Structure and conservation of amyloid spines from the Candida albicans Als5 adhesin including similarity to human LARKS

Candida Als family adhesins mediate adhesion to biological and abiotic substrates, as well as fungal cell aggregation and fungal-bacterial co-aggregation. The activity of at least two family members, Als5 and Als1, is dependent on amyloid-like protein aggregation that is initiated by shear force. Each Als adhesin has a ~300-residue N-terminal Ig-like/invasin region. The following 108-residue, low complexity, threonine-rich (T) domain unfolds under shear to expose a critical amyloid-forming segment 322SNGIVIVATTRTV334 at the interface between the Ig-like/invasin domain 2 and the T domain of Candida albicans Als5. Amyloid prediction programs identified six potential amyloidogenic sequences in the Ig/invasin region and three others in the T domain of C. albicans Als5. Peptides derived from four of these sequences formed fibrils that bound thioflavin T, the amyloid indicator dye, and three of these revealed atomic-resolution structures of cross-b spines. These are the first atomic-level structures for fungal adhesins. One of these segments, from the T domain, revealed kinked b-sheets, similarly to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked segments) found in human functional amyloids. Based on the cross-b structures in Als proteins, we use evolutionary arguments to identify functional amyloidogenic sequences in other fungal adhesins. Thus, cross-b structures are often involved in fungal pathogenesis and potentially in antifungal therapy.

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Threonine-Rich Repeats Increase Fibronectin Binding in the Candida albicans Adhesin Als5p

Eukaryotic Cell ◽

10.1128/ec.00120-06 ◽

2006 ◽

Vol 5 (10) ◽

pp. 1664-1673 ◽

Cited By ~ 86

Author(s):

Jason M. Rauceo ◽

Richard De Armond ◽

Henry Otoo ◽

Peter C. Kahn ◽

Stephen A. Klotz ◽

...

Keyword(s):

Candida Albicans ◽

Tandem Repeats ◽

Surface Display ◽

Cell Aggregation ◽

Cellular Adhesion ◽

Variable Number ◽

Fungal Cell ◽

T Domain ◽

Α Helix

ABSTRACT Commensal and pathogenic states of Candida albicans depend on cell surface-expressed adhesins, including those of the Als family. Mature Als proteins consist of a 300-residue N-terminal region predicted to have an immunoglobulin (Ig)-like fold, a 104-residue conserved Thr-rich region (T), a central domain of a variable number of tandem repeats (TR) of a 36-residue Thr-rich sequence, and a heavily glycosylated C-terminal Ser/Thr-rich stalk region, also of variable length (N. K. Gaur and S. A. Klotz, Infect. Immun. 65: 5289-5294, 1997). Domain deletions in ALS5 were expressed in Saccharomyces cerevisiae to excrete soluble protein and for surface display. Far UV circular dichroism indicated that soluble Ig-T showed a single negative peak at 212 nm, consistent with previous data indicating that this region has high β-sheet content with very little α-helix. A truncation of Als5p with six tandem repeats (Ig-T-TR6) gave spectra with additional negative ellipticity at 200 nm and, at 227 to 240 nm, spectra characteristic of a structure with a similar fraction ofβ -sheet but with additional structural elements as well. Soluble Als5p Ig-T and Ig-T-TR6 fragments bound to fibronectin in vitro, but the inclusion of the TR region substantially increased affinity. Cellular adhesion assays with S. cerevisiae showed that the Ig-T domain mediated adherence to fibronectin and that TR repeats greatly increased cell-to-cell aggregation. Thus, the TR region of Als5p modulated the structure of the Ig-T region, augmented cell adhesion activity through increased binding to mammalian ligands, and simultaneously promoted fungal cell-cell interactions.

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Structure and Function of Glycosylated Tandem Repeats from Candida albicans Als Adhesins

Eukaryotic Cell ◽

10.1128/ec.00235-09 ◽

2009 ◽

Vol 9 (3) ◽

pp. 405-414 ◽

Cited By ~ 38

Author(s):

Aaron T. Frank ◽

Caleen B. Ramsook ◽

Henry N. Otoo ◽

Cho Tan ◽

Gregory Soybelman ◽

...

Keyword(s):

Candida Albicans ◽

Tandem Repeats ◽

Cell Aggregation ◽

Structure And Function ◽

Hydrophobic Surfaces ◽

Content Type ◽

Domain Structures ◽

Aggregation Activity ◽

Fungal Adhesins ◽

And Function

ABSTRACT Tandem repeat (TR) regions are common in yeast adhesins, but their structures are unknown, and their activities are poorly understood. TR regions in Candida albicans Als proteins are conserved glycosylated 36-residue sequences with cell-cell aggregation activity (J. M. Rauceo, R. De Armond, H. Otoo, P. C. Kahn, S. A. Klotz, N. K. Gaur, and P. N. Lipke, Eukaryot. Cell 5:1664–1673, 2006). Ab initio modeling with either Rosetta or LINUS generated consistent structures of three-stranded antiparallel β-sheet domains, whereas randomly shuffled sequences with the same composition generated various structures with consistently higher energies. O- and N-glycosylation patterns showed that each TR domain had exposed hydrophobic surfaces surrounded by glycosylation sites. These structures are consistent with domain dimensions and stability measurements by atomic force microscopy (D. Alsteen, V. Dupres, S. A. Klotz, N. K. Gaur, P. N. Lipke, and Y. F. Dufrene, ACS Nano 3:1677–1682, 2009) and with circular dichroism determination of secondary structure and thermal stability. Functional assays showed that the hydrophobic surfaces of TR domains supported binding to polystyrene surfaces and other TR domains, leading to nonsaturable homophilic binding. The domain structures are like “classic” subunit interaction surfaces and can explain previously observed patterns of promiscuous interactions between TR domains in any Als proteins or between TR domains and surfaces of other proteins. Together, the modeling techniques and the supporting data lead to an approach that relates structure and function in many kinds of repeat domains in fungal adhesins.

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Efg1 Controls Caspofungin-Induced Cell Aggregation of Candida albicans through the Adhesin Als1

Eukaryotic Cell ◽

10.1128/ec.05187-11 ◽

2011 ◽

Vol 10 (12) ◽

pp. 1694-1704 ◽

Cited By ~ 22

Author(s):

Christa Gregori ◽

Walter Glaser ◽

Ingrid E. Frohner ◽

Cristina Reinoso-Martín ◽

Steffen Rupp ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Cell Aggregation ◽

Large Cell ◽

Fungal Species ◽

Invasive Growth ◽

Cell Integrity ◽

Fungal Cell ◽

Content Type ◽

Cell Wall Remodeling

ABSTRACTEchinocandin drugs such as caspofungin (CASP), micafungin, and anidulafungin inhibit fungal cell wall biogenesis by blocking Fks1-mediated β-glucan deposition into the cell surface. Candins have become suitable drugs to treat life-threatening diseases caused by several fungal species, includingCandida albicans, that are pathogenic for humans.Here, we present the discovery of a novel CASP-induced flocculation phenotype ofC. albicans, which formed large cell aggregates in the presence of CASP. High concentrations of sugars such as mannose or glucose inhibit CASP-induced flocculation and improve survival ofC. albicanscells exposed to CASP. Notably, exposure ofC. albicanscells to CASP triggers Efg1-dependent expression of the adhesinALS1and induces invasive growth on agar plates. Indeed, cells lacking either Efg1 or Als1 show strongly diminished CASP-induced flocculation, and the absence of Efg1 leads to marked CASP hypersensitivity. On the other hand, CASP-induced invasive growth is enhanced in cells lacking Efg1. Hence, CASP stress drives an Efg1-dependent response, indicating that this multifunctional transcriptional regulator, which is otherwise involved in filamentation, white-to-opaque switching, and virulence, also modulates cell wall remodeling upon CASP challenge. Taken together, our data suggest that CASP-induced cell wall damage activates Efg1 in parallel with the known cell integrity stress signaling pathway to coordinate cell wall remodeling.

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Sublethal Injury and Resuscitation of Candida albicans after Amphotericin B Treatment

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.4.1200-1206.2003 ◽

2003 ◽

Vol 47 (4) ◽

pp. 1200-1206 ◽

Cited By ~ 13

Author(s):

Robert S. Liao ◽

Robert P. Rennie ◽

James A. Talbot

Keyword(s):

Cell Death ◽

Candida Albicans ◽

Amphotericin B ◽

Antifungal Agents ◽

Sybr Green ◽

Sequential Treatment ◽

Tetrazolium Salt ◽

Fungal Cell ◽

Sublethal Injury

ABSTRACT Amphotericin B treatment was previously shown to inhibit Candida albicans reproduction and reduce the fluorescence of vitality-specific dyes without causing a corresponding increase in the fluorescence of the mortality-specific dyes bis-(1,3-dibutylbarbituric acid)trimethine oxonol and SYBR Green Ι. In the present study, we have confirmed these results and have shown that the numbers of CFU are reduced by 99.9% by treatment with 0.5 μg of amphotericin B per ml for 10 h at 35°C. This reduction was not due to fungal cell death. First, the level of reduction of the tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide increased in the presence of concentrations of amphotericin B that caused greater than 90% reductions in the numbers of CFU. Second, fungal cells treated with amphotericin B at a concentration of 0.5 μg/ml were resuscitated by further incubation at 22°C for 15 h in the continued presence of amphotericin B. Third, recovery of the ability to replicate was prevented by sequential treatment with 20 μg of miconazole per ml, which also increased the fluorescence of mortality-specific dyes to near the maximal levels achieved with 0.9 μg of amphotericin B per ml. Sequential treatment with fluconazole and flucytosine did not increase the levels of staining with the mortality-specific dyes. Itraconazole was less effective than ketoconazole, which was less effective than miconazole. The practice of equating the loss of the capacity of C. albicans to form colonies with fungal cell death may give incorrect results in assays with amphotericin B, and the results of assays with caution with other antifungal agents that are lipophilic or that possess significant postantifungal effects may need to be interpreted.

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The newly synthesized thiazole derivatives as potential antifungal compounds against Candida albicans

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11477-7 ◽

2021 ◽

Author(s):

Anna Biernasiuk ◽

Anna Berecka-Rycerz ◽

Anna Gumieniczek ◽

Maria Malm ◽

Krzysztof Z. Łączkowski ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Antifungal Activity ◽

Cell Membrane ◽

Mode Of Action ◽

Thiazole Derivatives ◽

Fungal Cell ◽

Erythrocyte Lysis ◽

Low Cytotoxicity ◽

High Antifungal Activity

Abstract Recently, the occurrence of candidiasis has increased dramatically, especially in immunocompromised patients. Additionally, their treatment is often ineffective due to the resistance of yeasts to antimycotics. Therefore, there is a need to search for new antifungals. A series of nine newly synthesized thiazole derivatives containing the cyclopropane system, showing promising activity against Candida spp., has been further investigated. We decided to verify their antifungal activity towards clinical Candida albicans isolated from the oral cavity of patients with hematological malignancies and investigate the mode of action on fungal cell, the effect of combination with the selected antimycotics, toxicity to erythrocytes, and lipophilicity. These studies were performed by the broth microdilution method, test with sorbitol and ergosterol, checkerboard technique, erythrocyte lysis assay, and reversed phase thin-layer chromatography, respectively. All derivatives showed very strong activity (similar and even higher than nystatin) against all C. albicans isolates with minimal inhibitory concentration (MIC) = 0.008–7.81 µg/mL Their mechanism of action may be related to action within the fungal cell wall structure and/or within the cell membrane. The interactions between the derivatives and the selected antimycotics (nystatin, chlorhexidine, and thymol) showed additive effect only in the case of combination some of them and thymol. The erythrocyte lysis assay confirmed the low cytotoxicity of these compounds as compared to nystatin. The high lipophilicity of the derivatives was related with their high antifungal activity. The present studies confirm that the studied thiazole derivatives containing the cyclopropane system appear to be a very promising group of compounds in treatment of infections caused by C. albicans. However, this requires further studies in vivo. Key points • The newly thiazoles showed high antifungal activity and some of them — additive effect in combination with thymol. • Their mode of action may be related with the influence on the structure of the fungal cell wall and/or the cell membrane. • The low cytotoxicity against erythrocytes and high lipophilicity of these derivatives are their additional good properties. Graphical abstract

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A Label-Free Cellular Proteomics Approach to Decipher the Antifungal Action of DiMIQ, a Potent Indolo[2,3-b]Quinoline Agent, against Candida albicans Biofilms

International Journal of Molecular Sciences ◽

10.3390/ijms22010108 ◽

2020 ◽

Vol 22 (1) ◽

pp. 108

Author(s):

Robert Zarnowski ◽

Anna Jaromin ◽

Agnieszka Zagórska ◽

Eddie G. Dominguez ◽

Katarzyna Sidoryk ◽

...

Keyword(s):

Extracellular Matrix ◽

Candida Albicans ◽

Ribosomal Proteins ◽

Nucleotide Metabolism ◽

Antibiofilm Activity ◽

Label Free ◽

Xtt Assay ◽

Fungal Cell ◽

Select Amino Acid ◽

Cellular Oxidation

Candida albicans forms extremely drug-resistant biofilms, which present a serious threat to public health globally. Biofilm-based infections are difficult to treat due to the lack of efficient antifungal therapeutics, resulting in an urgent demand for the development of novel antibiofilm strategies. In this study, the antibiofilm activity of DiMIQ (5,11-dimethyl-5H-indolo[2,3-b]quinoline) was evaluated against C. albicans biofilms. DiMIQ is a synthetic derivative of indoquinoline alkaloid neocryptolepine isolated from a medicinal African plant, Cryptolepis sanguinolenta. Antifungal activity of DiMIQ was determined using the XTT assay, followed by cell wall and extracellular matrix profiling and cellular proteomes. Here, we demonstrated that DiMIQ inhibited C. albicans biofilm formation and altered fungal cell walls and the extracellular matrix. Cellular proteomics revealed inhibitory action against numerous translation-involved ribosomal proteins, enzymes involved in general energy producing processes and select amino acid metabolic pathways including alanine, aspartate, glutamate, valine, leucine and isoleucine. DiMIQ also stimulated pathways of cellular oxidation, metabolism of carbohydrates, amino acids (glycine, serine, threonine, arginine, phenylalanine, tyrosine, tryptophan) and nucleic acids (aminoacyl-tRNA biosynthesis, RNA transport, nucleotide metabolism). Our findings suggest that DiMIQ inhibits C. albicans biofilms by arresting translation and multidirectional pathway reshaping of cellular metabolism. Overall, this agent may provide a potent alternative to treating biofilm-associated Candida infections.

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Paradoxical Effect of Caspofungin: Reduced Activity against Candida albicans at High Drug Concentrations

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.9.3407-3411.2004 ◽

2004 ◽

Vol 48 (9) ◽

pp. 3407-3411 ◽

Cited By ~ 167

Author(s):

David A. Stevens ◽

Marife Espiritu ◽

Rachana Parmar

Keyword(s):

Candida Albicans ◽

Resistance Mechanisms ◽

Paradoxical Effect ◽

Fungal Cell ◽

High Drug ◽

Minimum Fungicidal Concentration ◽

Drug Concentrations ◽

High Concentrations ◽

Glucan Synthesis

ABSTRACT Resistance problems with caspofungin, an echinocandin inhibitor of fungal cell wall glucan synthesis, have been rare. We noted paradoxical turbid growth of Candida albicans isolates in broth in some high (supra-MIC) concentrations. Among isolates submitted for susceptibility testing and screened at drug concentrations up to 12.5 μg/ml, the frequency was 16%. Analysis of the turbid growth indicated slowing of growth in the presence of drug but with numbers of CFU up to 72% those of drug-free controls. Clearing of growth again by the highest drug concentrations produced a quadriphasic pattern in a tube dilution series. Cells growing at high drug concentrations were not resistant on retesting but showed the paradoxical effect of the parent. Among a selected series of isolates tested at concentrations up to 50 μg/ml, an additional 53% showed a “mini-paradoxical effect”: no turbid growth but incomplete killing at high concentrations (supra-minimum fungicidal concentration). These effects were reproducible; medium dependent in extent; noted in macro- and microdilution, in the presence or absence of serum, and on agar containing drug (but not when drug concentrations were not constant, as in agar diffusion); not seen with other echinocandins and less commonly in other Candida species; and not due to destruction of drug in tubes showing the effect. Cooperative enhancement of inhibition by a second drug could eradicate the effect. We postulate that high drug concentrations derepress or activate resistance mechanisms. The abilities of subpopulations to survive at high drug concentrations could have in vivo consequences.

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Conserved Processes and Lineage-Specific Proteins in Fungal Cell Wall Evolution

Eukaryotic Cell ◽

10.1128/ec.00044-07 ◽

2007 ◽

Vol 6 (12) ◽

pp. 2269-2277 ◽

Cited By ~ 34

Author(s):

Juan E. Coronado ◽

Saad Mneimneh ◽

Susan L. Epstein ◽

Wei-Gang Qiu ◽

Peter N. Lipke

Keyword(s):

Cell Wall ◽

Protein Function ◽

Phosphatidyl Inositol ◽

Low Complexity ◽

Open Reading Frames ◽

Specific Cell ◽

Glycosyl Hydrolases ◽

Fungal Cell ◽

Structural Wall ◽

Lineage Specificity

ABSTRACT The cell wall is a defining organelle that differentiates fungi from its sister clades in the opisthokont superkingdom. With a sensitive technique to align low-complexity protein sequences, we have identified 187 cell wall-related proteins in Saccharomyces cerevisiae and determined the presence or absence of homologs in 17 other fungal genomes. There were both conserved and lineage-specific cell wall proteins, and the degree of conservation was strongly correlated with protein function. Some functional classes were poorly conserved and lineage specific: adhesins, structural wall glycoprotein components, and unannotated open reading frames. These proteins are primarily those that are constituents of the walls themselves. On the other hand, glycosyl hydrolases and transferases, proteases, lipases, proteins in the glycosyl phosphatidyl-inositol-protein synthesis pathway, and chaperones were strongly conserved. Many of these proteins are also conserved in other eukaryotes and are associated with wall synthesis in plants. This gene conservation, along with known similarities in wall architecture, implies that the basic architecture of fungal walls is ancestral to the divergence of the ascomycetes and basidiomycetes. The contrasting lineage specificity of wall resident proteins implies diversification. Therefore, fungal cell walls consist of rapidly diversifying proteins that are assembled by the products of an ancestral and conserved set of genes.

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In Fungal Intracellular Pathogenesis, Form Determines Fate

mBio ◽

10.1128/mbio.02092-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 4

Author(s):

Robin C. May ◽

Arturo Casadevall

Keyword(s):

Candida Albicans ◽

Fungal Pathogen ◽

Membrane Integrity ◽

Pathogenic Fungi ◽

Morphological Transition ◽

Physical Damage ◽

Fungal Cell ◽

Morphological Transitions ◽

Pathogenic Microbes ◽

Phagosomal Membrane

ABSTRACT For pathogenic microbes to survive ingestion by macrophages, they must subvert powerful microbicidal mechanisms within the phagolysosome. After ingestion, Candida albicans undergoes a morphological transition producing hyphae, while the surrounding phagosome exhibits a loss of phagosomal acidity. However, how these two events are related has remained enigmatic. Now Westman et al. (mBio 9:e01226-18, 2018, https://doi.org/10.1128/mBio.01226-18) report that phagosomal neutralization results from disruption of phagosomal membrane integrity by the enlarging hyphae, directly implicating the morphological transition in physical damage that promotes intracellular survival. The C. albicans intracellular strategy shows parallels with another fungal pathogen, Cryptococcus neoformans, where a morphological changed involving capsular enlargement intracellularly is associated with loss of membrane integrity and death of the host cell. These similarities among distantly related pathogenic fungi suggest that morphological transitions that are common in fungi directly affect the outcome of the fungal cell-macrophage interaction. For this class of organisms, form determines fate in the intracellular environment.

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Spatial Distribution of a Porphyrin-Based Photosensitizer Reveals Mechanism of Photodynamic Inactivation of Candida albicans

Frontiers in Medicine ◽

10.3389/fmed.2021.641244 ◽

2021 ◽

Vol 8 ◽

Author(s):

Thomas Voit ◽

Fabian Cieplik ◽

Johannes Regensburger ◽

Karl-Anton Hiller ◽

Anita Gollmer ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Fungal Infections ◽

Cell Envelope ◽

Antimicrobial Photodynamic Therapy ◽

Fungal Cell ◽

Before And After ◽

Antifungal Action ◽

Complete Cell ◽

Further Development

The antimicrobial photodynamic therapy (aPDT) is a promising approach for the control of microbial and especially fungal infections such as mucosal mycosis. TMPyP [5,10,15, 20-tetrakis(1-methylpyridinium-4-yl)-porphyrin tetra p-toluenesulfonate] is an effective photosensitizer (PS) that is commonly used in aPDT. The aim of this study was to examine the localization of TMPyP in Candida albicans before and after irradiation with visible light to get information about the cellular mechanism of antifungal action of the photodynamic process using this PS. Immediately after incubation of C. albicans with TMPyP, fluorescence microscopy revealed an accumulation of the PS in the cell envelope. After irradiation with blue light the complete cell showed red fluorescence, which indicates, that aPDT is leading to a damage in the cell wall with following influx of PS into the cytosol. Incubation of C. albicans with Wheat Germ Agglutinin (WGA) could confirm the cell wall as primary binding site of TMPyP. The finding that the porphyrin accumulates in the fungal cell wall and does not enter the interior of the cell before irradiation makes it unlikely that resistances can emerge upon aPDT. The results of this study may help in further development and modification of PS in order to increase efficacy against fungal infections such as those caused by C. albicans.

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