A GDPase/UDPase Bifunctional Enzyme From Candida Albicans: Purification and Biochemical Characterization

Abstract The most frequently isolated human fungal pathogen is Candida albicans which is responsible for about 50% of all Candida infections. In healthy individuals, this organism resides as a part of the normal microbiota in equilibrium with the host. However, under certain conditions, particularly in immunocompromised patients, this opportunistic pathogen adheres to host cells causing serious systemic infections. Thus, much effort has been dedicated to the study of its physiology with emphasis on factors associated to pathogenicity. A representative analysis deals with the mechanisms of glycoprotein assembly as many cell surface antigens and other macromolecules that modulate the immune system fall within this chemical category. In this regard, studies of the terminal protein glycosylation stage which occurs in Golgi vesicles has led to the identification of nucleotidases that convert glycosyltransferase-generated dinucleotides into the corresponding mononucleotides, thus playing a double function: their activity prevent inhibition of further glycosyl transfer by the accumulation of dinucleotides and the resulting mononucleotides are exchanged by specific membrane transporters for equimolecular amounts of sugar donors from the cytosol. Here, using a simple protocol for protein separation we isolated a bifunctional nucleotidase from C. albicans active on GDP and UDP that was characterized in terms of its molecular mass, response to bivalent ions and other factors, substrate specificity and affinity. Results are discussed in terms of the similarities and differences of this nucleotidase with similar counterparts from other organisms thus contributing to the knowledge of a bifunctional diphosphatase not described before in C. albicans.

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A small molecule produced by Lactobacillus species blocks Candida albicans filamentation by inhibiting a DYRK1-family kinase

Nature Communications ◽

10.1038/s41467-021-26390-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jessie MacAlpine ◽

Martin Daniel-Ivad ◽

Zhongle Liu ◽

Junko Yano ◽

Nicole M. Revie ◽

...

Keyword(s):

Candida Albicans ◽

Small Molecule ◽

Biochemical Characterization ◽

Opportunistic Pathogen ◽

Vaginal Candidiasis ◽

Lactobacillus Species ◽

Microbiome Composition ◽

Human Host ◽

Bioassay Guided Fractionation ◽

Human Mucosa

AbstractThe fungus Candida albicans is an opportunistic pathogen that can exploit imbalances in microbiome composition to invade its human host, causing pathologies ranging from vaginal candidiasis to fungal sepsis. Bacteria of the genus Lactobacillus are colonizers of human mucosa and can produce compounds with bioactivity against C. albicans. Here, we show that some Lactobacillus species produce a small molecule under laboratory conditions that blocks the C. albicans yeast-to-filament transition, an important virulence trait. It remains unexplored whether the compound is produced in the context of the human host. Bioassay-guided fractionation of Lactobacillus-conditioned medium linked this activity to 1-acetyl-β-carboline (1-ABC). We use genetic approaches to show that filamentation inhibition by 1-ABC requires Yak1, a DYRK1-family kinase. Additional biochemical characterization of structurally related 1-ethoxycarbonyl-β-carboline confirms that it inhibits Yak1 and blocks C. albicans biofilm formation. Thus, our findings reveal Lactobacillus-produced 1-ABC can prevent the yeast-to-filament transition in C. albicans through inhibition of Yak1.

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Heterogeneous distribution of Candida albicans cell-surface antigens demonstrated with an Als1-specific monoclonal antibody

Microbiology ◽

10.1099/mic.0.043851-0 ◽

2010 ◽

Vol 156 (12) ◽

pp. 3645-3659 ◽

Cited By ~ 26

Author(s):

David A. Coleman ◽

Soon-Hwan Oh ◽

Xiaomin Zhao ◽

Lois L. Hoyer

Keyword(s):

Candida Albicans ◽

Cell Surface ◽

Spatial Localization ◽

Host Cells ◽

Yeast Cells ◽

Cell Surface Antigens ◽

Heterogeneous Distribution ◽

Germ Tubes

Despite an abundance of data describing expression of genes in the Candida albicans ALS (agglutinin-like sequence) gene family, little is known about the production of Als proteins on individual cells, their spatial localization or stability. Als proteins are most commonly discussed with respect to function in adhesion of C. albicans to host and abiotic surfaces. Development of a mAb specific for Als1, one of the eight large glycoproteins encoded by the ALS family, provided the opportunity to detect Als1 during growth of yeast and hyphae, both in vitro and in vivo, and to demonstrate the utility of the mAb in blocking C. albicans adhesion to host cells. Although most C. albicans yeast cells in a saturated culture are Als1-negative by indirect immunofluorescence, Als1 is detected on the surface of nearly all cells shortly after transfer into fresh growth medium. Als1 covers the yeast cell surface, with the exception of bud scars. Daughters of the inoculum cells, and sometimes granddaughters, also have detectable Als1, but Als1 is not detectable on cells from subsequent generations. On germ tubes and hyphae, most Als1 is localized proximal to the mother yeast. Once deposited on yeasts or hyphae, Als1 persists long after the culture has reached saturation. Growth stage-dependent production of Als1, coupled with its persistence on the cell surface, results in a heterogeneous population of cells within a C. albicans culture. Anti-Als1 immunolabelling patterns vary depending on the source of the C. albicans cells, with obvious differences between cells recovered from culture and those from a murine model of disseminated candidiasis. Results from this work highlight the temporal parallels for ALS1 expression and Als1 production in yeasts and germ tubes, the specialized spatial localization and persistence of Als1 on the C. albicans cell surface, and the differences in Als1 localization that occur in vitro and in vivo.

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Depletion of the Cullin Cdc53p Induces Morphogenetic Changes in Candida albicans

Eukaryotic Cell ◽

10.1128/ec.00332-08 ◽

2009 ◽

Vol 8 (5) ◽

pp. 756-767 ◽

Cited By ~ 16

Author(s):

Katharina Trunk ◽

Patrick Gendron ◽

André Nantel ◽

Sébastien Lemieux ◽

Terry Roemer ◽

...

Keyword(s):

Candida Albicans ◽

Essential Gene ◽

Hyphal Growth ◽

Growth Mode ◽

Environmental Signals ◽

Human Fungal Pathogen ◽

Ubiquitin Ligase Complex ◽

A Genome ◽

Ubiquitination Pathway ◽

Systemic Infections

ABSTRACT Candida albicans is an important opportunistic human fungal pathogen that can cause both mucosal and systemic infections in immunocompromised patients. Critical for the virulence of C. albicans is its ability to undergo a morphological transition from yeast to hyphal growth mode. Proper induction of filamentation is dependent on the ubiquitination pathway, which targets proteins for proteasome-mediated protein degradation or activates them for signaling events. In the present study, we evaluated the role of ubiquitination in C. albicans by impairing the function of the major ubiquitin-ligase complex SCF. This was done by depleting its backbone, the cullin Cdc53p (orf19.1674), using a tetracycline downregulatable promoter system. Cdc53p-depleted cells displayed an invasive phenotype and constitutive filamentation under conditions favoring yeast growth mode, both on solid and in liquid media. In addition, these cells exhibited an early onset of cell death, as judged from propidium iodide staining, suggesting that CDC53 is an essential gene in C. albicans. To identify Cdc53p-dependent pathways in C. albicans, a genome-wide expression analysis was carried out that revealed a total of 425 differentially expressed genes (fold change, ≥2; P ≤ 0.05) with 192 up- and 233 downregulated genes in the CDC53-repressed mutant compared to the control strain. GO term analysis identified biological processes significantly affected by Cdc53p depletion, including amino acid starvation response, with 14 genes being targets of the transcriptional regulator Gcn4p, and reductive iron transport. These results indicate that Cdc53p enables C. albicans to adequately respond to environmental signals.

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MNN5 Encodes an Iron-Regulated α-1,2-Mannosyltransferase Important for Protein Glycosylation, Cell Wall Integrity, Morphogenesis, and Virulence in Candida albicans

Eukaryotic Cell ◽

10.1128/ec.5.2.238-247.2006 ◽

2006 ◽

Vol 5 (2) ◽

pp. 238-247 ◽

Cited By ~ 33

Author(s):

Chen Bai ◽

Xiao-Li Xu ◽

Fong-Yee Chan ◽

Raymond Teck Ho Lee ◽

Yue Wang

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Iron Homeostasis ◽

Pathogenic Fungus ◽

Hyphal Growth ◽

Protein Glycosylation ◽

Host Cells ◽

Microbial Pathogens ◽

Solid Media ◽

Wide Range

ABSTRACT The cell walls of microbial pathogens mediate physical interactions with host cells and hence play a key role in infection. Mannosyltransferases have been shown to determine the cell wall properties and virulence of the pathogenic fungus Candida albicans. We previously identified a C. albicans α-1,2-mannosyltransferase, Mnn5, for its novel ability to enhance iron usage in Saccharomyces cerevisiae. Here we have studied the enzymatic properties of purified Mnn5 and characterized its function in its natural host. Mnn5 catalyzes the transfer of mannose to both α-1,2- and α-1,6-mannobiose, and this activity requires Mn2+ as a cofactor and is regulated by the Fe2+ concentration. An mnn5Δ mutant showed a lowered ability to extend O-linked, and possibly also N-linked, mannans, hypersensitivity to cell wall-damaging agents, and a reduction of cell wall mannosylphosphate content, phenotypes typical of many fungal mannosyltransferase mutants. The mnn5Δ mutant also exhibited some unique defects, such as impaired hyphal growth on solid media and attenuated virulence in mice. An unanticipated phenotype was the mnn5Δ mutant's resistance to killing by the iron-chelating protein lactoferrin, rendering it the first protein found that mediates lactoferrin killing of C. albicans. In summary, MNN5 deletion impairs a wide range of cellular events, most likely due to its broad substrate specificity. Of particular interest was the observed role of iron in regulating the enzymatic activity, suggesting an underlying relationship between Mnn5 activity and cellular iron homeostasis.

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Recent advances on Candida albicans biology and virulence

F1000Research ◽

10.12688/f1000research.9617.1 ◽

2016 ◽

Vol 5 ◽

pp. 2582 ◽

Cited By ~ 19

Author(s):

Adnane Sellam ◽

Malcolm Whiteway

Keyword(s):

Candida Albicans ◽

Drug Targets ◽

Opportunistic Pathogen ◽

Antifungal Drugs ◽

Human Fungal Pathogen ◽

Recent Developments ◽

Immunosuppressed Patients ◽

Technical Advances ◽

Life Threatening ◽

Human Flora

Candida albicans is an important human fungal pathogen, in terms of both its clinical significance and its use as an experimental model for scientific investigation. Although this opportunistic pathogen is a natural component of the human flora, it can cause life-threatening infections in immunosuppressed patients. There are currently a limited number of antifungal molecules and drug targets, and increasing resistance to the front-line therapeutics, demonstrating a clear need for new antifungal drugs. Understanding the biology of this pathogen is an important prerequisite for identifying new drug targets for antifungal therapeutics. In this review, we highlight some recent developments that help us to understand how virulence traits are regulated at the molecular level, in addition to technical advances that improve the ability of genome editing in C. albicans.

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Inhibition of Candida albicans extracellular enzyme activity by selected natural substances and their application inCandidainfection

Canadian Journal of Microbiology ◽

10.1139/w08-029 ◽

2008 ◽

Vol 54 (6) ◽

pp. 435-440 ◽

Cited By ~ 19

Author(s):

M. Yordanov ◽

P. Dimitrova ◽

S. Patkar ◽

L. Saso ◽

N. Ivanovska

Keyword(s):

Enzyme Activity ◽

Candida Albicans ◽

Extracellular Enzymes ◽

Indole Alkaloid ◽

Extracellular Enzyme ◽

Protein Glycosylation ◽

Host Cells ◽

Natural Substances ◽

Secreted Aspartyl Proteinase

Extracellular enzymes secreted by Candida albicans are claimed to be virulence factors responsible for penetration of the yeast into host cells. Substances able to inhibit lipolytic and proteinase activities of the fungus might be of therapeutic use in some pathologic conditions caused by C. albicans. In the present work, we have tested the influence of the flavonoid compounds apigenin and kaempferol, the indole alkaloid ibogaine, and the protoberberine alkaloid berberine on the in vitro enzyme activity of C. albicans. The substances showed complex suppressive effects concerning the processes of adherence to epithelial cells, secreted aspartyl proteinase activity, and the rate of cell wall protein glycosylation. Apigenin and kaempferol were administered in systemic C. albicans infection, demonstrating an increased number of survivors by kaempferol. The application of apigenin, kaempferol, ibogaine, and berberine in cutaneous infection suppressed the symptoms and accelerated elimination of the yeast from the site of inoculation.

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Diagnosis and treatment of mucosa Candida spp. infections – a review article

Annales Universitatis Mariae Curie-Sklodowska sectio C – Biologia ◽

10.17951/c.2018.73.1.61-68 ◽

2019 ◽

Vol 73 (1) ◽

pp. 61 ◽

Cited By ~ 1

Author(s):

Marta Dąbrowska ◽

Monika Sienkiewicz ◽

Paweł Kwiatkowski ◽

Michał Dąbrowski

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Fungal Infections ◽

Opportunistic Pathogen ◽

Review Article ◽

Immune Disorders ◽

Candida Spp ◽

Common Cause ◽

Invasive Infections ◽

Systemic Infections

<p>Candida albicans is the most common cause of fungal infections worldwide. Non-albicans Candida species play an important role in vulvovaginal candidiasis and invasive infections. Most cases of infections are endogenous. In case of patients with immune disorders this opportunistic pathogen causes both surface, systemic infections, and candidemia. Symptoms depend on the area affected. Candidiasis are treated with antimycotics; these include clotrimazole, nystatin, fluconazole, voriconazole, amphotericin B, and echinocandins. The emergence of drug resistance and the side effects of currently available antifungals are becoming a major problem in the management of Candida spp. infection.</p>

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Sur7 Promotes Plasma Membrane Organization and Is Needed for Resistance to Stressful Conditions and to the Invasive Growth and Virulence of Candida albicans

mBio ◽

10.1128/mbio.00254-11 ◽

2011 ◽

Vol 3 (1) ◽

Cited By ~ 32

Author(s):

Lois M. Douglas ◽

Hong X. Wang ◽

Sabine Keppler-Ross ◽

Neta Dean ◽

James B. Konopka

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Plasma Membrane ◽

Fungal Pathogen ◽

Antifungal Drugs ◽

Membrane Organization ◽

Content Type ◽

Human Fungal Pathogen ◽

Membrane Compartment ◽

Systemic Infections

ABSTRACTThe human fungal pathogenCandida albicanscauses lethal systemic infections because of its ability to grow and disseminate in a host. TheC. albicansplasma membrane is essential for virulence by acting as a protective barrier and through its key roles in interfacing with the environment, secretion of virulence factors, morphogenesis, and cell wall synthesis. Difficulties in studying hydrophobic membranes have limited the understanding of how plasma membrane organization contributes to its function and to the actions of antifungal drugs. Therefore, the role of the recently discovered plasma membrane subdomains termed the membrane compartment containing Can1 (MCC) was analyzed by assessing the virulence of asur7Δ mutant. Sur7 is an integral membrane protein component of the MCC that is needed for proper localization of actin, morphogenesis, cell wall synthesis, and responding to cell wall stress. MCC domains are stable 300-nm-sized punctate patches that associate with a complex of cytoplasmic proteins known as an eisosome. Analysis of virulence-related properties of asur7Δ mutant revealed defects in intraphagosomal growth in macrophages that correlate with increased sensitivity to oxidation and copper. Thesur7Δ mutant was also strongly defective in pathogenesis in a mouse model of systemic candidiasis. The mutant cells showed a decreased ability to initiate an infection and greatly diminished invasive growth into kidney tissues. These studies on Sur7 demonstrate that the plasma membrane MCC domains are critical for virulence and represent an important new target for the development of novel therapeutic strategies.IMPORTANCECandida albicans, the most common human fungal pathogen, causes lethal systemic infections by growing and disseminating in a host. The plasma membrane plays key roles in enablingC. albicansto growin vivo, and it is also the target of the most commonly used antifungal drugs. However, plasma membrane organization is poorly understood because of the experimental difficulties in studying hydrophobic components. Interestingly, recent studies have identified a novel type of plasma membrane subdomain in fungi known as the membrane compartment containing Can1 (MCC). Cells lacking the MCC-localized protein Sur7 display broad defects in cellular organization and response to stressin vitro. Consistent with this,C. albicanscells lacking theSUR7gene were more susceptible to attack by macrophages than cells with the gene and showed greatly reduced virulence in a mouse model of systemic infection. Thus, Sur7 and other MCC components represent novel targets for antifungal therapy.

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