C.Albicans Enolase
Description
Metabolic Role
As a glycolytic enzyme, CaEno1 is essential for energy production. Knockout mutants show reduced viability and attenuated virulence in Galleria mellonella infection models .
Extracellular Roles
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Adhesion and Colonization:
CaEno1 mediates intestinal mucosal adhesion, a critical step for systemic candidiasis. Pretreating intestinal epithelium with recombinant enolase reduces C. albicans adhesion by 70% (0.5 mg/ml) . Anti-enolase antibodies inhibit adhesion by 48% . -
Biofilm Formation:
Enolase is enriched in C. albicans biofilms and hyphae, where it interacts with Als3, a surface adhesin. This interaction facilitates plasminogen recruitment, promoting tissue invasion . -
Immune Evasion and Activation:
CaEno1 binds B cells via MyD88-dependent signaling, inducing proliferation and antifungal antibody secretion (e.g., IgG2b) . Concurrently, it inactivates complement proteins, aiding immune evasion .
Diagnostic Applications
CaEno1 is detectable in serum during invasive candidiasis. A one-step DAS-ELISA using monoclonal antibodies achieves a sensitivity of 0.1 ng/ml, outperforming (1,3)-β-D-glucan assays :
| Diagnostic Method | Sensitivity | Specificity | Time to Detection |
|---|---|---|---|
| DAS-ELISA (CaEno1) | 95% | 98% | 2–7 days post-infection |
| (1,3)-β-D-glucan (G-test) | 80% | 85% | 1–3 days post-infection |
| Blood culture | 60% | 100% | 24–48 hours |
Therapeutic Targeting
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Baicalein (BE): Inhibits CaEno1 enolase activity () by binding D263/S269/K273, disrupting glycolysis .
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Recombinant enolase vaccines: Antibodies against CaEno1 reduce fungal burden in murine models .
Evolutionary and Clinical Significance
CaEno1’s dual roles exemplify "moonlighting" in fungi, where a single gene product performs unrelated functions . Its extracellular stability (short half-life unless secreted) suggests active secretion mechanisms rather than cell lysis . Clinically, CaEno1 is a biomarker for candidemia and a promising target for antifungal therapies .
Product Specs
Q&A
What is the primary function of C. albicans enolase in cellular metabolism?
Enolase (2-phospho-D-glycerate hydrolase, EC 4.2.1.11) in C. albicans is a cytosolic enzyme that catalyzes the interconversion between 2-phosphoglycerate and phosphoenolpyruvate during glycolysis (forward reaction) and gluconeogenesis (reverse reaction) . This reaction is a critical step in central carbon metabolism. Notably, enolase is encoded by a single locus in C. albicans, making its dual role as both a glycolytic enzyme and an extracellular protein a remarkable example of gene sharing in fungi .
What is meant by the "moonlighting" function of C. albicans enolase?
The term "moonlighting" refers to proteins that perform multiple, unrelated functions with a single polypeptide chain. C. albicans enolase exemplifies this concept through its dual roles:
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Primary function: Catalyzing a step in glycolysis within the cytoplasm
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Secondary functions: When localized to the cell surface or secreted extracellularly, enolase mediates adhesion to host tissues, binds to extracellular matrix proteins, and interacts with human plasminogen
This functional versatility enables C. albicans to utilize a single protein for both metabolic processes and host-pathogen interactions, despite enolase lacking a classical secretion signal .
Where is enolase located in C. albicans cells and how is this determined?
Enolase in C. albicans exhibits a dual localization pattern:
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Primarily in the cytosol as a glycolytic enzyme
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Significantly present on the cell surface and in extracellular biofilms
Researchers use multiple methodologies to confirm this dual localization:
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Flow cytometry comparing permeabilized and non-permeabilized cells to distinguish between intracellular and surface-exposed enolase
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Immunoblot analysis of conditioned media to detect secreted enolase
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Confocal microscopy with immunofluorescent labeling to visualize surface-exposed enolase
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Enolase decay assays to distinguish between actively secreted forms and those released by cell lysis
These techniques have demonstrated that extracellular enolase is not merely an artifact of cell lysis but represents functional secretion of a stable form, as cytosolic enolase released by lysis has a very short half-life outside the cells .
How does C. albicans enolase differ structurally from human enolase?
Crystal structure analysis reveals specific differences between C. albicans enolase (CaEno1) and human enolase (hEno1):
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The primary binding site for the antifungal compound Baicalein (BE) on CaEno1 is located between amino acids D261 and W274
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Three residues (D263, S269, and K273) play critical roles in interaction with BE
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Both positions S269 and K273 have different amino acids in human Eno1
These structural differences are significant because they enable selective targeting of the fungal enzyme without affecting the human counterpart, potentially reducing side effects in antifungal therapies targeting enolase .
What is the molecular basis for C. albicans enolase interactions with human proteins?
Surface plasmon resonance measurements have quantified the binding affinities between purified C. albicans enolase and human proteins:
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C. albicans enolase binds tightly to human vitronectin, fibronectin, and plasminogen with dissociation constants in the 10⁻⁷-10⁻⁸ M range
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In contrast, S. cerevisiae enolase binds these human proteins much more weakly
Chemical cross-linking methods have mapped the interaction sites:
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An internal motif 235DKAGYKGKVGIAMDVASSEFYKDGK259 in C. albicans enolase contributes to binding all three human proteins tested
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The binding sites for these human proteins extensively overlap yet are well-separated from the catalytic activity center
This spatial separation allows enolase to maintain its enzymatic function while simultaneously engaging in protein-protein interactions with host molecules.
How is C. albicans enolase related to pathogenicity?
C. albicans enolase contributes to pathogenicity through multiple mechanisms:
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Adhesion to host tissues: Enolase mediates attachment to intestinal mucosa, the major translocation site of C. albicans. Experimental evidence shows that pretreatment of intestinal epithelium with recombinant C. albicans enolase inhibits fungal adhesion by 70% at 0.5 mg/ml, while pretreatment of C. albicans with anti-enolase antibodies reduces adhesion by 48% with 20 μg antiserum .
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Interactions with host proteins: Enolase binds to extracellular matrix components and plasma proteins, potentially contributing to tissue invasion and modulation of host defense mechanisms .
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Biofilm formation: Enolase is present in C. albicans biofilms, which represent an essential mechanism for fungi to resist antifungal drugs .
These functions collectively enhance C. albicans virulence by facilitating colonization, invasion, and persistence in host tissues.
What mechanisms regulate the surface exposure of enolase in C. albicans?
The regulation of enolase surface exposure involves several mechanisms:
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Als3-mediated attachment: The agglutinin-like sequence protein Als3 serves as a crucial binding partner for surface display of enolase. Specific regions of Als3 are essential for this interaction, including:
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Morphological regulation: Surface-exposed enolase is more abundant on hyphal forms compared to yeast-like cells, suggesting coordination with morphogenesis pathways .
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Active secretion: Despite lacking a classical secretion signal, enolase appears to be actively secreted rather than passively released by cell lysis, as demonstrated by stability studies of extracellular enolase .
Understanding these regulatory mechanisms provides potential targets for disrupting enolase surface exposure without affecting its essential metabolic functions.
How does hyphal morphogenesis affect enolase distribution in C. albicans?
Hyphal morphogenesis significantly impacts enolase distribution:
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Differential expression: Higher abundance of enolase is observed at the surface of C. albicans hyphal forms compared to yeast-like cells .
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Als3 dependence: Als3, which mediates enolase attachment to the cell surface, is predominantly expressed in hyphal forms, providing a molecular explanation for the increased surface enolase in this morphology .
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Functional implications: Increased surface exposure of enolase on hyphal forms may enhance adhesion to host tissues during invasive growth, contributing to the greater virulence generally associated with the hyphal morphology .
This morphology-dependent regulation represents an important aspect of C. albicans pathogenicity, as the hyphal form is typically associated with tissue invasion.
What experimental approaches are used to study the extracellular functions of C. albicans enolase?
Researchers employ multiple complementary techniques:
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Recombinant protein expression: The C. albicans enolase gene can be PCR amplified using specific primers (e.g., EnoexpresF: 5'-CGGGATCCATGTCTTACGCCACTAAAATC-3' and EnoexpresR: 5'-ATAGTTTAGCGGCCGCTTACAATTGAGAAGCCTTT-3'), cloned into expression vectors, and purified for functional studies .
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Adhesion assays: Using intestinal mucosa disks to quantify how pretreatment with recombinant enolase or anti-enolase antibodies affects C. albicans adhesion .
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Enolase decay assays: Comparing the stability of cytosolic versus secreted enolase under different conditions to distinguish between active secretion and passive release .
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Surface plasmon resonance: Quantifying binding kinetics between purified enolase and human proteins like vitronectin, fibronectin, and plasminogen .
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Crystallography: Determining three-dimensional structures to identify binding sites for potential inhibitors and differences from human enolase .
These approaches collectively provide insights into the multifunctional nature of C. albicans enolase and its roles in pathogenicity.
How can researchers differentiate between cytosolic enolase released by cell lysis and actively secreted enolase?
Distinguishing between passive release and active secretion requires several experimental approaches:
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Enolase decay assays: Comparing the stability of enolase in different environments. In one protocol, C. albicans cultures are separated into two batches:
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Batch 1: Cells and media together
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Batch 2: Conditioned media separated from cells
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Samples are taken at various time points (0 min, 30 min, 1, 2, 3, 4, and 5 h), concentrated, and analyzed by immunoblotting. Actively secreted enolase shows greater stability than cytosolic enolase released by lysis .
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Cell integrity markers: Monitoring other cytosolic enzymes that are not actively secreted as controls for cell lysis.
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Flow cytometry: Comparing permeabilized versus non-permeabilized cells to distinguish between intracellular and surface-exposed enolase .
These methods have demonstrated that extracellular enolase is not simply an artifact of cell lysis but represents functional secretion of a stable form.
How can C. albicans enolase be targeted for antifungal therapy?
C. albicans enolase represents a promising target for novel antifungal strategies:
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Small molecule inhibitors: Compounds like Baicalein (BE) disrupt glycolysis by targeting Eno1. Crystal structure analysis identified the binding site between amino acids D261 and W274, with D263, S269, and K273 playing critical roles .
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Selective targeting: Structural differences between C. albicans and human enolase can be exploited for selective inhibition. Positions S269 and K273 in C. albicans enolase differ from human Eno1, offering potential for species-specific targeting .
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Adhesion inhibitors: Since enolase mediates adhesion to host tissues, compounds that block this function could prevent colonization. In experimental models, pretreatment with recombinant enolase or anti-enolase antibodies significantly reduces adhesion to intestinal epithelium .
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Disrupting surface localization: Targeting the Als3-enolase interaction could reduce surface exposure without affecting cytosolic function, potentially limiting virulence while minimizing selection pressure for resistance .
These approaches offer novel strategies for antifungal development that differ from conventional drugs targeting cell wall or membrane components.
What is the potential of targeting the Als3-enolase interaction for antifungal development?
The Als3-enolase interaction represents a promising target for novel antifungal strategies:
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Specificity: Targeting protein-protein interactions specific to C. albicans may reduce side effects compared to broader-spectrum antifungals.
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Reduced resistance potential: Disrupting surface localization of enolase rather than its essential enzymatic function could limit selection pressure for resistance.
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Identified binding domains: Research has defined specific regions of Als3 essential for enolase binding:
These defined regions provide structural targets for rational drug design of small molecules or peptides that could interfere with the Als3-enolase interaction.
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Downstream effects: Disrupting this interaction would not only reduce surface enolase but also limit subsequent binding of host plasma proteins, potentially attenuating virulence without directly affecting fungal viability .
Future research in this area could yield novel antifungal approaches that reduce virulence rather than targeting essential cellular functions.
Product Science Overview
Enolase is an immunodominant antigen in Candida albicans, meaning it is a primary target for the immune response during infection . It is abundantly expressed in the cytoplasm of C. albicans cells and is distributed in a ring-shaped pattern along the cell wall . Interestingly, enolase is also found on the surface of the fungal cells, where it can interact with host immune cells .
Recombinant enolase refers to the enolase protein that has been cloned, expressed, and purified using recombinant DNA technology. This involves inserting the enolase gene from Candida albicans into a suitable expression vector, which is then introduced into a host organism, such as Escherichia coli, to produce the protein . The recombinant enolase can be tagged with a His-tag to facilitate its purification and detection .
Recombinant enolase has several important applications in research and medicine:
- Diagnostic Marker: Enolase is considered a promising molecular marker for the diagnosis of invasive candidiasis, a severe fungal infection .
- Vaccine Development: Due to its immunogenic properties, enolase is being explored as a potential vaccine candidate against Candida infections .
- Immune Modulation: Studies have shown that enolase can bind to human CD4+ T cells and modulate immune responses, which could have implications for understanding fungal pathogenesis and developing new therapeutic strategies .
