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

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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Polymorphic centromere locations in the pathogenic yeast Candida parapsilosis

Genome Research ◽

10.1101/gr.257816.119 ◽

2020 ◽

Vol 30 (5) ◽

pp. 684-696

Author(s):

Mihaela Ola ◽

Caoimhe E. O'Brien ◽

Aisling Y. Coughlan ◽

Qinxi Ma ◽

Paul D. Donovan ◽

...

Keyword(s):

Candida Parapsilosis ◽

Pathogenic Yeast

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Unexpected mutual regulation underlies paralogue functional diversification and promotes epithelial tissue maturation in Tribolium

Communications Biology ◽

10.1038/s42003-020-01250-3 ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Daniela Gurska ◽

Iris M. Vargas Jentzsch ◽

Kristen A. Panfilio

Keyword(s):

Feedback Loop ◽

Single Species ◽

Epithelial Tissue ◽

Rna Seq ◽

Developmental Progression ◽

Late Embryogenesis ◽

Mutual Regulation ◽

Transcriptional Landscape

Abstract Insect Hox3/zen genes represent an evolutionary hotspot for changes in function and copy number. Single orthologues are required either for early specification or late morphogenesis of the extraembryonic tissues, which protect the embryo. The tandemly duplicated zen paralogues of the beetle Tribolium castaneum present a unique opportunity to investigate both functions in a single species. We dissect the paralogues’ expression dynamics (transcript and protein) and transcriptional targets (RNA-seq after RNAi) throughout embryogenesis. We identify an unexpected role of Tc-Zen2 in repression of Tc-zen1, generating a negative feedback loop that promotes developmental progression. Tc-Zen2 regulation is dynamic, including within co-expressed multigene loci. We also show that extraembryonic development is the major event within the transcriptional landscape of late embryogenesis and provide a global molecular characterization of the extraembryonic serosal tissue. Altogether, we propose that paralogue mutual regulation arose through multiple instances of zen subfunctionalization, leading to their complementary extant roles.

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Genetic manipulation of the pathogenic yeast Candida parapsilosis

Current Genetics ◽

10.1007/s00294-002-0326-7 ◽

2002 ◽

Vol 42 (1) ◽

pp. 27-35 ◽

Cited By ~ 22

Author(s):

Jozef Nosek ◽

Ľubica Adamíkovâ ◽

Júlia Zemanová ◽

Ľubonír Tomáška ◽

Rachel Zufferey ◽

...

Keyword(s):

Candida Parapsilosis ◽

Genetic Manipulation ◽

Pathogenic Yeast

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Correlation between Biofilm Formation and the Hypoxic Response in Candida parapsilosis

Eukaryotic Cell ◽

10.1128/ec.00350-08 ◽

2009 ◽

Vol 8 (4) ◽

pp. 550-559 ◽

Cited By ~ 57

Author(s):

Tristan Rossignol ◽

Chen Ding ◽

Alessandro Guida ◽

Christophe d'Enfert ◽

Desmond G. Higgins ◽

...

Keyword(s):

Cell Wall ◽

Biofilm Formation ◽

Candida Parapsilosis ◽

Transcriptional Profile ◽

Open Reading Frames ◽

Hypoxic Response ◽

Hypoxic Conditions ◽

Expression Of Genes ◽

Biofilm Development ◽

Reading Frames

ABSTRACT The ability of Candida parapsilosis to form biofilms on indwelling medical devices is correlated with virulence. To identify genes that are important for biofilm formation, we used arrays representing approximately 4,000 open reading frames (ORFs) to compare the transcriptional profile of biofilm cells growing in a microfermentor under continuous flow conditions with that of cells in planktonic culture. The expression of genes involved in fatty acid and ergosterol metabolism and in glycolysis, is upregulated in biofilms. The transcriptional profile of C. parapsilosis biofilm cells resembles that of Candida albicans cells grown under hypoxic conditions. We therefore subsequently used whole-genome arrays (representing 5,900 ORFs) to determine the hypoxic response of C. parapsilosis and showed that the levels of expression of genes involved in the ergosterol and glycolytic pathways, together with several cell wall genes, are increased. Our results indicate that there is substantial overlap between the hypoxic responses of C. parapsilosis and C. albicans and that this may be important for biofilm development. Knocking out an ortholog of the cell wall gene RBT1, whose expression is induced both in biofilms and under conditions of hypoxia in C. parapsilosis, reduces biofilm development.

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Unexpected mutual regulation underlies paralogue functional diversification and promotes maturation of a protective epithelial tissue

10.1101/427245 ◽

2018 ◽

Cited By ~ 1

Author(s):

Daniela Gurska ◽

Iris M. Vargas Jentzsch ◽

Kristen A. Panfilio

Keyword(s):

Feedback Loop ◽

Single Species ◽

Epithelial Tissue ◽

Rna Seq ◽

Developmental Progression ◽

Late Embryogenesis ◽

Mutual Regulation ◽

Transcriptional Landscape

ABSTRACTInsect Hox3/zen genes represent an evolutionary hotspot for changes in function and copy number. Single orthologues are required either for early specification or late morphogenesis of the extraembryonic tissues, which protect the embryo. The tandemly duplicated zen paralogues of the beetle Tribolium castaneum present a unique opportunity to investigate both functions in a single species. We dissect the paralogues’ expression dynamics (transcript and protein) and transcriptional targets (RNA-seq after RNAi) throughout embryogenesis. We identify an unexpected role of Tc-Zen2 in repression of Tc-zen1, generating a negative feedback loop that promotes developmental progression. Tc-Zen2 regulation is dynamic, including within co-expressed multigene loci. We also show that extraembryonic development is the major event within the transcriptional landscape of late embryogenesis and provide a global molecular characterization of the extraembryonic serosal tissue. Altogether, we propose that paralogue mutual regulation arose progressively and drove multiple instances of zen subfunctionalization, leading to complementary extant roles.

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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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Transcriptional landscape of the human cell cycle

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1617636114 ◽

2017 ◽

Vol 114 (13) ◽

pp. 3473-3478 ◽

Cited By ~ 54

Author(s):

Yin Liu ◽

Sujun Chen ◽

Su Wang ◽

Fraser Soares ◽

Martin Fischer ◽

...

Keyword(s):

Cell Cycle ◽

Steady State ◽

Histone Modification ◽

Human Cell ◽

Rna Seq ◽

Motif Analysis ◽

Chip Sequencing ◽

Histone 3 ◽

M Phase ◽

Transcriptional Landscape

Steady-state gene expression across the cell cycle has been studied extensively. However, transcriptional gene regulation and the dynamics of histone modification at different cell-cycle stages are largely unknown. By applying a combination of global nuclear run-on sequencing (GRO-seq), RNA sequencing (RNA-seq), and histone-modification Chip sequencing (ChIP-seq), we depicted a comprehensive transcriptional landscape at the G0/G1, G1/S, and M phases of breast cancer MCF-7 cells. Importantly, GRO-seq and RNA-seq analysis identified different cell-cycle–regulated genes, suggesting a lag between transcription and steady-state expression during the cell cycle. Interestingly, we identified genes actively transcribed at early M phase that are longer in length and have low expression and are accompanied by a global increase in active histone 3 lysine 4 methylation (H3K4me2) and histone 3 lysine 27 acetylation (H3K27ac) modifications. In addition, we identified 2,440 cell-cycle–regulated enhancer RNAs (eRNAs) that are strongly associated with differential active transcription but not with stable expression levels across the cell cycle. Motif analysis of dynamic eRNAs predicted Kruppel-like factor 4 (KLF4) as a key regulator of G1/S transition, and this identification was validated experimentally. Taken together, our combined analysis characterized the transcriptional and histone-modification profile of the human cell cycle and identified dynamic transcriptional signatures across the cell cycle.

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