scholarly journals High-Resolution Genome-Wide Occupancy in Candida spp. Using ChEC-seq

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Faiza Tebbji ◽  
Inès Khemiri ◽  
Adnane Sellam
Keyword(s):  
Public Health ◽  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
High Resolution ◽  
Transcriptional Regulator ◽  
Candida Auris ◽  
Content Type ◽  
Genome Wide ◽  
Public Health Threat

ABSTRACT To persist in their dynamic human host environments, fungal pathogens must sense and adapt by modulating their gene expression to fulfill their cellular needs. Understanding transcriptional regulation on a global scale would uncover cellular processes linked to persistence and virulence mechanisms that could be targeted for antifungal therapeutics. Infections associated with the yeast Candida albicans, a highly prevalent fungal pathogen, and the multiresistant related species Candida auris are becoming a serious public health threat. To define the set of a gene regulated by a transcriptional regulator in C. albicans, chromatin immunoprecipitation (ChIP)-based techniques, including ChIP with microarray technology (ChIP-chip) or ChIP-DNA sequencing (ChIP-seq), have been widely used. Here, we describe a new set of PCR-based micrococcal nuclease (MNase)-tagging plasmids for C. albicans and other Candida spp. to determine the genome-wide location of any transcriptional regulator of interest using chromatin endogenous cleavage (ChEC) coupled to high-throughput sequencing (ChEC-seq). The ChEC procedure does not require protein-DNA cross-linking or sonication, thus avoiding artifacts related to epitope masking or the hyper-ChIPable euchromatic phenomenon. In a proof-of-concept application of ChEC-seq, we provided a high-resolution binding map of the SWI/SNF chromatin remodeling complex, a master regulator of fungal fitness in C. albicans, in addition to the transcription factor Nsi1 that is an ortholog of the DNA-binding protein Reb1 for which genome-wide occupancy was previously established in Saccharomyces cerevisiae. The ChEC-seq procedure described here will allow a high-resolution genomic location definition which will enable a better understanding of transcriptional regulatory circuits that govern fungal fitness and drug resistance in these medically important fungi. IMPORTANCE Systemic fungal infections caused by Candida albicans and the “superbug” Candida auris are becoming a serious public health threat. The ability of these yeasts to cause disease is linked to their faculty to modulate the expression of genes that mediate their escape from the immune surveillance and their persistence in the different unfavorable niches within the host. Comprehensive knowledge on gene expression control of fungal fitness is consequently an interesting framework for the identification of essential infection processes that could be hindered by chemicals as potential therapeutics. Here, we expanded the use of ChEC-seq, a technique that was initially developed in the yeast model Saccharomyces cerevisiae to identify genes that are modulated by a transcriptional regulator, in pathogenic yeasts from the genus Candida. This robust technique will allow a better characterization of key gene expression regulators and their contribution to virulence and antifungal resistance in these pathogenic yeasts.

scholarly journals High resolution genome-wide occupancy in Candida spp. using ChEC-seq

2020 ◽  
Author(s):  
Faiza Tebbji ◽  
Inès Khemiri ◽  
Adnane Sellam
Keyword(s):  
Public Health ◽  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
High Resolution ◽  
Transcriptional Regulator ◽  
Candida Spp ◽  
Health Threat ◽  
Genome Wide ◽  
Public Health Threat

AbstractTo persist in their hostile and dynamic human host environments, fungal pathogens has to sense and adapt by modulating their gene expression to fulfil their cellular needs. Understanding transcriptional regulation on a global scale would uncover cellular processes linked to persistence and virulence mechanisms that could be targeted for antifungal therapeutics. Infections associated with the yeast Candida albicans, a highly prevalent fungal pathogen, and the multi-resistant related species C. auris are becoming a serious public health threat. To define the set of a gene regulated by a transcriptional regulator in C. albicans, Chromatin Immuno-Precipitation (ChIP) based techniques including ChIP-chip or ChIP-seq has been widely used. Here, we describe a new set of PCR-based MNase-tagging plasmids for C. albicans and other Candida spp. to determine genome-wide location of any transcriptional regulator of interest using Chromatin endogenous cleavage (ChEC) coupled to high-throughput sequencing (ChEC-seq). The ChEC procedure does not require protein-DNA crosslinking or sonication avoiding thus artefacts related to epitope masking or the hyper-ChIPable euchromatic phenomenon. In a proof-of-concept application of ChEC-seq, we provided a high-resolution binding map of the SWI/SNF chromatin remodeling complex, a master regulator of fungal fitness in C. albicans in addition to the transcription factor NsiI that is an ortholog of the DNA-binding protein Reb1 for which genome-wide occupancy were previously established in Saccharomyces cerevisiae. The ChEC-seq procedure described here will allow a high-resolution genomic location definition which will enable a better understanding of transcriptional regulatory circuits that govern fungal fitness and drug resistance in these medically important fungi.ImportanceSystemic fungal infections caused by Candida albicans and the ‘superbug’ C. auris are becoming a serious public health threat. The ability of these yeasts to cause disease is linked to their faculty to modulate the expression of genes that mediate their escape from the immune surveillance and their persistence in the different unfavourable niches within the host. Comprehensive knowledge on gene expression control of fungal fitness is consequently an interesting framework for the identification of essential infection processes that could be hindered by chemicals as potential therapeutics. Here, we expanded the use of ChEC-seq, a technique that was initially developed in the yeast model Saccharomyces cerevisiae to identify genes that are modulated by a transcriptional regulator, to the pathogenic yeasts C. albicans and C. auris. This robust technique will allow a better characterization of key gene expression regulators and their contribution to virulence and antifungal resistance in these pathogenic yeasts.

scholarly journals Reconfiguration of Transcriptional Control of Lysine Biosynthesis in Candida albicans Involves a Central Role for the Gcn4 Transcriptional Activator

mSphere ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Yumnam Priyadarshini ◽  
Krishnamurthy Natarajan
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Fungal Pathogen ◽  
Biosynthetic Pathway ◽  
Transcriptional Regulator ◽  
Lysine Biosynthesis ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Starvation Conditions

ABSTRACT Microbes evolve rapidly so as to reconfigure their gene expression to adapt to the metabolic demands in diverse environmental niches. Here, we explored how conditions of nutrient deprivation regulate lysine biosynthesis in the human fungal pathogen Candida albicans. We show that although both Saccharomyces cerevisiae and C. albicans respond to lysine deprivation by transcriptional upregulation of lysine biosynthesis, the regulatory factors required for this control have been reconfigured in these species. We found that Gcn4 is an essential and direct transcriptional regulator of the expression of lysine biosynthetic genes under lysine starvation conditions in C. albicans. Our results therefore suggest that the regulation of the lysine biosynthetic pathway in Candida clade genomes involves gain of function by the master transcriptional regulator Gcn4, coincident with the neofunctionalization of the S. cerevisiae pathway-specific regulator Lys14. Evolution of transcriptional control is essential for organisms to cope with diversification into a spectrum of environments, including environments with limited nutrients. Lysine biosynthesis in fungi occurs in eight enzymatic steps. In Saccharomyces cerevisiae, amino acid starvation elicits the induction of LYS gene expression, mediated by the master regulator Gcn4 and the pathway-specific transcriptional regulator Lys14. Here, we have shown that the activation of LYS gene expression in the human fungal pathogen Candida albicans is predominantly controlled by Gcn4 under amino acid starvation conditions. Multiple lines of study showed that the four C. albicans LYS14-like genes have no role in the regulation of lysine biosynthesis. Whereas Gcn4 is dispensable for the growth of S. cerevisiae under lysine deprivation conditions, it is an essential regulator required for the growth of C. albicans under these conditions, as gcn4 deletion caused lysine auxotrophy. Gcn4 is required for the induction of increased LYS2 and LYS9 mRNA but not for the induction of increased LYS4 mRNA. Under lysine or isoleucine-valine deprivation conditions, Gcn4 recruitment to LYS2 and LYS9 promoters was induced in C. albicans. Indeed, in contrast to the S. cerevisiae LYS gene promoters, all LYS gene promoters in C. albicans harbored a Gcn4 binding site but not all harbored the S. cerevisiae Lys14 binding site, indicating the evolutionary divergence of cis-regulatory motifs. Thus, the transcriptional rewiring of the lysine biosynthetic pathway in C. albicans involves not only neofunctionalization of the four LYS14-like genes but the attendant strengthening of control by Gcn4, indicating a coordinated response with a much broader scope for control of amino acid biosynthesis in this human pathogen. IMPORTANCE Microbes evolve rapidly so as to reconfigure their gene expression to adapt to the metabolic demands in diverse environmental niches. Here, we explored how conditions of nutrient deprivation regulate lysine biosynthesis in the human fungal pathogen Candida albicans. We show that although both Saccharomyces cerevisiae and C. albicans respond to lysine deprivation by transcriptional upregulation of lysine biosynthesis, the regulatory factors required for this control have been reconfigured in these species. We found that Gcn4 is an essential and direct transcriptional regulator of the expression of lysine biosynthetic genes under lysine starvation conditions in C. albicans. Our results therefore suggest that the regulation of the lysine biosynthetic pathway in Candida clade genomes involves gain of function by the master transcriptional regulator Gcn4, coincident with the neofunctionalization of the S. cerevisiae pathway-specific regulator Lys14.

scholarly journals Chromatin Profiling of the Repetitive and Nonrepetitive Genomes of the Human Fungal Pathogen Candida albicans

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Robert Jordan Price ◽  
Esther Weindling ◽  
Judith Berman ◽  
Alessia Buscaino
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Histone Modifications ◽  
Chromatin Structure ◽  
Fungal Pathogen ◽  
Histone H3 ◽  
Content Type ◽  
Chromatin States ◽  
Genome Wide ◽  
Chromatin Profiling

ABSTRACT Eukaryotic genomes are packaged into chromatin structures that play pivotal roles in regulating all DNA-associated processes. Histone posttranslational modifications modulate chromatin structure and function, leading to rapid regulation of gene expression and genome stability, key steps in environmental adaptation. Candida albicans, a prevalent fungal pathogen in humans, can rapidly adapt and thrive in diverse host niches. The contribution of chromatin to C. albicans biology is largely unexplored. Here, we generated the first comprehensive chromatin profile of histone modifications (histone H3 trimethylated on lysine 4 [H3K4me3], histone H3 acetylated on lysine 9 [H3K9Ac], acetylated lysine 16 on histone H4 [H4K16Ac], and γH2A) across the C. albicans genome and investigated its relationship to gene expression by harnessing genome-wide sequencing approaches. We demonstrated that gene-rich nonrepetitive regions are packaged into canonical euchromatin in association with histone modifications that mirror their transcriptional activity. In contrast, repetitive regions are assembled into distinct chromatin states; subtelomeric regions and the ribosomal DNA (rDNA) locus are assembled into heterochromatin, while major repeat sequences and transposons are packaged in chromatin that bears features of euchromatin and heterochromatin. Genome-wide mapping of γH2A, a marker of genome instability, identified potential recombination-prone genomic loci. Finally, we present the first quantitative chromatin profiling in C. albicans to delineate the role of the chromatin modifiers Sir2 and Set1 in controlling chromatin structure and gene expression. This report presents the first genome-wide chromatin profiling of histone modifications associated with the C. albicans genome. These epigenomic maps provide an invaluable resource to understand the contribution of chromatin to C. albicans biology and identify aspects of C. albicans chromatin organization that differ from that of other yeasts. IMPORTANCE The fungus Candida albicans is an opportunistic pathogen that normally lives on the human body without causing any harm. However, C. albicans is also a dangerous pathogen responsible for millions of infections annually. C. albicans is such a successful pathogen because it can adapt to and thrive in different environments. Chemical modifications of chromatin, the structure that packages DNA into cells, can allow environmental adaptation by regulating gene expression and genome organization. Surprisingly, the contribution of chromatin modification to C. albicans biology is still largely unknown. For the first time, we analyzed C. albicans chromatin modifications on a genome-wide basis. We demonstrate that specific chromatin states are associated with distinct regions of the C. albicans genome and identify the roles of the chromatin modifiers Sir2 and Set1 in shaping C. albicans chromatin and gene expression.

scholarly journals Impact of Candida auris Infection in a Neutropenic Murine Model

2019 ◽  
Vol 64 (3) ◽  
Author(s):  
Steven R. Torres ◽  
Amber Pichowicz ◽  
Fernando Torres-Velez ◽  
Renjie Song ◽  
Navjot Singh ◽  
...  
Keyword(s):  
Public Health ◽  
Innate Immunity ◽  
Monoclonal Antibodies ◽  
Multidrug Resistance ◽  
Murine Models ◽  
Global Public Health ◽  
Candida Auris ◽  
Innate And Adaptive Immunity ◽  
Content Type ◽  
Public Health Threat

ABSTRACT Candida auris has become a global public health threat due to its multidrug resistance and persistence. Currently, there are limited murine models to study C. auris infection. Those models use a combination of cyclophosphamide and cortisone acetate, suppressing both innate and adaptive immunity. Here, we compare C. auris infection in two neutrophil-depleted murine models in which innate immunity is targeted using the monoclonal antibodies 1A8 and RB6-8C5.

scholarly journals Assessment of the In Vitro and In Vivo Antifungal Activity of NSC319726 against Candida auris

2021 ◽  
Author(s):  
Jizhou Li ◽  
Alix T. Coste ◽  
Daniel Bachmann ◽  
Dominique Sanglard ◽  
Frederic Lamoth
Keyword(s):  
Public Health ◽  
Antifungal Activity ◽  
Invasive Candidiasis ◽  
Multidrug Resistant ◽  
Candida Auris ◽  
Content Type ◽  
Health Threat ◽  
Public Health Threat

Candida auris is emerging as a major public health threat because of its ability to cause nosocomial outbreaks of severe invasive candidiasis. Management of C. auris infection is difficult because of its frequent multidrug-resistant profile for currently licensed antifungals.

scholarly journals Candida auris Cell Wall Mannosylation Contributes to Neutrophil Evasion through Pathways Divergent from Candida albicans and Candida glabrata

mSphere ◽  
2021 ◽  
Author(s):  
Mark V. Horton ◽  
Chad J. Johnson ◽  
Robert Zarnowski ◽  
Brody D. Andes ◽  
Taylor J. Schoen ◽  
...  
Keyword(s):  
Public Health ◽  
Fungal Pathogen ◽  
Candida Glabrata ◽  
Mortality Rates ◽  
Invasive Disease ◽  
Drug Resistant ◽  
Global Public Health ◽  
Candida Auris ◽  
Content Type ◽  
Public Health Threat

The emerging fungal pathogen Candida auris presents a global public health threat. Therapeutic options are often limited for this frequently drug-resistant pathogen, and mortality rates for invasive disease are high.

scholarly journals Cell Wall-Related Bionumbers and Bioestimates of Saccharomyces cerevisiae and Candida albicans

2013 ◽  
Vol 13 (1) ◽  
pp. 2-9 ◽  
Author(s):  
Frans M. Klis ◽  
Chris G. de Koster ◽  
Stanley Brul
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Candida Albicans ◽  
Cell Size ◽  
Pathogenic Fungus ◽  
Turgor Pressure ◽  
Hyphal Growth ◽  
Biological Research ◽  
Content Type ◽  
Starting Point

ABSTRACTBionumbers and bioestimates are valuable tools in biological research. Here we focus on cell wall-related bionumbers and bioestimates of the budding yeastSaccharomyces cerevisiaeand the polymorphic, pathogenic fungusCandida albicans. We discuss the linear relationship between cell size and cell ploidy, the correlation between cell size and specific growth rate, the effect of turgor pressure on cell size, and the reason why using fixed cells for measuring cellular dimensions can result in serious underestimation ofin vivovalues. We further consider the evidence that individual buds and hyphae grow linearly and that exponential growth of the population results from regular formation of new daughter cells and regular hyphal branching. Our calculations show that hyphal growth allowsC. albicansto cover much larger distances per unit of time than the yeast mode of growth and that this is accompanied by strongly increased surface expansion rates. We therefore predict that the transcript levels of genes involved in wall formation increase during hyphal growth. Interestingly, wall proteins and polysaccharides seem barely, if at all, subject to turnover and replacement. A general lesson is how strongly most bionumbers and bioestimates depend on environmental conditions and genetic background, thus reemphasizing the importance of well-defined and carefully chosen culture conditions and experimental approaches. Finally, we propose that the numbers and estimates described here offer a solid starting point for similar studies of other cell compartments and other yeast species.

scholarly journals The Cyclic AMP Receptor Protein Regulates Quorum Sensing and Global Gene Expression in Yersinia pestis during Planktonic Growth and Growth in Biofilms

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Jeremy T. Ritzert ◽  
George Minasov ◽  
Ryan Embry ◽  
Matthew J. Schipma ◽  
Karla J. F. Satchell
Keyword(s):  
Gene Expression ◽  
Quorum Sensing ◽  
Carbon Source ◽  
Cyclic Amp ◽  
Yersinia Pestis ◽  
Carbon Sources ◽  
Transcriptional Regulator ◽  
Receptor Protein ◽  
Content Type ◽  
Bacterial Physiology

ABSTRACT Cyclic AMP (cAMP) receptor protein (Crp) is an important transcriptional regulator of Yersinia pestis. Expression of crp increases during pneumonic plague as the pathogen depletes glucose and forms large biofilms within lungs. To better understand control of Y. pestis Crp, we determined a 1.8-Å crystal structure of the protein-cAMP complex. We found that compared to Escherichia coli Crp, C helix amino acid substitutions in Y. pestis Crp did not impact the cAMP dependency of Crp to bind DNA promoters. To investigate Y. pestis Crp-regulated genes during plague pneumonia, we performed RNA sequencing on both wild-type and Δcrp mutant bacteria growing in planktonic and biofilm states in minimal media with glucose or glycerol. Y. pestis Crp was found to dramatically alter expression of hundreds of genes in a manner dependent upon carbon source and growth state. Gel shift assays confirmed direct regulation of the malT and ptsG promoters, and Crp was then linked to Y. pestis growth on maltose as a sole carbon source. Iron regulation genes ybtA and fyuA were found to be indirectly regulated by Crp. A new connection between carbon source and quorum sensing was revealed as Crp was found to regulate production of acyl-homoserine lactones (AHLs) through direct and indirect regulation of genes for AHL synthetases and receptors. AHLs were subsequently identified in the lungs of Y. pestis-infected mice when crp expression was highest in Y. pestis biofilms. Thus, in addition to the well-studied pla gene, other Crp-regulated genes likely have important functions during plague infection. IMPORTANCE Bacterial pathogens have evolved extensive signaling pathways to translate environmental signals into changes in gene expression. While Crp has long been appreciated for its role in regulating metabolism of carbon sources in many bacterial species, transcriptional profiling has revealed that this protein regulates many other aspects of bacterial physiology. The plague pathogen Y. pestis requires this global regulator to survive in blood, skin, and lungs. During disease progression, this organism adapts to changes within these niches. In addition to regulating genes for metabolism of nonglucose sugars, we found that Crp regulates genes for virulence, metal acquisition, and quorum sensing by direct or indirect mechanisms. Thus, this single transcriptional regulator, which responds to changes in available carbon sources, can regulate multiple critical behaviors for causing disease.

scholarly journals Genome‐wide effect of tetracycline, doxycycline and 4‐epidoxycycline on gene expression in Saccharomyces cerevisiae

Yeast ◽  
2020 ◽  
Vol 37 (7-8) ◽  
pp. 389-396
Author(s):  
Guadalupe Sanchez ◽  
Samuel C. Linde ◽  
Joseph D. Coolon
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Genome Wide

scholarly journals Probiotic Yeasts Inhibit Virulence of Non-albicans Candida Species

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Lohith Kunyeit ◽  
Nawneet K. Kurrey ◽  
K. A. Anu-Appaiah ◽  
Reeta P. Rao
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Tropicalis ◽  
Biofilm Formation ◽  
Candida Glabrata ◽  
Candida Parapsilosis ◽  
Candida Krusei ◽  
Probiotic Properties ◽  
Candida Auris ◽  
Content Type ◽  
Alternative Approach

ABSTRACT Systemic infections of Candida species pose a significant threat to public health. Toxicity associated with current therapies and emergence of resistant strains present major therapeutic challenges. Here, we report exploitation of the probiotic properties of two novel, food-derived yeasts, Saccharomyces cerevisiae (strain KTP) and Issatchenkia occidentalis (strain ApC), as an alternative approach to combat widespread opportunistic fungal infections. Both yeasts inhibit virulence traits such as adhesion, filamentation, and biofilm formation of several non-albicans Candida species, including Candida tropicalis, Candida krusei, Candida glabrata, and Candida parapsilosis as well as the recently identified multidrug-resistant species Candida auris. They inhibit adhesion to abiotic surfaces as well as cultured colon epithelial cells. Furthermore, probiotic treatment blocks the formation of biofilms of individual non-albicans Candida strains as well as mixed-culture biofilms of each non-albicans Candida strain in combination with Candida albicans. The probiotic yeasts attenuated non-albicans Candida infections in a live animal. In vivo studies using Caenorhabditis elegans suggest that exposure to probiotic yeasts protects nematodes from infection with non-albicans Candida strains compared to worms that were not exposed to the probiotic yeasts. Furthermore, application of probiotic yeasts postinfection with non-albicans Candida alleviated pathogenic colonization of the nematode gut. The probiotic properties of these novel yeasts are better than or comparable to those of the commercially available probiotic yeast Saccharomyces boulardii, which was used as a reference strain throughout this study. These results indicate that yeasts derived from food sources could serve as an effective alternative to antifungal therapy against emerging pathogenic Candida species. IMPORTANCE Non-albicans Candida-associated infections have emerged as a major risk factor in the hospitalized and immunecompromised patients. Besides, antifungal-associated complications occur more frequently with these non-albicans Candida species than with C. albicans. Therefore, as an alternative approach to combat these widespread non-albicans Candida-associated infections, here we showed the probiotic effect of two yeasts, Saccharomyces cerevisiae (strain KTP) and Issatchenkia occidentalis (ApC), in preventing adhesion and biofilm formation of five non-albicans Candida strains, Candida tropicalis, Candida krusei, Candida glabrata, Candida parapsilosis, and Candida auris. The result would influence the current trend of the conversion of conventional antimicrobial therapy into beneficial probiotic microbe-associated antimicrobial treatment.

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