scholarly journals G 1 /S Transcription Factor Orthologues Swi4p and Swi6p Are Important but Not Essential for Cell Proliferation and Influence Hyphal Development in the Fungal Pathogen Candida albicans

2011 ◽  
Vol 10 (3) ◽  
pp. 384-397 ◽  
Author(s):  
Bahira Hussein ◽  
Hao Huang ◽  
Amandeep Glory ◽  
Amin Osmani ◽  
Susan Kaminskyj ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Candida Albicans ◽  
Fungal Pathogen ◽  
Expression Patterns ◽  
Control Point ◽  
Yeast Cells ◽  
Content Type ◽  
Hyphal Development ◽  
Critical Control Point ◽  
Transcription Profiles

ABSTRACT The G 1 /S transition is a critical control point for cell proliferation and involves essential transcription complexes termed SBF and MBF in Saccharomyces cerevisiae or MBF in Schizosaccharomyces pombe . In the fungal pathogen Candida albicans , G 1 /S regulation is not clear. To gain more insight into the G 1 /S circuitry, we characterized Swi6p, Swi4p and Mbp1p, the closest orthologues of SBF (Swi6p and Swi4p) and MBF (Swi6p and Mbp1p) components in S. cerevisiae . The mbp1 Δ/Δ cells showed minor growth defects, whereas swi4 Δ/Δ and swi6 Δ/Δ yeast cells dramatically increased in size, suggesting a G 1 phase delay. G ene s et e nrichment a nalysis (GSEA) of transcription profiles revealed that genes associated with G 1 /S phase were significantly enriched in cells lacking Swi4p and Swi6p. These expression patterns suggested that Swi4p and Swi6p have repressing as well as activating activity. Intriguingly, swi4 Δ / Δ swi6 Δ/Δ and swi4 Δ / Δ mbp1 Δ/Δ strains were viable, in contrast to the situation in S. cerevisiae , and showed pleiotropic phenotypes that included multibudded yeast, pseudohyphae, and intriguingly, true hyphae. Consistently, GSEA identified strong enrichment of genes that are normally modulated during C. albicans -host cell interactions. Since Swi4p and Swi6p influence G 1 phase progression and SBF binding sites are lacking in the C. albicans genome, these factors may contribute to MBF activity. Overall, the data suggest that the putative G 1 /S regulatory machinery of C. albican s contains novel features and underscore the existence of a relationship between G 1 phase and morphogenetic switching, including hyphal development, in the pathogen.

scholarly journals Candida albicans CUG Mistranslation Is a Mechanism To Create Cell Surface Variation

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
Author(s):  
Isabel Miranda ◽  
Ana Silva-Dias ◽  
Rita Rocha ◽  
Rita Teixeira-Santos ◽  
Carolina Coelho ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Cell Surface ◽  
Fungal Pathogen ◽  
Single Gene ◽  
Yeast Cells ◽  
Phenotypic Switching ◽  
Leucine Incorporation ◽  
Host Immune System ◽  
Content Type ◽  
Human Fungal Pathogen

ABSTRACT In the human fungal pathogen Candida albicans, the CUG codon is translated 97% of the time as serine and 3% of the time as leucine, which potentially originates an array of proteins resulting from the translation of a single gene. Genes encoding cell surface proteins are enriched in CUG codons; thus, CUG mistranslation may influence the interactions of the organism with the host. To investigate this, we compared a C. albicans strain that misincorporates 28% of leucine at CUGs with a wild-type parental strain. The first strain displayed increased adherence to inert and host molecules. In addition, it was less susceptible to phagocytosis by murine macrophages, probably due to reduced exposure of cell surface β-glucans. To prove that these phenotypes occurred due to serine/leucine exchange, the C. albicans adhesin and invasin ALS3 was expressed in Saccharomyces cerevisiae in its two natural isoforms (Als3p-Leu and Als3p-Ser). The cells with heterologous expression of Als3p-Leu showed increased adherence to host substrates and flocculation. We propose that CUG mistranslation has been maintained during the evolution of C. albicans due to its potential to generate cell surface variability, which significantly alters fungus-host interactions. IMPORTANCE The translation of genetic information into proteins is a highly accurate cellular process. In the human fungal pathogen Candida albicans, a unique mistranslation event involving the CUG codon occurs. The CUG codon is mainly translated as serine but can also be translated as leucine. Leucine and serine are two biochemically distinct amino acids, hydrophobic and hydrophilic, respectively. The increased rate of leucine incorporation at CUG decoding triggers C. albicans virulence attributes, such as morphogenesis, phenotypic switching, and adhesion. Here, we show that CUG mistranslation masks the fungal cell wall molecule β-glucan that is normally recognized by the host immune system, delaying its response. Furthermore, we demonstrate that two different proteins of the adhesin Als3 generated by CUG mistranslation confer increased hydrophobicity and adhesion ability on yeast cells. Thus, CUG mistranslation functions as a mechanism to create protein diversity with differential activities, constituting an advantage for a mainly asexual microorganism. This could explain its preservation during evolution.

scholarly journals Regulation of the Candida albicans Hypha-Inducing Transcription Factor Ume6 by the CDK1 Cyclins Cln3 and Hgc1

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Sigal Mendelsohn ◽  
Mariel Pinsky ◽  
Ziva Weissman ◽  
Daniel Kornitzer
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Candida Albicans ◽  
Fungal Pathogen ◽  
Molecular Basis ◽  
Yeast Cells ◽  
Industrialized Countries ◽  
Content Type ◽  
Hyphal Morphogenesis ◽  
Multicellular Organisms

ABSTRACT The yeast to hypha (mold) morphogenetic switch of Candida albicans plays a role in its virulence and constitutes a diagnostic trait for this organism, the most prevalent systemic fungal pathogen in industrialized countries. It has long been known that hyphae are most efficiently induced from stationary cultures. Here, a molecular basis for this observation is provided. The G1 cyclin Cln3, an essential promoter of yeast proliferation, was found to suppress hyphal induction. Suppression of hyphal induction is achieved by inhibition of the activity of the central activator of hyphal morphogenesis, the transcription factor Ume6. Thus, levels of Cln3 control the switch between proliferation of C. albicans as individual yeast cells and development into extended hyphae, a switch that may preface the proliferation/differentiation switch in multicellular organisms. The ability to switch between proliferation as yeast cells and development into hyphae is a hallmark of Candida albicans. The switch to hyphal morphogenesis depends on external inducing conditions, but its efficiency is augmented in stationary-phase cells. Ume6, a transcription factor that is itself transcriptionally induced under hypha-promoting conditions, is both necessary and sufficient for hyphal morphogenesis. We found that Ume6 is regulated posttranslationally by the cell cycle kinase Cdc28/Cdk1, which reduces Ume6 activity via different mechanisms using different cyclins. Together with the cyclin Hgc1, Cdk1 promotes degradation of Ume6 via the SCFCDC4 ubiquitin ligase. Since HGC1 is a key transcriptional target of Ume6, this results in a negative-feedback loop between Hgc1 and Ume6. In addition, we found that Cln3, a G1 cyclin that is essential for cell cycle progression and yeast proliferation, suppresses hyphal morphogenesis and that Cln3 suppresses Ume6 activity both in the heterologous Saccharomyces cerevisiae system and in C. albicans itself. This activity of Cln3 may provide the basis for the antagonistic relationship between yeast proliferation and hyphal development in C. albicans. IMPORTANCE The yeast to hypha (mold) morphogenetic switch of Candida albicans plays a role in its virulence and constitutes a diagnostic trait for this organism, the most prevalent systemic fungal pathogen in industrialized countries. It has long been known that hyphae are most efficiently induced from stationary cultures. Here, a molecular basis for this observation is provided. The G1 cyclin Cln3, an essential promoter of yeast proliferation, was found to suppress hyphal induction. Suppression of hyphal induction is achieved by inhibition of the activity of the central activator of hyphal morphogenesis, the transcription factor Ume6. Thus, levels of Cln3 control the switch between proliferation of C. albicans as individual yeast cells and development into extended hyphae, a switch that may preface the proliferation/differentiation switch in multicellular organisms.

scholarly journals Orthologues of the Anaphase-Promoting Complex/Cyclosome Coactivators Cdc20p and Cdh1p Are Important for Mitotic Progression and Morphogenesis in Candida albicans

2011 ◽  
Vol 10 (5) ◽  
pp. 696-709 ◽  
Author(s):  
Hsini Chou ◽  
Amandeep Glory ◽  
Catherine Bachewich
Keyword(s):  
Candida Albicans ◽  
Fungal Pathogen ◽  
Regulatory Networks ◽  
Yeast Cells ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Content Type ◽  
Mitotic Cyclin ◽  
Late Mitosis ◽  
A Minor

ABSTRACTThe conserved anaphase-promoting complex/cyclosome (APC/C) system mediates protein degradation during mitotic progression. Conserved coactivators Cdc20p and Cdh1p regulate the APC/C during early to late mitosis and G1phase.Candida albicansis an important fungal pathogen of humans, and it forms highly polarized cells when mitosis is blocked through depletion of the polo-like kinase Cdc5p or other treatments. However, the mechanisms governing mitotic progression and associated polarized growth in the pathogen are poorly understood. In order to gain insights into these processes, we characterizedC. albicansorthologues of Cdc20p and Cdh1p. Cdc20p-depleted cells were blocked in early or late mitosis with elevated levels of Cdc5p and the mitotic cyclin Clb2p, suggesting that Cdc20p is essential and has some conserved functions during mitosis. However, the yeast cells formed highly polarized buds in contrast to the large doublets ofS. cerevisiaecdc20mutants, implying a distinct role in morphogenesis. In comparison,cdh1Δ/cdh1Δcells were viable but showed enrichment of Clb2p and Cdc5p, suggesting that Cdh1p may influence mitotic exit. Thecdh1Δ/cdh1Δphenotype was pleiotropic, consisting of normal or enlarged yeast, pseudohyphae, and some elongated buds, whereasS. cerevisiaecdh1Δyeast cells were reduced in size. Thus,C. albicansCdh1p may have some distinct functions. Finally, absence of Cdh1p or Cdc20p had a minor or no effect on hyphal development, respectively. Overall, the results suggest that Cdc20p and Cdh1p may be APC/C activators that are important for mitosis but also morphogenesis inC. albicans. Their novel features imply additional variations in function and underscore rewiring in the emerging mitotic regulatory networks of the pathogen.

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 Altered Dynamics of Candida albicans Phagocytosis by Macrophages and PMNs When Both Phagocyte Subsets Are Present

mBio ◽  
2013 ◽  
Vol 4 (6) ◽  
Author(s):  
Fiona M. Rudkin ◽  
Judith M. Bain ◽  
Catriona Walls ◽  
Leanne E. Lewis ◽  
Neil A. R. Gow ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Immune System ◽  
Innate Immune System ◽  
Video Microscopy ◽  
Innate Immune ◽  
Live Cell ◽  
Yeast Cells ◽  
Polymorphonuclear Cells ◽  
Fungal Cell ◽  
Content Type

ABSTRACT An important first line of defense against Candida albicans infections is the killing of fungal cells by professional phagocytes of the innate immune system, such as polymorphonuclear cells (PMNs) and macrophages. In this study, we employed live-cell video microscopy coupled with dynamic image analysis tools to provide insights into the complexity of C. albicans phagocytosis when macrophages and PMNs were incubated with C. albicans alone and when both phagocyte subsets were present. When C. albicans cells were incubated with only one phagocyte subtype, PMNs had a lower overall phagocytic capacity than macrophages, despite engulfing fungal cells at a higher rate once fungal cells were bound to the phagocyte surface. PMNs were more susceptible to C. albicans-mediated killing than macrophages, irrespective of the number of C. albicans cells ingested. In contrast, when both phagocyte subsets were studied in coculture, the two cell types phagocytosed and cleared C. albicans at equal rates and were equally susceptible to killing by the fungus. The increase in macrophage susceptibility to C. albicans-mediated killing was a consequence of macrophages taking up a higher proportion of hyphal cells under these conditions. In the presence of both PMNs and macrophages, C. albicans yeast cells were predominantly cleared by PMNs, which migrated at a greater speed toward fungal cells and engulfed bound cells more rapidly. These observations demonstrate that the phagocytosis of fungal pathogens depends on, and is modified by, the specific phagocyte subsets present at the site of infection. IMPORTANCE Extensive work investigating fungal cell phagocytosis by macrophages and PMNs of the innate immune system has been carried out. These studies have been informative but have examined this phenomenon only when one phagocyte subset is present. The current study employed live-cell video microscopy to break down C. albicans phagocytosis into its component parts and examine the effect of a single phagocyte subset, versus a mixed phagocyte population, on these individual stages. Through this approach, we identified that the rate of fungal cell engulfment and rate of phagocyte killing altered significantly when both macrophages and PMNs were incubated in coculture with C. albicans compared to the rate of either phagocyte subset incubated alone with the fungus. This research highlights the significance of studying pathogen-host cell interactions with a combination of phagocytes in order to gain a greater understanding of the interactions that occur between cells of the host immune system in response to fungal invasion.

scholarly journals The Quorum-Sensing Molecules Farnesol/Homoserine Lactone and Dodecanol Operate via Distinct Modes of Action in Candida albicans

2011 ◽  
Vol 10 (8) ◽  
pp. 1034-1042 ◽  
Author(s):  
Rebecca A. Hall ◽  
Kara J. Turner ◽  
James Chaloupka ◽  
Fabien Cottier ◽  
Luisa De Sordi ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Quorum Sensing ◽  
Signaling Pathway ◽  
Homoserine Lactone ◽  
Burkholderia Cenocepacia ◽  
Camp Signaling ◽  
Mammalian Host ◽  
Content Type ◽  
Hyphal Development ◽  
Natural Flora

ABSTRACTLiving as a commensal,Candida albicansmust adapt and respond to environmental cues generated by the mammalian host and by microbes comprising the natural flora. These signals have opposing effects onC. albicans, with host cues promoting the yeast-to-hyphal transition and bacteria-derived quorum-sensing molecules inhibiting hyphal development. Hyphal development is regulated through modulation of the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, and it has been postulated that quorum-sensing molecules can affect filamentation by inhibiting the cAMP pathway. Here, we show that both farnesol and 3-oxo-C12-homoserine lactone, a quorum-sensing molecule secreted byPseudomonas aeruginosa, block hyphal development by affecting cAMP signaling; they both directly inhibited the activity of theCandidaadenylyl cyclase, Cyr1p. In contrast, the 12-carbon alcohol dodecanol appeared to modulate hyphal development and the cAMP signaling pathway without directly affecting the activity of Cyr1p. Instead, we show that dodecanol exerted its effects through a mechanism involving theC. albicanshyphal repressor, Sfl1p. Deletion ofSFL1did not affect the response to farnesol but did interfere with the response to dodecanol. Therefore, quorum sensing inC. albicansis mediated via multiple mechanisms of action. Interestingly, our experiments raise the possibility that theBurkholderia cenocepaciadiffusible signal factor, BDSF, also mediates its effects via Sfl1p, suggesting that dodecanol's mode of action, but not farnesol or 3-oxo-C12-homoserine lactone, may be used by other quorum-sensing molecules.

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.

scholarly journals The “Finger,” a Unique Multicellular Morphology of Candida albicans Induced by CO2and Dependent upon the Ras1-Cyclic AMP Pathway

2012 ◽  
Vol 11 (10) ◽  
pp. 1257-1267 ◽  
Author(s):  
Karla J. Daniels ◽  
Claude Pujol ◽  
Thyagarajan Srikantha ◽  
David R. Soll
Keyword(s):  
Candida Albicans ◽  
Cyclic Amp ◽  
Mutational Analysis ◽  
Cell Monolayer ◽  
Yeast Cells ◽  
Content Type ◽  
Dispersal Mechanism ◽  
Basal Bulb ◽  
High Doses

ABSTRACTMost experiments exploring the basic biology of pathogenic microbes are performedin vitrounder conditions that do not usually mimic those of their host niche. Hence, developmental programs initiated by specific host cues may be missedin vitro. We have tested the effects of growing low-density agar cultures of the yeast pathogenCandida albicansin concentrations of CO2found in the gastrointestinal tract. It is demonstrated that in physiological concentrations of CO2at 37°C, yeast cells form a heretofore undescribed multicellular “finger” morphology distinct from a previously described stalk-like structure induced by high doses of UV irradiation that kills more than 99.99% of cells. The finger extends aerially, is uniform in diameter, and is visible to the naked eye, attaining lengths of 3 mm. It is composed of a basal yeast cell monolayer adhering to a semispherical crater formed in the agar and connected to a basal bulb of yeast cells at a fragile interface. The bulb extends into the long shaft. We propose that a single, centrally located hypha extending the length of the shaft forms buds at compartment junctions that serve as the source of the yeast cells in the shaft. A mutational analysis reveals finger formation is dependent upon the pathway Ras1→Cdc35→cyclic AMP (cAMP) (PDE2—|)→Tpk2→Tec1. Because of the mechanically fragile interface and the compactness of bulb and shaft, we suggest that the finger may function as a multicellular dispersal mechanism produced in host niches containing high levels of CO2.

scholarly journals In Vivo Indicators of Cytoplasmic, Vacuolar, and Extracellular pH Using pHluorin2 in Candida albicans

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Hélène Tournu ◽  
Arturo Luna-Tapia ◽  
Brian M. Peters ◽  
Glen E. Palmer
Keyword(s):  
Candida Albicans ◽  
Intracellular Ph ◽  
Fungal Pathogen ◽  
Fluorescent Protein ◽  
Extracellular Ph ◽  
Adaptive Responses ◽  
Ph Homeostasis ◽  
Content Type ◽  
A Cell

ABSTRACT Candida albicans is an opportunistic fungal pathogen that colonizes the reproductive and gastrointestinal tracts of its human host. It can also invade the bloodstream and deeper organs of immunosuppressed individuals, and thus it encounters enormous variations in external pH in vivo. Accordingly, survival within such diverse niches necessitates robust adaptive responses to regulate intracellular pH. However, the impact of antifungal drugs upon these adaptive responses, and on intracellular pH in general, is not well characterized. Furthermore, the tools and methods currently available to directly monitor intracellular pH in C. albicans, as well as other fungal pathogens, have significant limitations. To address these issues, we developed a new and improved set of pH sensors based on the pH-responsive fluorescent protein pHluorin. This includes a cytoplasmic sensor, a probe that localizes inside the fungal vacuole (an acidified compartment that plays a central role in intracellular pH homeostasis), and a cell surface probe that can detect changes in extracellular pH. These tools can be used to monitor pH within single C. albicans cells or in cell populations in real time through convenient and high-throughput assays. Environmental or chemically induced stresses often trigger physiological responses that regulate intracellular pH. As such, the capacity to detect pH changes in real time and within live cells is of fundamental importance to essentially all aspects of biology. In this respect, pHluorin, a pH-sensitive variant of green fluorescent protein, has provided an invaluable tool to detect such responses. Here, we report the adaptation of pHluorin2 (PHL2), a substantially brighter variant of pHluorin, for use with the human fungal pathogen Candida albicans. As well as a cytoplasmic PHL2 indicator, we describe a version that specifically localizes within the fungal vacuole, an acidified subcellular compartment with important functions in nutrient storage and pH homeostasis. In addition, by means of a glycophosphatidylinositol-anchored PHL2-fusion protein, we generated a cell surface pH sensor. We demonstrated the utility of these tools in several applications, including accurate intracellular and extracellular pH measurements in individual cells via flow cytometry and in cell populations via a convenient plate reader-based protocol. The PHL2 tools can also be used for endpoint as well as time course experiments and to conduct chemical screens to identify drugs that alter normal pH homeostasis. These tools enable observation of the highly dynamic intracellular pH shifts that occur throughout the fungal growth cycle, as well as in response to various chemical treatments. IMPORTANCE Candida albicans is an opportunistic fungal pathogen that colonizes the reproductive and gastrointestinal tracts of its human host. It can also invade the bloodstream and deeper organs of immunosuppressed individuals, and thus it encounters enormous variations in external pH in vivo. Accordingly, survival within such diverse niches necessitates robust adaptive responses to regulate intracellular pH. However, the impact of antifungal drugs upon these adaptive responses, and on intracellular pH in general, is not well characterized. Furthermore, the tools and methods currently available to directly monitor intracellular pH in C. albicans, as well as other fungal pathogens, have significant limitations. To address these issues, we developed a new and improved set of pH sensors based on the pH-responsive fluorescent protein pHluorin. This includes a cytoplasmic sensor, a probe that localizes inside the fungal vacuole (an acidified compartment that plays a central role in intracellular pH homeostasis), and a cell surface probe that can detect changes in extracellular pH. These tools can be used to monitor pH within single C. albicans cells or in cell populations in real time through convenient and high-throughput assays.

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