungal Virulence ◽

Content Type ◽

Fungal Meningitis

ABSTRACTCryptococcus neoformansandCryptococcus gattiiare globally distributed human fungal pathogens and the leading causes of fungal meningitis. Recent studies reveal thatmyo-inositol is an important factor for fungal sexual reproduction. ThatC. neoformanscan utilizemyo-inositol as a sole carbon source and the existence of abundant inositol in the human central nervous system suggest that inositol is important forCryptococcusdevelopment and virulence. In accord with this central importance of inositol, an expandedmyo-inositol transporter (ITR) gene family has been identified inCryptococcus. This gene family contains two phylogenetically distinct groups, with a total of 10 or more members inC. neoformansand at least six members in the sibling speciesC. gattii. These inositol transporter genes are differentially expressed under inositol-inducing conditions based on quantitative real-time PCR analyses. Expression ofITRgenes in aSaccharomyces cerevisiaeitr1 itr2mutant lacking inositol transport can complement the slow-growth phenotype of this strain, confirming thatITRgenes arebona fideinositol transporters. Gene mutagenesis studies reveal that the Itr1 and Itr1A transporters are important formyo-inositol stimulation of mating and that functional redundancies among themyo-inositol transporters likely exist. Deletion of the inositol 1-phosphate synthase geneINO1in anitr1oritr1amutant background compromised virulence in a murine inhalation model, indicating the importance of inositol sensing and acquisition for fungal infectivity. Our study provides a platform for further understanding the roles of inositol in fungal physiology and virulence.IMPORTANCECryptococcus neoformansis an AIDS-associated human fungal pathogen that causes over 1 million cases of meningitis annually and is the leading cause of fungal meningitis in immunosuppressed patients. The initial cryptococcal infection is caused predominantly via inhalation of sexual spores or desiccated yeast cells from the environment. How this fungus completes its sexual cycle and produces infectious spores in nature and why it frequently infects the central nervous system to cause fatal meningitis are critical questions that remain to be understood. In this study, we demonstrate that inositol acquisition is important not only for fungal sexual reproduction but also for fungal virulence. We identified an expanded inositol transporter gene family that contains over 10 members, important for both fungal sexual reproduction and virulence. Our work contributes to our understanding of how fungi respond to the environmental inositol availability and its impact on sexual reproduction and virulence.

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