scholarly journals High-Throughput Nano-Biofilm Microarray for Antifungal Drug Discovery

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
Author(s):  
Anand Srinivasan ◽  
Kai P. Leung ◽  
Jose L. Lopez-Ribot ◽  
Anand K. Ramasubramanian
Keyword(s):  
Cell Culture ◽  
Candida Albicans ◽  
Drug Discovery ◽  
High Throughput ◽  
Fungal Pathogen ◽  
Microarray Platform ◽  
Microbial Cell ◽  
Content Type ◽  
Petri Dishes ◽  
Opportunistic Fungal Pathogen

ABSTRACT Micro- and nanoscale technologies have radically transformed biological research from genomics to tissue engineering, with the relative exception of microbial cell culture, which is still largely performed in microtiter plates and petri dishes. Here, we present nanoscale culture of the opportunistic fungal pathogen Candida albicans on a microarray platform. The microarray consists of 1,200 individual cultures of 30 nl of C. albicans biofilms (“nano-biofilms”) encapsulated in an inert alginate matrix. We demonstrate that these nano-biofilms are similar to conventional macroscopic biofilms in their morphological, architectural, growth, and phenotypic characteristics. We also demonstrate that the nano-biofilm microarray is a robust and efficient tool for accelerating the drug discovery process: (i) combinatorial screening against a collection of 28 antifungal compounds in the presence of immunosuppressant FK506 (tacrolimus) identified six drugs that showed synergistic antifungal activity, and (ii) screening against the NCI challenge set small-molecule library identified three heretofore-unknown hits. This cell-based microarray platform allows for miniaturization of microbial cell culture and is fully compatible with other high-throughput screening technologies. IMPORTANCE Microorganisms are typically still grown in petri dishes, test tubes, and Erlenmeyer flasks in spite of the latest advances in miniaturization that have benefitted other allied research fields, including genomics and proteomics. Culturing microorganisms in small scale can be particularly valuable in cutting down time, cost, and reagent usage. This paper describes the development, characterization, and application of nanoscale culture of an opportunistic fungal pathogen, Candida albicans. Despite a more than 2,000-fold reduction in volume, the growth characteristics and drug response profiles obtained from the nanoscale cultures were comparable to the industry standards. The platform also enabled rapid identification of new drug candidates that were effective against C. albicans biofilms, which are a major cause of mortality in hospital-acquired infections.

scholarly journals Ascorbic Acid Inhibition of Candida albicans Hsp90-Mediated Morphogenesis Occurs via the Transcriptional Regulator Upc2

2014 ◽  
Vol 13 (10) ◽  
pp. 1278-1289 ◽  
Author(s):  
Frédérique Van Hauwenhuyse ◽  
Alessandro Fiori ◽  
Patrick Van Dijck
Keyword(s):  
Ascorbic Acid ◽  
Candida Albicans ◽  
Fungal Pathogen ◽  
Transcriptional Regulator ◽  
Hsp90 Inhibitor ◽  
Ergosterol Biosynthesis ◽  
Content Type ◽  
Temperature Changes ◽  
Opportunistic Fungal Pathogen ◽  
Hsp90 Function

ABSTRACTMorphogenetic transitions of the opportunistic fungal pathogenCandida albicansare influenced by temperature changes, with induction of filamentation upon a shift from 30 to 37°C. Hsp90 was identified as a major repressor of an elongated cell morphology at low temperatures, as treatment with specific inhibitors of Hsp90 results in elongated growth forms at 30°C. Elongated growth resulting from a compromised Hsp90 is considered neither hyphal nor pseudohyphal growth. It has been reported that ascorbic acid (vitamin C) interferes with the yeast-to-hypha transition inC. albicans. In the present study, we show that ascorbic acid also antagonizes the morphogenetic change caused by hampered Hsp90 function. Further analysis revealed that Upc2, a transcriptional regulator of genes involved in ergosterol biosynthesis, and Erg11, the target of azole antifungals, whose expression is in turn regulated by Upc2, are required for this antagonism. Ergosterol levels correlate with elongated growth and are reduced in cells treated with the Hsp90 inhibitor geldanamycin (GdA) and restored by cotreatment with ascorbic acid. In addition, we show that Upc2 appears to be required for ascorbic acid-mediated inhibition of the antifungal activity of fluconazole. These results identify Upc2 as a major regulator of ascorbic acid-induced effects inC. albicansand suggest an association between ergosterol content and elongated growth upon Hsp90 compromise.

scholarly journals Improved Gene Ontology Annotation for Biofilm Formation, Filamentous Growth, and Phenotypic Switching in Candida albicans

2012 ◽  
Vol 12 (1) ◽  
pp. 101-108 ◽  
Author(s):  
Diane O. Inglis ◽  
Marek S. Skrzypek ◽  
Martha B. Arnaud ◽  
Jonathan Binkley ◽  
Prachi Shah ◽  
...  
Keyword(s):  
Gene Ontology ◽  
Candida Albicans ◽  
Gene Product ◽  
Fungal Pathogen ◽  
Biofilm Formation ◽  
Filamentous Growth ◽  
Phenotypic Switching ◽  
Gene Products ◽  
Content Type ◽  
Opportunistic Fungal Pathogen

ABSTRACTThe opportunistic fungal pathogenCandida albicansis a significant medical threat, especially for immunocompromised patients. Experimental research has focused on specific areas ofC. albicansbiology, with the goal of understanding the multiple factors that contribute to its pathogenic potential. Some of these factors include cell adhesion, invasive or filamentous growth, and the formation of drug-resistant biofilms. The Gene Ontology (GO) (www.geneontology.org) is a standardized vocabulary that theCandidaGenome Database (CGD) (www.candidagenome.org) and other groups use to describe the functions of gene products. To improve the breadth and accuracy of pathogenicity-related gene product descriptions and to facilitate the description of as yet uncharacterized but potentially pathogenicity-related genes inCandidaspecies, CGD undertook a three-part project: first, the addition of terms to the biological process branch of the GO to improve the description of fungus-related processes; second, manual recuration of gene product annotations in CGD to use the improved GO vocabulary; and third, computational ortholog-based transfer of GO annotations from experimentally characterized gene products, using these new terms, to uncharacterized orthologs in otherCandidaspecies. Through genome annotation and analysis, we identified candidate pathogenicity genes in seven non-C. albicans Candidaspecies and in one additionalC. albicansstrain, WO-1. We also defined a set ofC. albicansgenes at the intersection of biofilm formation, filamentous growth, pathogenesis, and phenotypic switching of this opportunistic fungal pathogen, which provides a compelling list of candidates for further experimentation.

scholarly journals Candida albicans ENT2 Contributes to Efficient Endocytosis, Cell Wall Integrity, Filamentation, and Virulence

mSphere ◽  
2021 ◽  
Author(s):  
Christiane Rollenhagen ◽  
Harrison Agyeman ◽  
Susan Eszterhas ◽  
Samuel A. Lee
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fungal Pathogen ◽  
Clinical Importance ◽  
Hospitalized Patients ◽  
Cell Wall Integrity ◽  
Content Type ◽  
Invasive Infections ◽  
Opportunistic Fungal Pathogen ◽  
Considerable Morbidity

The opportunistic fungal pathogen Candida albicans is an important cause of invasive infections in hospitalized patients and a source of considerable morbidity and mortality. Despite its clinical importance, we still need to improve our ability to diagnose and treat this common pathogen.

scholarly journals Unique, Diverged, and Conserved Mitochondrial Functions InfluencingCandida albicansRespiration

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Nuo Sun ◽  
Rebecca S. Parrish ◽  
Richard A. Calderone ◽  
William A. Fonzi
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Complex I ◽  
Electron Transport Chain ◽  
Complex Iii ◽  
Respiratory Metabolism ◽  
Content Type ◽  
Transport Chain ◽  
Opportunistic Fungal Pathogen ◽  
Lineage Specific Genes

ABSTRACTCandida albicansis an opportunistic fungal pathogen of major clinical concern. The virulence of this pathogen is intimately intertwined with its metabolism. Mitochondria, which have a central metabolic role, have undergone many lineage-specific adaptations in association with their eukaryotic host. A screen for lineage-specific genes identified seven such genes specific to the CTG clade of fungi, of whichC. albicansis a member. Each is required for respiratory growth and is integral to expression of complex I, III, or IV of the electron transport chain. Two genes,NUO3andNUO4, encode supernumerary subunits of complex I, whereasNUE1andNUE2have nonstructural roles in expression of complex I. Similarly, the other three genes have nonstructural roles in expression of complex III (QCE1) or complex IV (COE1andCOE2). In addition to these novel additions, an alternative functional assignment was found for the mitochondrial protein encoded byMNE1.MNE1was required for complex I expression inC. albicans, whereas the distantly relatedSaccharomyces cerevisiaeortholog participates in expression of complex III. Phenotypic analysis of deletion mutants showed that fermentative metabolism is unable to support optimal growth rates or yields ofC. albicans. However, yeast-hypha morphogenesis, an important virulence attribute, did not require respiratory metabolism under hypoxic conditions. The inability to respire also resulted in hypersensitivity to the antifungal fluconazole and in attenuated virulence in aGalleria mellonellainfection model. The results show that lineage-specific adaptations have occurred inC. albicansmitochondria and highlight the significance of respiratory metabolism in the pathobiology ofC. albicans.IMPORTANCECandida albicansis an opportunistic fungal pathogen of major clinical concern. The virulence of this pathogen is intimately intertwined with its metabolic behavior, and mitochondria have a central role in that metabolism. Mitochondria have undergone many evolutionary changes, which include lineage-specific adaptations in association with their eukaryotic host. Seven lineage-specific genes required for electron transport chain function were identified in the CTG clade of fungi, of whichC. albicansis a member. Additionally, examination of several highly diverged orthologs encoding mitochondrial proteins demonstrated functional reassignment for one of these. Deficits imparted by deletion of these genes revealed the critical role of respiration in virulence attributes of the fungus and highlight important evolutionary adaptations inC. albicansmetabolism.

scholarly journals A High-ThroughputCandida albicansTwo-Hybrid System

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
Author(s):  
Floris Schoeters ◽  
Carol A. Munro ◽  
Christophe d’Enfert ◽  
Patrick Van Dijck
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
High Throughput ◽  
Protein Interactions ◽  
Fungal Pathogen ◽  
Model Organism ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Content Type ◽  
Two Hybrid

ABSTRACTCandida albicansis a human fungal pathogen that does not follow the universal codon usage, as it translates the CUG codon into serine rather than leucine. This makes it difficult to study protein-protein interactions using the standard yeast two-hybrid (Y2H) system in the model organismSaccharomyces cerevisiae. Due to the lack of adapted tools, only a small number of protein-protein interactions (PPIs) have been detected or studied usingC. albicans-optimized tools despite the importance of PPIs to understand cell biology. However, with the sequencing of the whole genome ofC. albicans, the availability of an ORFeome collection containing 5,099 open reading frames (ORFs) in Gateway-adapted donor vectors, and the creation of a Gateway-compatibleC. albicans-specific two-hybrid (C2H) system, it became possible to study protein-protein interactions on a larger scale usingC. albicansitself as the model organism. Erroneous translations are hereby eliminated compared to using theS. cerevisiaeY2H system. Here, we describe the technical adaptations and the first application of the C2H system for a high-throughput screen, thus making it possible to screen thousands of PPIs at once inC. albicansitself. This first, small-scale high-throughput screen, using Pho85 as a bait protein against 1,646 random prey proteins, yielded one interacting partner (Pcl5). The interaction found with the high-throughput setup was further confirmed with a low-throughput C2H experiment and with a coimmunoprecipitation (co-IP) experiment.IMPORTANCECandida albicansis a major fungal pathogen, and due to the rise of fungal infections and emerging resistance to the limited antifungals available, it is important to develop novel and more specific antifungals. Protein-protein interactions (PPIs) can be applied as very specific drug targets. However, because of the aberrant codon usage ofC. albicans, the traditional yeast two-hybrid system inSaccharomyces cerevisiaeis difficult to use, and only a limited number of PPIs have been described inC. albicans. To overcome this, aC. albicanstwo-hybrid (C2H) system was developed in 2010. The current work describes, for the first time, the application of the C2H system in a high-throughput setup. We hereby show the usefulness of the C2H system to investigate and detect PPIs inC. albicans, making it possible to further elucidate protein networks inC. albicans, which has the potential to lead to the development of novel antifungals which specifically disrupt PPIs important for virulence.

scholarly journals An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Namkha Nguyen ◽  
Morgan M. F. Quail ◽  
Aaron D. Hernday
Keyword(s):  
Candida Albicans ◽  
Genome Editing ◽  
Fungal Pathogen ◽  
Gene Knockout ◽  
Strain Engineering ◽  
Molecular Genetic Analysis ◽  
Content Type ◽  
Highly Efficient ◽  
Discovery Research ◽  
Gene Knockouts

ABSTRACT Candida albicans is the most common fungal pathogen of humans. Historically, molecular genetic analysis of this important pathogen has been hampered by the lack of stable plasmids or meiotic cell division, limited selectable markers, and inefficient methods for generating gene knockouts. The recent development of clustered regularly interspaced short palindromic repeat(s) (CRISPR)-based tools for use with C. albicans has opened the door to more efficient genome editing; however, previously reported systems have specific limitations. We report the development of an optimized CRISPR-based genome editing system for use with C. albicans. Our system is highly efficient, does not require molecular cloning, does not leave permanent markers in the genome, and supports rapid, precise genome editing in C. albicans. We also demonstrate the utility of our system for generating two independent homozygous gene knockouts in a single transformation and present a method for generating homozygous wild-type gene addbacks at the native locus. Furthermore, each step of our protocol is compatible with high-throughput strain engineering approaches, thus opening the door to the generation of a complete C. albicans gene knockout library. IMPORTANCE Candida albicans is the major fungal pathogen of humans and is the subject of intense biomedical and discovery research. Until recently, the pace of research in this field has been hampered by the lack of efficient methods for genome editing. We report the development of a highly efficient and flexible genome editing system for use with C. albicans. This system improves upon previously published C. albicans CRISPR systems and enables rapid, precise genome editing without the use of permanent markers. This new tool kit promises to expedite the pace of research on this important fungal pathogen.

scholarly journals Aspergillus fumigatus Acetate Utilization Impacts Virulence Traits and Pathogenicity

mBio ◽  
2021 ◽  
Author(s):  
Laure Nicolas Annick Ries ◽  
Patricia Alves de Castro ◽  
Lilian Pereira Silva ◽  
Clara Valero ◽  
Thaila Fernanda dos Reis ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Fungal Pathogen ◽  
Carbon Sources ◽  
Body Fluids ◽  
Peripheral Tissues ◽  
Content Type ◽  
Virulence Traits ◽  
Acetate Utilization ◽  
Opportunistic Fungal Pathogen

Aspergillus fumigatus is an opportunistic fungal pathogen in humans. During infection, A. fumigatus is predicted to use host carbon sources, such as acetate, present in body fluids and peripheral tissues, to sustain growth and promote colonization and invasion.

scholarly journals Melanin Externalization in Candida albicans Depends on Cell Wall Chitin Structures

2010 ◽  
Vol 9 (9) ◽  
pp. 1329-1342 ◽  
Author(s):  
Claire A. Walker ◽  
Beatriz L. Gómez ◽  
Héctor M. Mora-Montes ◽  
Kevin S. Mackenzie ◽  
Carol A. Munro ◽  
...  
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Fungal Pathogen ◽  
Cell Walls ◽  
Chitin Synthesis ◽  
Melanin Production ◽  
Content Type ◽  
Encoding Gene ◽  
Chitinase Genes

ABSTRACT The fungal pathogen Candida albicans produces dark-pigmented melanin after 3 to 4 days of incubation in medium containing l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. Expression profiling of C. albicans revealed very few genes significantly up- or downregulated by growth in l-DOPA. We were unable to determine a possible role for melanin in the virulence of C. albicans. However, we showed that melanin was externalized from the fungal cells in the form of electron-dense melanosomes that were free or often loosely bound to the cell wall exterior. Melanin production was boosted by the addition of N-acetylglucosamine to the medium, indicating a possible association between melanin production and chitin synthesis. Melanin externalization was blocked in a mutant specifically disrupted in the chitin synthase-encoding gene CHS2. Melanosomes remained within the outermost cell wall layers in chs3Δ and chs2Δ chs3Δ mutants but were fully externalized in chs8Δ and chs2Δ chs8Δ mutants. All the CHS mutants synthesized dark pigment at equivalent rates from mixed membrane fractions in vitro, suggesting it was the form of chitin structure produced by the enzymes, not the enzymes themselves, that was involved in the melanin externalization process. Mutants with single and double disruptions of the chitinase genes CHT2 and CHT3 and the chitin pathway regulator ECM33 also showed impaired melanin externalization. We hypothesize that the chitin product of Chs3 forms a scaffold essential for normal externalization of melanosomes, while the Chs8 chitin product, probably produced in cell walls in greater quantity in the absence of CHS2, impedes externalization.

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.

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