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

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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Quinacrine Inhibits Candida albicans Growth and Filamentation at Neutral pH

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03083-14 ◽

2014 ◽

Vol 58 (12) ◽

pp. 7501-7509 ◽

Cited By ~ 14

Author(s):

Vibhati V. Kulkarny ◽

Alba Chavez-Dozal ◽

Hallie S. Rane ◽

Maximillian Jahng ◽

Stella M. Bernardo ◽

...

Keyword(s):

Candida Albicans ◽

Bloodstream Infections ◽

High Dose ◽

Antibiofilm Activity ◽

Dependent Manner ◽

Xtt Assay ◽

Content Type ◽

Prevention And Treatment ◽

Ph Dependent

ABSTRACTCandida albicansis a common cause of catheter-related bloodstream infections (CR-BSI), in part due to its strong propensity to form biofilms. Drug repurposing is an approach that might identify agents that are able to overcome antifungal drug resistance within biofilms. Quinacrine (QNC) is clinically active against the eukaryotic protozoan parasitesPlasmodiumandGiardia. We sought to investigate the antifungal activity of QNC againstC. albicansbiofilms.C. albicansbiofilms were incubated with QNC at serially increasing concentrations (4 to 2,048 μg/ml) and assessed using a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay in a static microplate model. Combinations of QNC and standard antifungals were assayed using biofilm checkerboard analyses. To define a mechanism of action, QNC was assessed for the inhibition of filamentation, effects on endocytosis, and pH-dependent activity. High-dose QNC was effective for the prevention and treatment ofC. albicansbiofilmsin vitro. QNC with fluconazole had no interaction, while the combination of QNC and either caspofungin or amphotericin B demonstrated synergy. QNC was most active against planktonic growth at alkaline pH. QNC dramatically inhibited filamentation. QNC accumulated within vacuoles as expected and caused defects in endocytosis. A tetracycline-regulatedVMA3mutant lacking vacuolar ATPase (V-ATPase) function demonstrated increased susceptibility to QNC. These experiments indicate that QNC is active againstC. albicansgrowth in a pH-dependent manner. Although QNC activity is not biofilm specific, QNC is effective in the prevention and treatment of biofilms. QNC antibiofilm activity likely occurs via several independent mechanisms: vacuolar alkalinization, inhibition of endocytosis, and impaired filamentation. Further investigation of QNC for the treatment and prevention of biofilm-relatedCandidaCR-BSI is warranted.

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Antibiofilm Activity on Candida albicans and Mechanism of Action on Biomembrane Models of the Antimicrobial Peptide Ctn[15–34]

International Journal of Molecular Sciences ◽

10.3390/ijms21218339 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8339

Author(s):

Francisca Lidiane Linhares de Aguiar ◽

Nuno C. Santos ◽

Carolina Sidrim de Paula Cavalcante ◽

David Andreu ◽

Gandhi Radis Baptista ◽

...

Keyword(s):

Candida Albicans ◽

Antimicrobial Peptide ◽

Mechanism Of Action ◽

Laser Scanning ◽

Fluorescent Dyes ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Antibiofilm Activity ◽

Membrane Composition ◽

Fungal Cell

Ctn[15–34], the C-terminal fragment of crotalicidin, an antimicrobial peptide from the South American rattlesnake Crotalus durissus terrificus venom, displays remarkable anti-infective and anti-proliferative activities. Herein, its activity on Candida albicans biofilms and its interaction with the cytoplasmic membrane of the fungal cell and with a biomembrane model in vitro was investigated. A standard C. albicans strain and a fluconazole-resistant clinical isolate were exposed to the peptide at its minimum inhibitory concentration (MIC) (10 µM) and up to 100 × MIC to inhibit biofilm formation and its eradication. A viability test using XTT and fluorescent dyes, confocal laser scanning microscopy, and atomic force microscopy (AFM) were used to observe the antibiofilm effect. To evaluate the importance of membrane composition on Ctn[15–34] activity, C. albicans protoplasts were also tested. Fluorescence assays using di-8-ANEPPS, dynamic light scattering, and zeta potential measurements using liposomes, protoplasts, and C. albicans cells indicated a direct mechanism of action that was dependent on membrane interaction and disruption. Overall, Ctn[15–34] showed to be an effective antifungal peptide, displaying antibiofilm activity and, importantly, interacting with and disrupting fungal plasma membrane.

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Sox9 Determines Translational Capacity During Early Chondrogenic Differentiation of ATDC5 Cells by Regulating Expression of Ribosome Biogenesis Factors and Ribosomal Proteins

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.686096 ◽

2021 ◽

Vol 9 ◽

Author(s):

Marjolein M. J. Caron ◽

Maxime Eveque ◽

Berta Cillero-Pastor ◽

Ron M. A. Heeren ◽

Bas Housmans ◽

...

Keyword(s):

Extracellular Matrix ◽

Early Phase ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Chondrogenic Differentiation ◽

Immediate Early ◽

Label Free ◽

Sox9 Expression ◽

Network Analyses ◽

Cartilage Extracellular Matrix

IntroductionIn addition to the well-known cartilage extracellular matrix-related expression of Sox9, we demonstrated that chondrogenic differentiation of progenitor cells is driven by a sharply defined bi-phasic expression of Sox9: an immediate early and a late (extracellular matrix associated) phase expression. In this study, we aimed to determine what biological processes are driven by Sox9 during this early phase of chondrogenic differentiation.MaterialsSox9 expression in ATDC5 cells was knocked down by siRNA transfection at the day before chondrogenic differentiation or at day 6 of differentiation. Samples were harvested at 2 h and 7 days of differentiation. The transcriptomes (RNA-seq approach) and proteomes (Label-free proteomics approach) were compared using pathway and network analyses. Total protein translational capacity was evaluated with the SuNSET assay, active ribosomes were evaluated with polysome profiling, and ribosome modus was evaluated with bicistronic reporter assays.ResultsEarly Sox9 knockdown severely inhibited chondrogenic differentiation weeks later. Sox9 expression during the immediate early phase of ATDC5 chondrogenic differentiation regulated the expression of ribosome biogenesis factors and ribosomal protein subunits. This was accompanied by decreased translational capacity following Sox9 knockdown, and this correlated to lower amounts of active mono- and polysomes. Moreover, cap- versus IRES-mediated translation was altered by Sox9 knockdown. Sox9 overexpression was able to induce reciprocal effects to the Sox9 knockdown.ConclusionHere, we identified an essential new function for Sox9 during early chondrogenic differentiation. A role for Sox9 in regulation of ribosome amount, activity, and/or composition may be crucial in preparation for the demanding proliferative phase and subsequent cartilage extracellular matrix production of chondroprogenitors in the growth plate in vivo.

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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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Chromatin-Associated Proteins Revealed by SILAC-Proteomic Analysis Exhibit a High Likelihood of Requirement for Growth Fitness under DNA Damage Stress

International Journal of Proteomics ◽

10.1155/2012/630409 ◽

2012 ◽

Vol 2012 ◽

pp. 1-12

Author(s):

Han Wang ◽

Pornpimol Tipthara ◽

Lei Zhu ◽

Suk Yean Poon ◽

Kai Tang ◽

...

Keyword(s):

Dna Damage ◽

Proteomic Analysis ◽

Ribosomal Proteins ◽

Cell Extract ◽

Label Free ◽

High Likelihood ◽

Nonhistone Proteins ◽

Quantitative Proteomic Analysis ◽

Proteomic Approach ◽

Associated Functions

Chromatin-associated nonhistone proteins (CHRAPs) are readily collected from the DNaseI digested crude chromatin preparation. In this study, we show that the absolute abundance-based label-free quantitative proteomic analysis fail to identify potential CHRAPs from the CHRAP-prep. This is because that the most-highly abundant cytoplasmic proteins such as ribosomal proteins are not effectively depleted in the CHRAP-prep. Ribosomal proteins remain the top-ranked abundant proteins in the CHRAP-prep. On the other hand, we show that relative abundance-based SILAC-mediated quantitative proteomic analysis is capable of discovering the potential CHRAPs in the CHRAP-prep when compared to the whole-cell-extract. Ribosomal proteins are depleted from the top SILAC ratio-ranked proteins. In contrast, nucleus-localized proteins or potential CHRAPs are enriched in the top SILAC-ranked proteins. Consistent with this, gene-ontology analysis indicates that CHRAP-associated functions such as transcription, regulation of chromatin structures, and DNA replication and repair are significantly overrepresented in the top SILAC-ranked proteins. Some of the novel CHRAPs are confirmed using the traditional method. Notably, phenotypic assessment reveals that the top SILAC-ranked proteins exhibit the high likelihood of requirement for growth fitness under DNA damage stress. Taken together, our results indicate that the SILAC-mediated proteomic approach is capable of determining CHRAPs without prior knowledge.

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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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Effect of different periods of preconditioning with saliva on Candida albicans adhesion to a denture base resin by crystal violet staining and XTT assay

Journal of Investigative and Clinical Dentistry ◽

10.1111/j.2041-1626.2010.00013.x ◽

2010 ◽

Vol 1 (2) ◽

pp. 114-119 ◽

Cited By ~ 5

Author(s):

Camila Andrade Zamperini ◽

Patrícia Cristiane dos Santos Schiavinato ◽

Ana Lucia Machado ◽

Eunice Teresinha Giampaolo ◽

Ana Claudia Pavarina ◽

...

Keyword(s):

Candida Albicans ◽

Crystal Violet ◽

Crystal Violet Staining ◽

Xtt Assay ◽

Denture Base ◽

Base Resin ◽

Denture Base Resin

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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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