scholarly journals Effects of Depleting the Essential Central Metabolic Enzyme Fructose-1,6-Bisphosphate Aldolase on the Growth and Viability of Candida albicans: Implications for Antifungal Drug Target Discovery

2006 ◽  
Vol 5 (8) ◽  
pp. 1371-1377 ◽  
Author(s):  
Alexandra Rodaki ◽  
Tim Young ◽  
Alistair J. P. Brown
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Drug Target ◽  
Antifungal Drug ◽  
Metabolic Enzyme ◽  
Systemic Candidiasis ◽  
Wild Type ◽  
Drug Target Discovery ◽  
Conditional Mutant ◽  
Fructose 1,6 Bisphosphate

ABSTRACT The central metabolic enzyme fructose-1,6-bisphosphate aldolase (Fba1p) catalyzes a reversible reaction required for both glycolysis and gluconeogenesis. Fba1p is a potential antifungal target because it is essential in yeast and because fungal and human aldolases differ significantly. To test the validity of Fba1p as an antifungal target, we have examined the effects of depleting this enzyme in the major fungal pathogen Candida albicans. Using a methionine/cysteine-conditional mutant (MET3-FBA1/fba1), we have shown that Fba1p is required for the growth of C. albicans. However, Fba1p must be depleted to below 5% of wild-type levels before growth is blocked. Furthermore, Fba1p depletion exerts static rather than cidal effects upon C. albicans. Fba1p is a relatively abundant and stable protein in C. albicans, and hence, Fba1p levels decay relatively slowly following MET3-FBA1 shutoff. Taken together, our observations can account for our observation that the virulence of MET3-FBA1/fba1 cells is only partially attenuated in the mouse model of systemic candidiasis. We conclude that an antifungal drug directed against Fba1p would have to be potent to be effective.

scholarly journals Molecular Elucidation of Riboflavin Production and Regulation in Candida albicans, toward a Novel Antifungal Drug Target

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Liesbeth Demuyser ◽  
Ilse Palmans ◽  
Paul Vandecruys ◽  
Patrick Van Dijck
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Drug Target ◽  
Fungal Infections ◽  
Antifungal Drug ◽  
Model Systems ◽  
High Morbidity ◽  
Riboflavin Production ◽  
Content Type ◽  
Novel Strategy

ABSTRACT Candida albicans is a major cause of fungal infections, both superficial and invasive. The economic costs as well as consequences for patient welfare are substantial. Only a few treatment options are available due to the high resemblance between fungal targets and host molecules, as both are eukaryotes. Riboflavin is a yellow pigment, also termed vitamin B2. Unlike animals, fungi can synthesize this essential component themselves, thereby leading us to appreciate that targeting riboflavin production is a promising novel strategy against fungal infections. Here, we report that the GTP cyclohydrolase encoded by C. albicans RIB1 (CaRIB1) is essential and rate-limiting for production of riboflavin in the fungal pathogen. We confirm the high potential of CaRib1 as an antifungal drug target, as its deletion completely impairs in vivo infectibility by C. albicans in model systems. Furthermore, the stimulating effect of iron deprivation and PKA activation on riboflavin production seems to involve CaRib1 and the upstream transcription factor CaSef1. Gathering insights in the synthesis mechanism of riboflavin in pathogenic fungi, like C. albicans, will allow us to design a novel strategy and specifically target this process to combat fungal infections. IMPORTANCE Candida albicans is an important fungal pathogen causing common superficial infections as well as invasive diseases with an extremely high morbidity and mortality. Antifungal therapies are limited in efficiency and availability. In this research, we describe the regulation of riboflavin production in C. albicans. Since riboflavin biosynthesis is essential to this organism, we can appreciate that targeting it would be a promising new strategy to combat these fungal infections. We provide evidence that one particular enzyme in the production process, CaRib1, would be most promising as an antifungal drug target, as it plays a central role in regulation and proves to be essential in a mouse model of systemic infection.

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.

Immunoelectron microscopic detection of mannan-antigen on yeast cells of Candida albicans

1992 ◽  
Vol 50 (1) ◽  
pp. 822-823
Author(s):  
Mercedes Edwards ◽  
Robert Trimble ◽  
William Samsonoff
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fungal Pathogen ◽  
Yeast Cells ◽  
Systemic Candidiasis ◽  
Worldwide Distribution ◽  
Circulating Antigen ◽  
Infection Mechanisms ◽  
Opportunistic Fungal Pathogen

Candida albicans (Ca) is an opportunistic fungal pathogen of worldwide distribution. In recent years, infections by this fungus have received increased attention due to their prevalence in immunocomproimsed hosts (e.g. AIDS patients). However, Ca infection mechanisms are still to be ascertained. Investigations on the antigenicity of this organism have found that mannan, a component of its cell wall, is a major circulating antigen in systemic candidiasis. A few previous reports using electron microscopy, have indicated that mannan is located at the cell wall, but the resolution of the published micrographs did not afford a conclusive determination of the antigen distribution.

scholarly journals Arginine-Induced Germ Tube Formation in Candida albicans Is Essential for Escape from Murine Macrophage Line RAW 264.7

2009 ◽  
Vol 77 (4) ◽  
pp. 1596-1605 ◽  
Author(s):  
Suman Ghosh ◽  
Dhammika H. M. L. P. Navarathna ◽  
David D. Roberts ◽  
Jake T. Cooper ◽  
Audrey L. Atkin ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Germ Tube ◽  
Tube Formation ◽  
Raw 264.7 ◽  
Dependent Manner ◽  
Wild Type ◽  
Normal Flora ◽  
Opportunistic Fungal Pathogen ◽  
Germ Tubes

ABSTRACT The opportunistic fungal pathogen Candida albicans is a part of the normal flora but it also causes systemic candidiasis if it reaches the bloodstream. Upon being phagocytized by macrophages, an important component of innate immunity, C. albicans rapidly upregulates a set of arginine biosynthetic genes. Arginine, urea, and CO2 induced hyphae in a density-dependent manner in wild-type, cph1/cph1, and rim101/rim101 strains but not in efg1/efg1 or cph1/cph1 efg1/efg1 strains. Arginase (Car1p) converts arginine to urea, which in turn is degraded by urea amidolyase (Dur1,2p) to produce CO2, a signal for hyphal switching. We used a dur1,2/dur1,2 mutant (KWN6) and the complemented strain, KWN8 (dur1,2/dur1,2::DUR1,2/DUR1,2) to study germ tube formation. KWN6 could not make germ tubes in the presence of arginine or urea but did in the presence of 5% CO2, which bypasses Dur1,2p. We also tested the effect of arginine on the interaction between the macrophage line RAW 264.7 and several strains of C. albicans. Arginine activated an Efg1p-dependent yeast-to-hypha switch, enabling wild-type C. albicans and KWN8 to escape from macrophages within 6 h, whereas KWN6 was defective in this regard. Additionally, two mutants that cannot synthesize arginine, BWP17 and SN152, were defective in making hyphae inside the macrophages, whereas the corresponding arginine prototrophs, DAY286 and SN87, formed germ tubes and escaped from macrophages. Therefore, metabolism of arginine by C. albicans controls hyphal switching and provides an important mechanism for escaping host defense.

scholarly journals The SUMO protease Ulp2 regulates genome stability and drug resistance in the human fungal pathogen Candida albicans

2021 ◽  
Author(s):  
Marzia Rizzo ◽  
Natthapon Soisangwan ◽  
Jan Soetaert ◽  
Samuel Vega-Estevez ◽  
Anna Selmecki ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Candida Albicans ◽  
Fungal Pathogen ◽  
Genome Instability ◽  
Antifungal Drugs ◽  
Antifungal Drug ◽  
Genome Plasticity ◽  
Sumo Protease ◽  
Human Fungal Pathogen ◽  
Life Threatening

AbstractStress-induced genome instability in microbial organisms is emerging as a critical regulatory mechanism for driving rapid and reversible adaption to drastic environmental changes. In Candida albicans, a human fungal pathogen that causes life-threatening infections, genome plasticity confers increased virulence and antifungal drug resistance. Discovering the mechanisms regulating C. albicans genome plasticity is a priority to understand how this and other microbial pathogens establish life-threatening infections and develop resistance to antifungal drugs. We identified the SUMO protease Ulp2 as a critical regulator of C. albicans genome integrity through genetic screening. Deletion of ULP2 leads to hypersensitivity to genotoxic agents and increased genome instability. This increased genome diversity causes reduced fitness under standard laboratory growth conditions but enhances adaptation to stress, making ulp2Δ/Δ cells more likely to thrive in the presence of antifungal drugs. Whole-genome sequencing indicates that ulp2Δ/Δ cells counteract antifungal drug-induced stress by developing segmental aneuploidies of chromosome R and chromosome I. We demonstrate that intrachromosomal repetitive elements drive the formation of complex novel genotypes with adaptive power.

scholarly journals Altered Expression of Selectable Marker URA3 in Gene-Disrupted Candida albicans Strains Complicates Interpretation of Virulence Studies

1998 ◽  
Vol 66 (11) ◽  
pp. 5301-5306 ◽  
Author(s):  
Jennifer Lay ◽  
L. Keith Henry ◽  
Julie Clifford ◽  
Yigal Koltin ◽  
Christine E. Bulawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Virulence Factors ◽  
Virulence Factor ◽  
Fungal Pathogen ◽  
Selectable Marker ◽  
Activity Levels ◽  
Wild Type ◽  
Altered Expression ◽  
Genes Encoding

ABSTRACT The ura-blaster technique for the disruption of Candida albicans genes has been employed in a number of studies to identify possible genes encoding virulence factors of this fungal pathogen. In this study, the URA3-encoded orotidine 5′-monophosphate (OMP) decarboxylase enzyme activities of C. albicans strains with ura-blaster-mediated genetic disruptions were measured. All strains harboring genetic lesions via the ura-blaster construct showed reduced OMP decarboxylase activities compared to that of the wild type when assayed. The activity levels in different gene disruptions varied, suggesting a positional effect on the level of gene expression. Because the URA3 gene ofC. albicans has previously been identified as a virulence factor for this microorganism, our results suggest that decreased virulence observed in strains constructed with the ura-blaster cassette cannot accurately be attributed, in all cases, to the targeted genetic disruption. Although revised methods for validating aURA3-disrupted gene as a target for antifungal drug development could be devised, it is clearly desirable to replaceURA3 with a different selectable marker that does not influence virulence.

scholarly journals The Golgi GDPase of the Fungal Pathogen Candida albicans Affects Morphogenesis, Glycosylation, and Cell Wall Properties

2002 ◽  
Vol 1 (3) ◽  
pp. 420-431 ◽  
Author(s):  
Ana B. Herrero ◽  
Daniela Uccelletti ◽  
Carlos B. Hirschberg ◽  
Angel Dominguez ◽  
Claudia Abeijon
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fungal Pathogen ◽  
Solid Medium ◽  
Chain Extension ◽  
Wild Type ◽  
Adhesive Properties ◽  
Hyphal Formation ◽  
Cell Wall Properties ◽  
Increased Susceptibility

ABSTRACT Cell wall mannoproteins are largely responsible for the adhesive properties and immunomodulation ability of the fungal pathogen Candida albicans. The outer chain extension of yeast mannoproteins occurs in the lumen of the Golgi apparatus. GDP-mannose must first be transported from the cytosol into the Golgi lumen, where mannose is transferred to mannans. GDP is hydrolyzed by a GDPase, encoded by GDA1, to GMP, which then exits the Golgi lumen in a coupled, equimolar exchange with cytosolic GDP-mannose. We isolated and disrupted the C. albicans homologue of the Saccharomyces cerevisiae GDA1 gene in order to investigate its role in protein mannosylation and pathogenesis. CaGda1p shares four apyrase conserved regions with other nucleoside diphosphatases. Membranes prepared from the C. albicans disrupted gda1/gda1 strain had a 90% decrease in the ability to hydrolyze GDP compared to wild type. The gda1/gda1 mutants showed a severe defect in O-mannosylation and reduced cell wall phosphate content. Other cell wall-related phenotypes are present, such as elevated chitin levels and increased susceptibility to attack by β-1,3-glucanases. Our results show that the C. albicans organism contains β-mannose at their nonreducing end, differing from S. cerevisiae, which has only α-linked mannose residues in its O-glycans. Mutants lacking both alleles of GDA1 grow at the same rate as the wild type but are partially blocked in hyphal formation in Lee solid medium and during induction in liquid by changes in temperature and pH. However, the mutants still form normal hyphae in the presence of serum and N-acetylglucosamine and do not change their adherence to HeLa cells. Taken together, our data are in agreement with the hypothesis that several pathways regulate the yeast-hypha transition. Gda1/gda1 cells offer a model for discriminating among them.

scholarly journals Cytocidal amino acid starvation of Saccharomyces cerevisiae and Candida albicans acetolactate synthase (ilv2Δ) mutants is influenced by the carbon source and rapamycin

Microbiology ◽  
2010 ◽  
Vol 156 (3) ◽  
pp. 929-939 ◽  
Author(s):  
Joanne M. Kingsbury ◽  
John H. McCusker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Carbon Source ◽  
Drug Target ◽  
Acetolactate Synthase ◽  
Antifungal Drug ◽  
Tor Pathway ◽  
Fold Reduction ◽  
Isoleucine Biosynthesis

The isoleucine and valine biosynthetic enzyme acetolactate synthase (Ilv2p) is an attractive antifungal drug target, since the isoleucine and valine biosynthetic pathway is not present in mammals, Saccharomyces cerevisiae ilv2Δ mutants do not survive in vivo, Cryptococcus neoformans ilv2 mutants are avirulent, and both S. cerevisiae and Cr. neoformans ilv2 mutants die upon isoleucine and valine starvation. To further explore the potential of Ilv2p as an antifungal drug target, we disrupted Candida albicans ILV2, and demonstrated that Ca. albicans ilv2Δ mutants were significantly attenuated in virulence, and were also profoundly starvation-cidal, with a greater than 100-fold reduction in viability after only 4 h of isoleucine and valine starvation. As fungicidal starvation would be advantageous for drug design, we explored the basis of the starvation-cidal phenotype in both S. cerevisiae and Ca. albicans ilv2Δ mutants. Since the mutation of ILV1, required for the first step of isoleucine biosynthesis, did not suppress the ilv2Δ starvation-cidal defects in either species, the cidal phenotype was not due to α-ketobutyrate accumulation. We found that starvation for isoleucine alone was more deleterious in Ca. albicans than in S. cerevisiae, and starvation for valine was more deleterious than for isoleucine in both species. Interestingly, while the target of rapamycin (TOR) pathway inhibitor rapamycin further reduced S. cerevisiae ilv2Δ starvation viability, it increased Ca. albicans ilv1Δ and ilv2Δ viability. Furthermore, the recovery from starvation was dependent on the carbon source present during recovery for S. cerevisiae ilv2Δ mutants, reminiscent of isoleucine and valine starvation inducing a viable but non-culturable-like state in this species, while Ca. albicans ilv1Δ and ilv2Δ viability was influenced by the carbon source present during starvation, supporting a role for glucose wasting in the Ca. albicans cidal phenotype.

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.

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