Mitochondrial Carriers Link the Catabolism of Hydroxyaromatic Compounds to the Central Metabolism in Candida parapsilosis

Abstract The pathogenic yeast Candida parapsilosis metabolizes hydroxyderivatives of benzene and benzoic acid to compounds channeled into central metabolism, including the mitochondrially localized tricarboxylic acid cycle, via the 3-oxoadipate and gentisate pathways. The orchestration of both catabolic pathways with mitochondrial metabolism as well as their evolutionary origin is not fully understood. Our results show that the enzymes involved in these two pathways operate in the cytoplasm with the exception of the mitochondrially targeted 3-oxoadipate CoA-transferase (Osc1p) and 3-oxoadipyl-CoA thiolase (Oct1p) catalyzing the last two reactions of the 3-oxoadipate pathway. The cellular localization of the enzymes indicates that degradation of hydroxyaromatic compounds requires a shuttling of intermediates, cofactors, and products of the corresponding biochemical reactions between cytosol and mitochondria. Indeed, we found that yeast cells assimilating hydroxybenzoates increase the expression of genes SFC1, LEU5, YHM2, and MPC1 coding for succinate/fumarate carrier, coenzyme A carrier, oxoglutarate/citrate carrier, and the subunit of pyruvate carrier, respectively. A phylogenetic analysis uncovered distinct evolutionary trajectories for sparsely distributed gene clusters coding for enzymes of both pathways. Whereas the 3-oxoadipate pathway appears to have evolved by vertical descent combined with multiple losses, the gentisate pathway shows a striking pattern suggestive of horizontal gene transfer to the evolutionarily distant Mucorales.

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Transcriptome and proteome profiling reveals complex adaptations of Candida parapsilosis cells assimilating hydroxyaromatic carbon sources

10.1101/2021.09.13.460007 ◽

2021 ◽

Author(s):

Andrea Cillingová ◽

Renáta Tóth ◽

Anna Mojáková ◽

Igor Zeman ◽

Romana Vrzoňová ◽

...

Keyword(s):

Candida Parapsilosis ◽

Expression Profiles ◽

Glyoxylate Cycle ◽

Carbon Sources ◽

Gene Clusters ◽

Fungal Species ◽

Yeast Cells ◽

Gentisate Pathway ◽

Proteomic Investigation ◽

Pathway Regulation

Many fungal species utilize hydroxyderivatives of benzene and benzoic acid as carbon sources. The yeast Candida parapsilosis metabolizes these compounds via the 3-oxoadipate and gentisate pathways, whose components are encoded by two metabolic gene clusters. In this study, we determine the chromosome level assembly of the C. parapsilosis strain CLIB214 and use it for transcriptomic and proteomic investigation of cells cultivated on hydroxyaromatic substrates. We demonstrate that the genes coding for enzymes and plasma membrane transporters involved in the 3-oxoadipate and gentisate pathways are highly upregulated and their expression is controlled in a substrate-specific manner. However, regulatory proteins involved in this process are not known. Using the knockout mutants, we show that putative transcriptional factors encoded by the genes OTF1 and GTF1 located within these gene clusters function as transcriptional activators of the 3-oxoadipate and gentisate pathway, respectively. We also show that the activation of both pathways is accompanied by upregulation of genes for the enzymes involved in β-oxidation of fatty acids, glyoxylate cycle, amino acid metabolism, and peroxisome biogenesis. Transcriptome and proteome profiles of the cells grown on 4-hydroxybenzoate and 3-hydroxybenzoate, which are metabolized via the 3-oxoadipate and gentisate pathway, respectively, reflect their different connection to central metabolism. Yet we find that the expression profiles differ also in the cells assimilating 4-hydroxybenzoate and hydroquinone, which are both metabolized in the same pathway. This finding is consistent with the phenotype of the Otf1p-lacking mutant, which exhibits impaired growth on hydroxybenzoates, but still utilizes hydroxybenzenes, thus indicating that additional, yet unidentified transcription factor could be involved in the 3-oxoadipate pathway regulation. Moreover, we propose that bicarbonate ions resulting from decarboxylation of hydroxybenzoates also contribute to differences in the cell responses to hydroxybenzoates and hydroxybenzenes. Finally, our phylogenetic analysis highlights evolutionary paths leading to metabolic adaptations of yeast cells assimilating hydroxyaromatic substrates.

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Gentisate and 3-oxoadipate pathways in the yeast Candida parapsilosis: identification and functional analysis of the genes coding for 3-hydroxybenzoate 6-hydroxylase and 4-hydroxybenzoate 1-hydroxylase

Microbiology ◽

10.1099/mic.0.048215-0 ◽

2011 ◽

Vol 157 (7) ◽

pp. 2152-2163 ◽

Cited By ~ 26

Author(s):

Zuzana Holesova ◽

Michaela Jakubkova ◽

Ivana Zavadiakova ◽

Igor Zeman ◽

Lubomir Tomaska ◽

...

Keyword(s):

Functional Analysis ◽

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

Candida Parapsilosis ◽

Intracellular Localization ◽

Cultivation Medium ◽

Pathogenic Yeast ◽

Gentisate Pathway ◽

Derivatives Of

The pathogenic yeast Candida parapsilosis degrades various hydroxy derivatives of benzenes and benzoates by the gentisate and 3-oxoadipate pathways. We identified the genes MNX1, MNX2, MNX3, GDX1, HDX1 and FPH1 that code for enzymes involved in these pathways in the complete genome sequence of C. parapsilosis. Next, we demonstrated that MNX1, MNX2, MNX3 and GDX1 are inducible and transcriptionally controlled by hydroxyaromatic substrates present in cultivation media. Our results indicate that MNX1 and MNX2 code for flavoprotein monooxygenases catalysing the first steps in the 3-oxoadipate and gentisate pathways, respectively (i.e. 4-hydroxybenzoate 1-hydroxylase and 3-hydroxybenzoate 6-hydroxylase). Moreover, we found that the two pathways differ by their intracellular localization. The enzymes of the 3-oxoadipate pathway, Mnx1p and Mnx3p, localize predominantly in the cytosol. In contrast, intracellular localization of the components of the gentisate pathway, Mnx2p and Gdx1p, depends on the substrate in the cultivation medium. In cells growing on glucose these proteins localize in the cytosol, whereas in media containing hydroxyaromatic compounds they associate with mitochondria. Finally, we showed that the overexpression of MNX1 or MNX2 increases the tolerance of C. parapsilosis cells to the antifungal drug terbinafine.

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Oxidative Stress-Induced Alteration of Plant Central Metabolism

Life ◽

10.3390/life11040304 ◽

2021 ◽

Vol 11 (4) ◽

pp. 304

Author(s):

Tatyana Savchenko ◽

Konstantin Tikhonov

Keyword(s):

Oxidative Stress ◽

Stress Responses ◽

Tricarboxylic Acid Cycle ◽

Strong Dependence ◽

Plant Stress ◽

Metabolite Profile ◽

Agricultural Practice ◽

Central Metabolism ◽

Metabolic Markers ◽

Sugar Derivatives

Oxidative stress is an integral component of various stress conditions in plants, and this fact largely determines the substantial overlap in physiological and molecular responses to biotic and abiotic environmental challenges. In this review, we discuss the alterations in central metabolism occurring in plants experiencing oxidative stress. To focus on the changes in metabolite profile associated with oxidative stress per se, we primarily analyzed the information generated in the studies based on the exogenous application of agents, inducing oxidative stress, and the analysis of mutants displaying altered oxidative stress response. Despite of the significant variation in oxidative stress responses among different plant species and tissues, the dynamic and transient character of stress-induced changes in metabolites, and the strong dependence of metabolic responses on the intensity of stress, specific characteristic changes in sugars, sugar derivatives, tricarboxylic acid cycle metabolites, and amino acids, associated with adaptation to oxidative stress have been detected. The presented analysis of the available data demonstrates the oxidative stress-induced redistribution of metabolic fluxes targeted at the enhancement of plant stress tolerance through the prevention of ROS accumulation, maintenance of the biosynthesis of indispensable metabolites, and production of protective compounds. This analysis provides a theoretical basis for the selection/generation of plants with improved tolerance to oxidative stress and the development of metabolic markers applicable in research and routine agricultural practice.

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Using RNA-seq to determine the transcriptional landscape and the hypoxic response of the pathogenic yeast Candida parapsilosis

BMC Genomics ◽

10.1186/1471-2164-12-628 ◽

2011 ◽

Vol 12 (1) ◽

Cited By ~ 50

Author(s):

Alessandro Guida ◽

Claudia Lindstädt ◽

Sarah L Maguire ◽

Chen Ding ◽

Desmond G Higgins ◽

...

Keyword(s):

Candida Parapsilosis ◽

Rna Seq ◽

Pathogenic Yeast ◽

Hypoxic Response ◽

Transcriptional Landscape

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Bacterial cell cycle control by citrate synthase independent of enzymatic activity

10.1101/799742 ◽

2019 ◽

Author(s):

Matthieu Bergé ◽

Julian Pezzatti ◽

Víctor González-Ruiz ◽

Laurence Degeorges ◽

Serge Rudaz ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Citrate Synthase ◽

Caulobacter Crescentus ◽

Tricarboxylic Acid Cycle ◽

Cell Cycle Control ◽

Tca Cycle ◽

Cell Cycle Regulators ◽

Central Metabolism ◽

Cycle Enzyme

ABSTRACTCoordination of cell cycle progression with central metabolism is fundamental to all cell types and likely underlies differentiation into dispersal cells in bacteria. How central metabolism is monitored to regulate cell cycle functions is poorly understood. A forward genetic selection for cell cycle regulators in the polarized alpha-proteobacterium Caulobacter crescentus unearthed the uncharacterized CitA citrate synthase, a TCA (tricarboxylic acid) cycle enzyme, as unprecedented checkpoint regulator of the G1→S transition. We show that loss of the CitA protein provokes a (p)ppGpp alarmone-dependent G1-phase arrest without apparent metabolic or energy insufficiency. While S-phase entry is still conferred when CitA is rendered catalytically inactive, the paralogous CitB citrate synthase has no overt role other than sustaining TCA cycle activity when CitA is absent. With eukaryotic citrate synthase paralogs known to fulfill regulatory functions, our work extends the moonlighting paradigm to citrate synthase coordinating central (TCA) metabolism with development and perhaps antibiotic tolerance in bacteria.

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Resistance to miltefosine results from amplification of the RTA3 floppase or inactivation of flippases in Candida parapsilosis

10.1101/2021.12.16.473093 ◽

2021 ◽

Author(s):

Sean Bergin ◽

Fang Zhao ◽

Adam P Ryan ◽

Carolin A Müller ◽

Conrad A Nieduszynski ◽

...

Keyword(s):

Plasma Membrane ◽

Copy Number ◽

Candida Parapsilosis ◽

Copy Number Variations ◽

Loss Of Function ◽

Adaptive Laboratory Evolution ◽

Pathogenic Yeast ◽

Lipid Asymmetry ◽

Evolution Experiment ◽

Almost All

Flippases and floppases are two classes of proteins that have opposing functions in the maintenance of lipid asymmetry of the plasma membrane. Flippases translocate lipids from the exoplasmic leaflet to the cytosolic leaflet, and floppases act in the opposite direction. Phosphatidylcholine (PC) is a major component of the eukaryotic plasma membrane and is asymmetrically distributed, being more abundant in the exoplasmic leaflet. Here we show that gene amplification of a putative PC floppase or double disruption of two PC flippases in the pathogenic yeast Candida parapsilosis results in resistance to miltefosine, an alkylphosphocholine drug that affects PC metabolism that has recently been granted orphan drug designation approval by the US FDA for treatment of invasive candidiasis. We analysed the genomes of 170 C. parapsilosis isolates and found that 107 of them have copy number variations (CNVs) at the RTA3 gene. RTA3 encodes a putative PC floppase whose deletion is known to increase the inward translocation of PC in Candida albicans. RTA3 copy number ranges from 2 to >40 across the C. parapsilosis isolates. Interestingly, 16 distinct CNVs with unique endpoints were identified, and phylogenetic analysis shows that almost all of them have originated only once. We found that increased copy number of RTA3 correlates with miltefosine resistance. Additionally, we conducted an adaptive laboratory evolution experiment in which two C. parapsilosis isolates were cultured in increasing concentrations of miltefosine over 26 days. Two genes, CPAR2_303950 and CPAR2_102700, gained homozygous protein-disrupting mutations in the evolved strains and code for putative PC flippases homologous to S. cerevisiae DNF1. Our results indicate that alteration of lipid asymmetry across the plasma membrane is a key mechanism of miltefosine resistance. We also find that C. parapsilosis is likely to gain resistance to miltefosine rapidly, because many isolates carry loss-of-function alleles in one of the flippase genes.

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Dal81 Regulates Expression of Arginine Metabolism Genes inCandida parapsilosis

mSphere ◽

10.1128/msphere.00028-18 ◽

2018 ◽

Vol 3 (2) ◽

Cited By ~ 3

Author(s):

Siobhan A. Turner ◽

Qinxi Ma ◽

Mihaela Ola ◽

Kontxi Martinez de San Vicente ◽

Geraldine Butler

Keyword(s):

Saccharomyces Cerevisiae ◽

Pathogenic Fungus ◽

Nitrogen Sources ◽

Nitrogen Utilization ◽

Nitrogen Regulation ◽

Transcriptional Level ◽

Pathogenic Yeast ◽

Secondary Sources ◽

Expression Of Genes ◽

Maximal Induction

ABSTRACTFungi can use a wide variety of nitrogen sources. In the absence of preferred sources such as ammonium, glutamate, and glutamine, secondary sources, including most other amino acids, are used. Expression of the nitrogen utilization pathways is very strongly controlled at the transcriptional level. Here, we investigated the regulation of nitrogen utilization in the pathogenic yeastCandida parapsilosis. We found that the functions of many regulators are conserved with respect toSaccharomyces cerevisiaeand other fungi. For example, the core GATA activatorsGAT1andGLN3have a conserved role innitrogencataboliterepression (NCR). There is one ortholog ofGZF3andDAL80, which represses expression of genes in preferred nitrogen sources. The regulatorsPUT3andUGA3are required for metabolism of proline and γ-aminobutyric acid (GABA), respectively. However, the role of the Dal81 transcription factor is distinctly different. InS. cerevisiae, Dal81 is a positive regulator of acquisition of nitrogen from GABA, allantoin, urea, and leucine, and it is required for maximal induction of expression of the relevant pathway genes. InC. parapsilosis, induction of GABA genes is independent of Dal81, and deletingDAL81has no effect on acquisition of nitrogen from GABA or allantoin. Instead, Dal81 represses arginine synthesis during growth under preferred nitrogen conditions.IMPORTANCEUtilization of nitrogen by fungi is controlled bynitrogencataboliterepression (NCR). Expression of many genes is switched off during growth on nonpreferred nitrogen sources. Gene expression is regulated through a combination of activation and repression. Nitrogen regulation has been studied best in the model yeastSaccharomyces cerevisiae. We found that although many nitrogen regulators have a conserved function inSaccharomycesspecies, some do not. The Dal81 transcriptional regulator has distinctly different functions inS. cerevisiaeandC. parapsilosis. In the former, it regulates utilization of nitrogen from GABA and allantoin, whereas in the latter, it regulates expression of arginine synthesis genes. Our findings make an important contribution to our understanding of nitrogen regulation in a human-pathogenic fungus.

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Steroid Degradation in Comamonas testosteroni TA441: Identification of the Entire β-Oxidation Cycle of the Cleaved B Ring

Applied and Environmental Microbiology ◽

10.1128/aem.01204-19 ◽

2019 ◽

Vol 85 (20) ◽

Cited By ~ 4

Author(s):

Masae Horinouchi ◽

Hiroyuki Koshino ◽

Michal Malon ◽

Hiroshi Hirota ◽

Toshiaki Hayashi

Keyword(s):

Gene Clusters ◽

Degradation Process ◽

Degradation Pathway ◽

Ring Cleavage ◽

Comamonas Testosteroni ◽

Content Type ◽

Coa Transferase ◽

Degrading Bacteria ◽

Globally Distributed

ABSTRACT Comamonas testosteroni TA441 degrades steroids via aromatization of the A ring, followed by degradation of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, mainly by β-oxidation. In this study, we revealed that 7β,9α-dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-coenzyme A (CoA) ester is dehydrogenated by (3S)-3-hydroxylacyl CoA-dehydrogenase, encoded by scdE (ORF27), and then the resultant 9α-hydroxy-7,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is converted by 3-ketoacyl-CoA transferase, encoded by scdF (ORF23). With these results, the whole cycle of β-oxidation on the side chain at C-8 of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid is clarified; 9-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is dehydrogenated at C-6 by ScdC1C2, followed by hydration by ScdD. 7β,9α-Dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-CoA ester then is dehydrogenated by ScdE to be converted to 9α-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid-CoA ester and acetyl-CoA by ScdF. ScdF is an ortholog of FadA6 in Mycobacterium tuberculosis H37Rv, which was reported as a 3-ketoacyl-CoA transferase involved in C ring cleavage. We also obtained results suggesting that ScdF is also involved in C ring cleavage, but further investigation is required for confirmation. ORF25 and ORF26, located between scdF and scdE, encode enzymes belonging to the amidase superfamily. Disrupting either ORF25 or ORF26 did not affect steroid degradation. Among the bacteria having gene clusters similar to those of tesB to tesR, some have both ORF25- and ORF26-like proteins or only an ORF26-like protein, but others do not have either ORF25- or ORF26-like proteins. ORF25 and ORF26 are not crucial for steroid degradation, yet they might provide clues to elucidate the evolution of bacterial steroid degradation clusters. IMPORTANCE Studies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain materials for steroid drugs. Steroid-degrading bacteria are globally distributed, and the role of bacterial steroid degradation in the environment as well as in relation to human health is attracting attention. The overall aerobic degradation of the four basic steroidal rings has been proposed; however, there is still much to be revealed to understand the complete degradation pathway. This study aims to uncover the whole steroid degradation process in Comamonas testosteroni TA441 as a model of steroid-degrading bacteria. C. testosteroni is one of the most studied representative steroid-degrading bacteria and is suitable for exploring the degradation pathway, because the involvement of degradation-related genes can be determined by gene disruption. Here, we elucidated the entire β-oxidation cycle of the cleaved B ring. This cycle is essential for the following C and D ring cleavage.

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A Kinetic Modelling of Enzyme Inhibitions in the Central Metabolism of Yeast Cells

Journal of Physics Conference Series ◽

10.1088/1742-6596/979/1/012066 ◽

2018 ◽

Vol 979 ◽

pp. 012066

Author(s):

Kasbawati ◽

A Kalondeng ◽

N Aris ◽

N Erawaty ◽

M I Azis

Keyword(s):

Kinetic Modelling ◽

Yeast Cells ◽

Central Metabolism

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Toxic, Radical Scavenging, and Antifungal Activity of Rhododendron tomentosum H. Essential Oils

Molecules ◽

10.3390/molecules25071676 ◽

2020 ◽

Vol 25 (7) ◽

pp. 1676

Author(s):

Asta Judzentiene ◽

Jurga Budiene ◽

Jurgita Svediene ◽

Rasa Garjonyte

Keyword(s):

Antifungal Activity ◽

Essential Oils ◽

Radical Scavenging Activity ◽

Radical Scavenging ◽

Fold Increase ◽

Yeast Cells ◽

Yeast Saccharomyces Cerevisiae ◽

Pathogenic Yeast ◽

Toxic Activity ◽

Agar Disc

The chemical composition of eight (seven shoot and one inflorescence) essential oils (EOs) of Rh. tomentosum H. plants growing in Eastern Lithuania is reported. The plant material was collected during different phases of vegetation (from April to October). The oils were obtained by hydrodistillation from air-dried aerial parts (leaves and inflorescences). In total, up to 70 compounds were identified by GC−MS and GC (flame-ionization detector, FID); they comprised 91.0 ± 4.7%–96.2 ± 3.1% of the oil content. Sesquiterpene hydrocarbons (54.1 ± 1.5%–76.1 ± 4.5%) were found to be the main fraction. The major compounds were palustrol (24.6 ± 2.6%–33.5 ± 4.4%) and ledol (18.0 ± 2.9%–29.0 ± 5.0%). Ascaridol isomers (7.0 ± 2.4%–14.0 ± 2.4% in three oils), myrcene (7.2 ± 0.3% and 10.1 ± 1.3%), lepalol (3.3 ± 0.3% and 7.9 ± 3.0%), and cyclocolorenone isomers (4.1 ± 2.5%) were determined as the third main constituents. The toxic activity of marsh rosemary inflorescence and shoot oils samples was evaluated using a brine shrimp (Artemia sp.) bioassay. LC50 average values (11.23–20.50 µg/mL) obtained after 24 h of exposure revealed that the oils were notably toxic. The oil obtained from shoots gathered in September during the seed-ripening stage and containing appreciable amounts of palustrol (26.0 ± 2.5%), ledol (21.5 ± 4.0%), and ascaridol (7.0 ± 2.4%) showed the highest toxic activity. Radical scavenging activity of Rh. tomentosum EOs depended on the plant vegetation stage. The highest activities were obtained for EOs isolated from young shoots collected in June (48.19 ± 0.1 and 19.89 ± 0.3 mmol/L TROLOX (6-hydroxy-2,5,7,8-tetra-methylchromane-2-carboxylic acid) equivalent obtained by, respectively, ABTS•+ (2,2′-amino-bis(ethylbenzothiazoline-6-sulfonic acid) diammonium salt) and DPPH•(2,2-diphenyl-1-picrylhydrazyl) assays). Agar disc diffusion assay against pathogenic yeast Candida parapsilosis revealed the potential antifungal activity of EOs. An alternative investigation of antifungal activity employed mediated amperometry at yeast Saccharomyces cerevisiae-modified electrodes. The subjection of yeast cells to vapors of EO resulted in a three to four-fold increase of electrode responses due to the disruption of yeast cell membranes.

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