scholarly journals Chronological Aging Is Associated with Biophysical and Chemical Changes in the Capsule of Cryptococcus neoformans

2011 ◽  
Vol 79 (12) ◽  
pp. 4990-5000 ◽  
Author(s):  
Radames J. B. Cordero ◽  
Bruno Pontes ◽  
Allan J. Guimarães ◽  
Luis R. Martinez ◽  
Johanna Rivera ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Cryptococcus Neoformans ◽  
Pathogenic Fungus ◽  
Growth Conditions ◽  
Cellular Aging ◽  
Phase Growth ◽  
Chronic Infections ◽  
Content Type ◽  
Chronological Aging ◽  
Dynamic Structures

ABSTRACTDoes the age of a microbial cell affect its virulence factors? To our knowledge, this question has not been addressed previously, but the answer is of great relevance for chronic infections where microbial cells persist and age in hosts.Cryptococcus neoformansis an encapsulated human-pathogenic fungus notorious for causing chronic infections where cells of variable age persist in tissue. The major virulence factor forC. neoformansis a polysaccharide (PS) capsule. To understand how chronological age could impact the cryptococcal capsule properties, we compared the elastic properties, permeabilities, zeta potentials, and glycosidic compositions of capsules from young and old cells and found significant differences in all parameters measured. Changes in capsular properties were paralleled by changes in PS molecular mass and density, as well as modified antigenic density and antiphagocytic properties. Remarkably, chronological aging under stationary-phase growth conditions was associated with the expression of α-1,3-glucans in the capsule, indicating a new structural capsular component. Our results establish that cryptococcal capsules are highly dynamic structures that change dramatically with chronological aging under prolonged stationary-phase growth conditions. Changes associated with cellular aging in chronic infections could contribute to the remarkable capacity of this fungus to persist in tissues by generating phenotypically and antigenically different capsules.

scholarly journals Peroxisomal and Mitochondrial β-Oxidation Pathways Influence the Virulence of the Pathogenic Fungus Cryptococcus neoformans

2012 ◽  
Vol 11 (8) ◽  
pp. 1042-1054 ◽  
Author(s):  
Matthias Kretschmer ◽  
Joyce Wang ◽  
James W. Kronstad
Keyword(s):  
Carbon Source ◽  
Cryptococcus Neoformans ◽  
Virulence Factor ◽  
Fungal Pathogens ◽  
Pathogenic Fungus ◽  
Subsequent Work ◽  
Content Type ◽  
Capsule Production ◽  
Human Pathogenic Fungus ◽  
Host Tissues

ABSTRACTAn understanding of the connections between metabolism and elaboration of virulence factors during host colonization by the human-pathogenic fungusCryptococcus neoformansis important for developing antifungal therapies. Lipids are abundant in host tissues, and fungal pathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. In addition, lipids are important signaling molecules in both fungi and mammals. In this report, we demonstrate that defects in the peroxisomal and mitochondrial β-oxidation pathways influence the growth ofC. neoformanson fatty acids as well as the virulence of the fungus in a mouse inhalation model of cryptococcosis. Disease attenuation may be due to the cumulative influence of altered carbon source acquisition or processing, interference with secretion, changes in cell wall integrity, and an observed defect in capsule production for the peroxisomal mutant. Altered capsule elaboration in the context of a β-oxidation defect was unexpected but is particularly important because this trait is a major virulence factor forC. neoformans. Additionally, analysis of mutants in the peroxisomal pathway revealed a growth-promoting activity forC. neoformans, and subsequent work identified oleic acid and biotin as candidates for such factors. Overall, this study reveals that β-oxidation influences virulence inC. neoformansby multiple mechanisms that likely include contributions to carbon source acquisition and virulence factor elaboration.

scholarly journals TheBeauveria bassianaGas3 β-Glucanosyltransferase Contributes to Fungal Adaptation to Extreme Alkaline Conditions

2018 ◽  
Vol 84 (15) ◽  
Author(s):  
Zhibing Luo ◽  
Tongbing Zhang ◽  
Pengfei Liu ◽  
Yuting Bai ◽  
Qiyan Chen ◽  
...  
Keyword(s):  
Cell Wall ◽  
Pathogenic Fungus ◽  
Growth Conditions ◽  
Alkaline Ph ◽  
Ph Responsive ◽  
Content Type ◽  
Cell Wall Remodeling ◽  
Alkaline Conditions ◽  
Increased Sensitivity ◽  
Ph Dependent

ABSTRACTFungal β-1,3-glucanosyltransferases are cell wall-remodeling enzymes implicated in stress response, cell wall integrity, and virulence, with most fungal genomes containing multiple members. The insect-pathogenic fungusBeauveria bassianadisplays robust growth over a wide pH range (pH 4 to 10). A random insertion mutant library screening for increased sensitivity to alkaline (pH 10) growth conditions resulted in the identification and mapping of a mutant to a β-1,3-glucanosyltransferase gene (Bbgas3).Bbgas3expression was pH dependent and regulated by the PacC transcription factor, which activates genes in response to neutral/alkaline growth conditions. Targeted gene knockout ofBbgas3resulted in reduced growth under alkaline conditions, with only minor effects of increased sensitivity to cell wall stress (Congo red and calcofluor white) and no significant effects on fungal sensitivity to oxidative or osmotic stress. The cell walls of ΔBbgas3aerial conidia were thinner than those of the wild-type and complemented strains in response to alkaline conditions, and β-1,3-glucan antibody and lectin staining revealed alterations in cell surface carbohydrate epitopes. The ΔBbgas3mutant displayed alterations in cell wall chitin and carbohydrate content in response to alkaline pH. Insect bioassays revealed impaired virulence for the ΔBbgas3mutant depending upon the pH of the media on which the conidia were grown and harvested. Unexpectedly, a decreased median lethal time to kill (LT50, i.e., increased virulence) was seen for the mutant using intrahemocoel injection assays using conidia grown at acidic pH (5.6). These data show that BbGas3 acts as a pH-responsive cell wall-remodeling enzyme involved in resistance to extreme pH (>9).IMPORTANCELittle is known about adaptations required for growth at high (>9) pH. Here, we show that a specific fungal membrane-remodeling β-1,3-glucanosyltransferase gene (Bbgas3) regulated by the pH-responsive PacC transcription factor forms a critical aspect of the ability of the insect-pathogenic fungusBeauveria bassianato grow at extreme pH. The loss ofBbgas3resulted in a unique decreased ability to grow at high pH, with little to no effects seen with respect to other stress conditions, i.e., cell wall integrity and osmotic and oxidative stress. However, pH-dependent alternations in cell wall properties and virulence were noted for the ΔBbgas3 mutant. These data provide a mechanistic insight into the importance of the specific cell wall structure required to stabilize the cell at high pH and link it to the PacC/Pal/Rim pH-sensing and regulatory system.

scholarly journals The Stationary-Phase Cells of Saccharomyces cerevisiae Display Dynamic Actin Filaments Required for Processes Extending Chronological Life Span

2015 ◽  
Vol 35 (22) ◽  
pp. 3892-3908 ◽  
Author(s):  
Pavla Vasicova ◽  
Renata Lejskova ◽  
Ivana Malcova ◽  
Jiri Hasek
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stationary Phase ◽  
Actin Filaments ◽  
Phase Cell ◽  
Content Type ◽  
Chronological Aging ◽  
Yeast Cultures ◽  
Actin Cables ◽  
Mitochondrial Networks

Stationary-growth-phaseSaccharomyces cerevisiaeyeast cultures consist of nondividing cells that undergo chronological aging. For their successful survival, the turnover of proteins and organelles, ensured by autophagy and the activation of mitochondria, is performed. Some of these processes are engaged in by the actin cytoskeleton. InS. cerevisiaestationary-phase cells, F actin has been shown to form static aggregates named actin bodies, subsequently cited to be markers of quiescence. Ourin vivoanalyses revealed that stationary-phase cultures contain cells with dynamic actin filaments, besides the cells with static actin bodies. The cells with dynamic actin displayed active endocytosis and autophagy and well-developed mitochondrial networks. Even more, stationary-phase cell cultures grown under calorie restriction predominantly contained cells with actin cables, confirming that the presence of actin cables is linked to successful adaptation to stationary phase. Cells with actin bodies were inactive in endocytosis and autophagy and displayed aberrations in mitochondrial networks. Notably, cells of the respiratory activity-deficientcox4Δ strain displayed the same mitochondrial aberrations and actin bodies only. Additionally, our results indicate that mitochondrial dysfunction precedes the formation of actin bodies and the appearance of actin bodies corresponds to decreased cell fitness. We conclude that the F-actin status reflects the extent of damage that arises from exponential growth.

scholarly journals Antagonistic Interactions between Yeast Chaperones Hsp104 and Hsp70 in Prion Curing

1999 ◽  
Vol 19 (2) ◽  
pp. 1325-1333 ◽  
Author(s):  
Gary P. Newnam ◽  
Renee D. Wegrzyn ◽  
Susan L. Lindquist ◽  
Yury O. Chernoff
Keyword(s):  
Heat Shock ◽  
Stationary Phase ◽  
Molecular Level ◽  
Growth Conditions ◽  
Nonsense Suppression ◽  
Phase Growth ◽  
Heterologous Promoter ◽  
Natural Induction ◽  
Hsp70 Family ◽  
Chaperone Protein

ABSTRACT The maintenance of [PSI], a prion-like form of the yeast release factor Sup35, requires a specific concentration of the chaperone protein Hsp104: either deletion or overexpression of Hsp104 will cure cells of [PSI]. A major puzzle of these studies was that overexpression of Hsp104 alone, from a heterologous promoter, cures cells of [PSI] very efficiently, yet the natural induction of Hsp104 with heat shock, stationary-phase growth, or sporulation does not. These observations pointed to a mechanism for protecting the genetic information carried by the [PSI] element from vicissitudes of the environment. Here, we show that simultaneous overexpression of Ssa1, a protein of the Hsp70 family, protects [PSI] from curing by overexpression of Hsp104. Ssa1 protein belongs to the Ssa subfamily, members of which are normally induced with Hsp104 during heat shock, stationary-phase growth, and sporulation. At the molecular level, excess Ssa1 prevents a shift of Sup35 protein from the insoluble (prion) to the soluble (cellular) state in the presence of excess Hsp104. Overexpression of Ssa1 also increases nonsense suppression by [PSI] when Hsp104 is expressed at its normal level. In contrast,hsp104 deletion strains lose [PSI] even in the presence of overproduced Ssa1. Overproduction of the unrelated chaperone protein Hsp82 (Hsp90) neither cured [PSI] nor antagonized the [PSI]-curing effect of overproduced Hsp104. Our results suggest it is the interplay between Hsp104 and Hsp70 that allows the maintenance of [PSI] under natural growth conditions.

scholarly journals The Pathogenic Fungus Cryptococcus neoformans Expresses Two Functional GDP-Mannose Transporters with Distinct Expression Patterns and Roles in Capsule Synthesis

2007 ◽  
Vol 6 (5) ◽  
pp. 776-785 ◽  
Author(s):  
Tricia R. Cottrell ◽  
Cara L. Griffith ◽  
Hong Liu ◽  
Ashley A. Nenninger ◽  
Tamara L. Doering
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Pathogenic Fungus ◽  
Expression Patterns ◽  
Growth Conditions ◽  
Phosphomannose Isomerase ◽  
Microarray Experiments ◽  
Life Threatening ◽  
Nucleotide Sugars ◽  
Intracellular Organelles

ABSTRACT Cryptococcus neoformans is a fungal pathogen that is responsible for life-threatening disease, particularly in the context of compromised immunity. This organism makes extensive use of mannose in constructing its cell wall, glycoproteins, and glycolipids. Mannose also comprises up to two-thirds of the main cryptococcal virulence factor, a polysaccharide capsule that surrounds the cell. The glycosyltransfer reactions that generate cellular carbohydrate structures usually require activated donors such as nucleotide sugars. GDP-mannose, the mannose donor, is produced in the cytosol by the sequential actions of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase. However, most mannose-containing glycoconjugates are synthesized within intracellular organelles. This topological separation necessitates a specific transport mechanism to move this key precursor across biological membranes to the appropriate site for biosynthetic reactions. We have discovered two GDP-mannose transporters in C. neoformans, in contrast to the single such protein reported previously for other fungi. Biochemical studies of each protein expressed in Saccharomyces cerevisiae show that both are functional, with similar kinetics and substrate specificities. Microarray experiments indicate that the two proteins Gmt1 and Gmt2 are transcribed with distinct patterns of expression in response to variations in growth conditions. Additionally, deletion of the GMT1 gene yields cells with small capsules and a defect in capsule induction, while deletion of GMT2 does not alter the capsule. We suggest that C. neoformans produces two GDP-mannose transporters to satisfy its enormous need for mannose utilization in glycan synthesis. Furthermore, we propose that the two proteins have distinct biological roles. This is supported by the different expression patterns of GMT1 and GMT2 in response to environmental stimuli and the dissimilar phenotypes that result when each gene is deleted.

scholarly journals The Monothiol Glutaredoxin Grx4 Regulates Iron Homeostasis and Virulence inCryptococcus neoformans

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Rodgoun Attarian ◽  
Guanggan Hu ◽  
Eddy Sánchez-León ◽  
Mélissa Caza ◽  
Daniel Croll ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogens ◽  
Iron Homeostasis ◽  
Pathogenic Fungus ◽  
Nutrient Sensing ◽  
Iron Availability ◽  
Virulence Determinants ◽  
Poor Growth ◽  
Content Type

ABSTRACTThe acquisition of iron and the maintenance of iron homeostasis are important aspects of virulence for the pathogenic fungusCryptococcus neoformans. In this study, we characterized the role of the monothiol glutaredoxin Grx4 in iron homeostasis and virulence inC. neoformans. Monothiol glutaredoxins are important regulators of iron homeostasis because of their conserved roles in [2Fe-2S] cluster sensing and trafficking. We initially identified Grx4 as a binding partner of Cir1, a master regulator of iron-responsive genes and virulence factor elaboration inC. neoformans. We confirmed that Grx4 binds Cir1 and demonstrated that iron repletion promotes the relocalization of Grx4 from the nucleus to the cytoplasm. We also found that agrx4mutant lacking the GRX domain displayed iron-related phenotypes similar to those of acir1Δ mutant, including poor growth upon iron deprivation. Importantly, thegrx4mutant 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 thegrx4mutant and the WT strain under low-iron and iron-replete conditions confirmed a central role for Grx4 in iron homeostasis. Dysregulation of iron-related metabolism was consistent withgrx4mutant phenotypes related to oxidative stress, mitochondrial function, and DNA repair. Overall, the phenotypes of thegrx4mutant lacking the GRX domain and the transcriptome sequencing (RNA-Seq) analysis of the mutant support the hypothesis that Grx4 functions as an iron sensor, in part through an interaction with Cir1, to extensively regulate iron homeostasis.IMPORTANCEFungal pathogens cause life-threatening diseases in humans, particularly in immunocompromised people, and there is a tremendous need for a greater understanding of pathogenesis to support new therapies. One prominent fungal pathogen,Cryptococcus neoformans, causes meningitis in people suffering from HIV/AIDS. In the present study, we focused on characterizing mechanisms by whichC. neoformanssenses iron availability because iron is both a signal and a key nutrient for proliferation of the pathogen in vertebrate hosts. Specifically, we characterized a monothiol glutaredoxin protein, Grx4, that functions as a sensor of iron availability and interacts with regulatory factors to control the ability ofC. neoformansto cause disease. Grx4 regulates key virulence factors, and a mutant is unable to cause disease in a mouse model of cryptococcosis. Overall, our study provides new insights into nutrient sensing and the role of iron in the pathogenesis of fungal diseases.

scholarly journals Methylation of yeast ribosomal protein S2 is elevated during stationary phase growth conditions

2014 ◽  
Vol 445 (3) ◽  
pp. 535-541 ◽  
Author(s):  
Daniel T. Ladror ◽  
Brian L. Frey ◽  
Mark Scalf ◽  
Mark E. Levenstein ◽  
Jacklyn M. Artymiuk ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Ribosomal Protein ◽  
Growth Conditions ◽  
Phase Growth

scholarly journals Mutations in the Saccharomyces cerevisiae opi3 gene: effects on phospholipid methylation, growth and cross-pathway regulation of inositol synthesis.

Genetics ◽  
1989 ◽  
Vol 122 (2) ◽  
pp. 317-330 ◽  
Author(s):  
P McGraw ◽  
S A Henry
Keyword(s):  
Stationary Phase ◽  
De Novo ◽  
Growth Conditions ◽  
Temperature Sensitive ◽  
Phase Growth ◽  
Gene Effects ◽  
Growth Defects ◽  
Pathway Regulation ◽  
Cloned Gene

Abstract We report the isolation of two new opi3 mutants by EMS mutagenesis, and construction of an insertion allele in vitro using the cloned gene. We have demonstrated that the opi3 mutations cause a deficiency in the two terminal phospholipid N-methyltransferase (PLMT) activities required for the de novo synthesis of PC (phosphatidylcholine). The opi3 mutants, under certain growth conditions, produce membrane virtually devoid of PC although, surprisingly, none of the mutants displays a strict auxotrophic requirement for choline. Although the opi3 mutants grow without supplements, we have shown that the atypical membrane affects the ability of the mutant strains to initiate log phase growth and to sustain viability at stationary phase. The commencement of log phase growth is enhanced by addition of choline or to a lesser extent DME (dimethylethanolamine), and retarded by addition of MME (monomethylethanolamine). The mutant cells lose viability at the stationary phase of the cell cycle in the absence of DME or choline, and are also temperature sensitive for growth at 37 degrees especially in media containing MME. These growth defects have been correlated to the presence of specific phospholipids in the membrane. The opi3 growth defects are suppressed by an unusual mutation in the phospholipid methylation pathway that perturbs the N-methyltransferase (PEMT) activity immediately preceding the reactions affected by the opi3 lesion. We believe this mutation, cho2-S, alters the substrate specificity of the PEMT. A secondary effect of opi3 mutations is disruption of the cross pathway regulation of the synthesis of the PI (phosphatidylinositol) precursor inositol. Synthesis of inositol is controlled through regulation of the INO1 gene which encodes inositol-1-phosphate synthase. This highly regulated gene is expressed constitutively in opi3 mutants. We have used the opi3 strains to demonstrate that synthesis of either PC or PD (phosphatidyldimethylethanolamine) will restore normal regulation of the INO1 gene.

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