Quinacrine Inhibits Candida albicans Growth and Filamentation at Neutral pH

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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In VitroAnalyses of the Effects of Heparin and Parabens on Candida albicans Biofilms and Planktonic Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05061-11 ◽

2011 ◽

Vol 56 (1) ◽

pp. 148-153 ◽

Cited By ~ 15

Author(s):

Marisa H. Miceli ◽

Stella M. Bernardo ◽

T. S. Neil Ku ◽

Carla Walraven ◽

Samuel A. Lee

Keyword(s):

Candida Albicans ◽

Metabolic Activity ◽

Biofilm Formation ◽

High Dose ◽

Dependent Manner ◽

Planktonic Cells ◽

Content Type ◽

Heparin Sodium ◽

The Individual

ABSTRACTInfections and thromboses are the most common complications associated with central venous catheters. Suggested strategies for prevention and management of these complications include the use of heparin-coated catheters, heparin locks, and antimicrobial lock therapy. However, the effects of heparin onCandida albicansbiofilms and planktonic cells have not been previously studied. Therefore, we sought to determine thein vitroeffect of a heparin sodium preparation (HP) on biofilms and planktonic cells ofC. albicans. Because HP contains two preservatives, methyl paraben (MP) and propyl paraben (PP), these compounds and heparin sodium without preservatives (Pure-H) were also tested individually. The metabolic activity of the mature biofilm after treatment was assessed using XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction and microscopy. Pure-H, MP, and PP caused up to 75, 85, and 60% reductions of metabolic activity of the mature preformedC. albicansbiofilms, respectively. Maximal efficacy against the mature biofilm was observed with HP (up to 90%) compared to the individual compounds (P< 0.0001). Pure-H, MP, and PP each inhibitedC. albicansbiofilm formation up to 90%. A complete inhibition of biofilm formation was observed with HP at 5,000 U/ml and higher. When tested against planktonic cells, each compound inhibited growth in a dose-dependent manner. These data indicated that HP, MP, PP, and Pure-H havein vitroantifungal activity againstC. albicansmature biofilms, formation of biofilms, and planktonic cells. Investigation of high-dose heparin-based strategies (e.g., heparin locks) in combination with traditional antifungal agents for the treatment and/or prevention ofC. albicansbiofilms is warranted.

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Disparate Candida albicans Biofilm Formation in Clinical Lipid Emulsions Due to Capric Acid-Mediated Inhibition

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01394-19 ◽

2019 ◽

Vol 63 (11) ◽

Author(s):

Hubertine M. E. Willems ◽

Jeremy S. Stultz ◽

Molly E. Coltrane ◽

Jabez P. Fortwendel ◽

Brian M. Peters

Keyword(s):

Candida Albicans ◽

Biofilm Formation ◽

Bloodstream Infections ◽

Capric Acid ◽

Lipid Emulsions ◽

Biofilm Growth ◽

Content Type ◽

Biofilm Model ◽

Hypha Formation

ABSTRACT Receipt of parenteral nutrition (PN) remains an independent risk factor for developing catheter-related bloodstream infections (CR-BSI) caused by fungi, including by the polymorphic fungus Candida albicans, which is notoriously adept at forming drug-resistant biofilm structures. Among a variety of macronutrients, PN solutions contain lipid emulsions to supply daily essential fats and are often delivered via central venous catheters (CVCs). Therefore, using an in vitro biofilm model system, we sought to determine whether various clinical lipid emulsions differentially impacted biofilm growth in C. albicans. We observed that the lipid emulsions Intralipid and Omegaven both stimulated C. albicans biofilm formation during growth in minimal medium or a macronutrient PN solution. Conversely, Smoflipid inhibited C. albicans biofilm formation by approximately 50%. Follow-up studies revealed that while Smoflipid did not impair C. albicans growth, it did significantly inhibit hypha formation and hyphal elongation. Moreover, growth inhibition could be recapitulated in Intralipid when supplemented with capric acid—a fatty acid present in Smoflipid but absent in Intralipid. Capric acid was also found to dose dependently inhibit C. albicans biofilm formation in PN solutions. This is the first study to directly compare different clinical lipid emulsions for their capacity to affect C. albicans biofilm growth. Results derived from this study necessitate further research regarding different lipid emulsions and rates of fungus-associated CR-BSIs.

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In Vitro Antibiofilm Activity of Eucarobustol E against Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02707-16 ◽

2017 ◽

Vol 61 (8) ◽

Cited By ~ 17

Author(s):

Rui-Huan Liu ◽

Zhi-Chun Shang ◽

Tian-Xiao Li ◽

Ming-Hua Yang ◽

Ling-Yi Kong

Keyword(s):

Candida Albicans ◽

Antifungal Susceptibility ◽

Hyphal Growth ◽

Negative Regulator ◽

Ergosterol Biosynthesis ◽

Pcr Analysis ◽

Antibiofilm Activity ◽

Biofilm Inhibition ◽

Content Type

ABSTRACT Formyl-phloroglucinol meroterpenoids (FPMs) are important types of natural products with various bioactivities. Our antifungal susceptibility assay showed that one of the Eucalyptus robusta-derived FPMs, eucarobustol E (EE), exerted a strong inhibitory effect against Candida albicans biofilms at a concentration of 16 μg/ml. EE was found to block the yeast-to-hypha transition and reduce the cellular surface hydrophobicity of the biofilm cells. RNA sequencing and real-time reverse transcription-PCR analysis showed that exposure to 16 μg/ml of EE resulted in marked reductions in the levels of expressions of genes involved in hyphal growth (EFG1, CPH1, TEC1, EED1, UME6, and HGC1) and cell surface protein genes (ALS3, HWP1, and SAP5). Interestingly, in response to EE, genes involved in ergosterol biosynthesis were downregulated, while the farnesol-encoding gene (DPP3) was upregulated, and these findings were in agreement with those from the quantification of ergosterol and farnesol. Combined with the obvious elevation of negative regulator genes (TUP1, NRG1), we speculated that EE's inhibition of carbon flow to ergosterol triggered the mechanisms of the negative regulation of hyphal growth and eventually led to biofilm inhibition.

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In VitroInteractions between Aspirin and Amphotericin B against Planktonic Cells and Biofilm Cells of Candida albicans and C. parapsilosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.06082-11 ◽

2012 ◽

Vol 56 (6) ◽

pp. 3250-3260 ◽

Cited By ~ 43

Author(s):

Yabin Zhou ◽

Ganggang Wang ◽

Yutang Li ◽

Yang Liu ◽

Yu Song ◽

...

Keyword(s):

Candida Albicans ◽

Amphotericin B ◽

Antimicrobial Agents ◽

Positive Interactions ◽

Antibiofilm Activity ◽

Data Generation ◽

Planktonic Cells ◽

Content Type ◽

Time Kill

ABSTRACTThe increase in drug resistance and invasion caused by biofilm formation brings enormous challenges to the management ofCandidainfection. Aspirin's antibiofilm activityin vitrowas discovered recently. The spectrophotometric method and the XTT {2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide} reduction assay used for data generation make it possible to evaluate fungal biofilm growth accurately. The combined use of the most commonly used methods, the fractional inhibitory concentration index (FICI) and a newly developed method, the ΔEmodel, which uses the concentration-effect relationship over the whole concentration range instead of using the MIC index alone, makes the interpretation of results more reliable. As an attractive tool for studying the pharmacodynamics of antimicrobial agents, time-kill curves can provide detailed information about antimicrobial efficacy as a function of both time and concentration. In the present study,in vitrointeractions between aspirin (acetylsalicylic acid [ASA]) and amphotericin B (AMB) against planktonic cells and biofilm cells ofCandida albicansandC. parapsilosiswere evaluated by the checkerboard microdilution method and the time-kill test. Synergistic and indifferent effects were found for the combination of ASA and AMB against planktonic cells, while strong synergy was found against biofilm cells analyzed by FICI. The ΔEmodel gave more consistent results with FICI. The positive interactions in concentration were also confirmed by the time-kill test. Moreover, this approach also revealed the pharmacodynamics changes of ASA and synergistic action on time. Our findings suggest a potential clinical use for combination therapy with ASA and AMB to augment activity against biofilm-associated infections.

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The Candida albicans ATO Gene Family Promotes Neutralization of the Macrophage Phagolysosome

Infection and Immunity ◽

10.1128/iai.00984-15 ◽

2015 ◽

Vol 83 (11) ◽

pp. 4416-4426 ◽

Cited By ~ 30

Author(s):

Heather A. Danhof ◽

Michael C. Lorenz

Keyword(s):

Amino Acids ◽

Candida Albicans ◽

Hyphal Growth ◽

Dominant Negative ◽

Dependent Manner ◽

Ph Change ◽

Content Type ◽

Ammonia Release ◽

Hyphal Formation

ABSTRACTCandida albicansis an opportunistic human fungal pathogen that causes a variety of diseases, ranging from superficial mucosal to life-threatening systemic infections, the latter particularly in patients with defects in innate immune function.C. albicanscells phagocytosed by macrophages undergo a dramatic change in their metabolism in which amino acids are a key nutrient. We have shown that amino acid catabolism allows the cell to neutralize the phagolysosome and initiate hyphal growth. We show here that members of the 10-geneATOfamily, which are induced by phagocytosis or the presence of amino acids in an Stp2-dependent manner and encode putative acetate or ammonia transporters, are important effectors of this pH changein vitroand in macrophages. When grown with amino acids as the sole carbon source, the deletion ofATO5or the expression of a dominant-negativeATO1G53Dallele results in a delay in alkalinization, a defect in hyphal formation, and a reduction in the amount of ammonia released from the cell. These strains also form fewer hyphae after phagocytosis, have a reduced ability to escape macrophages, and reside in more acidic phagolysosomal compartments than wild-type cells. Furthermore, overexpression of many of the 10ATOgenes accelerates ammonia release, and anato5Δ ATO1G53Ddouble mutant strain has additive alkalinization and ammonia release defects. Taken together, these results indicate that the Ato protein family is a key mediator of the metabolic changes that allowC. albicansto overcome the macrophage innate immunity barrier.

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Candida albicans Induces Arginine Biosynthetic Genes in Response to Host-Derived Reactive Oxygen Species

Eukaryotic Cell ◽

10.1128/ec.00290-12 ◽

2012 ◽

Vol 12 (1) ◽

pp. 91-100 ◽

Cited By ~ 40

Author(s):

Claudia Jiménez-López ◽

John R. Collette ◽

Kimberly M. Brothers ◽

Kelly M. Shepardson ◽

Robert A. Cramer ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Candida Albicans ◽

Amino Acid ◽

Reactive Oxygen ◽

Single Amino Acid ◽

Dependent Manner ◽

Oxygen Species ◽

Biosynthetic Genes ◽

Content Type

ABSTRACTThe interaction ofCandida albicanswith phagocytes of the host's innate immune system is highly dynamic, and its outcome directly impacts the progression of infection. While the switch to hyphal growth within the macrophage is the most obvious physiological response, much of the genetic response reflects nutrient starvation: translational repression and induction of alternative carbon metabolism. Changes in amino acid metabolism are not seen, with the striking exception of arginine biosynthesis, which is upregulated in its entirety during coculture with macrophages. Using single-cell reporters, we showed here that arginine biosynthetic genes are induced specifically in phagocytosed cells. This induction is lower in magnitude than during arginine starvationin vitroand is driven not by an arginine deficiency within the phagocyte but instead by exposure to reactive oxygen species (ROS). Curiously, these genes are induced in a narrow window of sublethal ROS concentrations.C. albicanscells phagocytosed by primary macrophages deficient in thegp91phoxsubunit of the phagocyte oxidase do not express theARGpathway, indicating that the induction is dependent on the phagocyte oxidative burst.C. albicans argpathway mutants are retarded in germ tube and hypha formation within macrophages but are not notably more sensitive to ROS. We also find that theARGpathway is regulated not by the general amino acid control response but by transcriptional regulators similar to theSaccharomyces cerevisiaeArgR complex. In summary, phagocytosis induces this single amino acid biosynthetic pathway in an ROS-dependent manner.

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Screening the Pathogen Box for Identification of Candida albicans Biofilm Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02006-16 ◽

2016 ◽

Vol 61 (1) ◽

Cited By ~ 26

Author(s):

Taissa Vila ◽

Jose L. Lopez-Ribot

Keyword(s):

Candida Albicans ◽

Biofilm Formation ◽

Antibiofilm Activity ◽

Potential Candidate ◽

Predominant Role ◽

Chemical Library ◽

Content Type ◽

High Level ◽

Level Resistance

ABSTRACT Candida albicans remains the main causative agent of candidiasis, one of the most frequent nosocomial infections, with unacceptably high mortality rates. Biofilm formation is a major risk factor for invasive candidiasis, as Candida biofilms display high-level resistance to most antifungal agents. In this work we have screened the Pathogen Box chemical library (Medicines for Malaria Venture [MMV], Switzerland) in search for inhibitors of C. albicans biofilm formation. Our initial screen identified seven hits, and additional dose-response assays confirmed the biofilm-inhibitory activity of six of these small molecules. Three compounds, MMV688768, MMV687273, and MMV687807, were also able to reduce the metabolic activity of cells within preformed biofilms. Interestingly, the most potent of these, compound MMV688768, displayed increased antibiofilm activity compared to its activity against planktonic cultures, indicating that it may affect processes with a predominant role during the biofilm mode of growth. This compound demonstrated a high selectivity index when its antibiofilm activity was compared with its toxicity in liver hepatocellular cells. In vitro combination assays showed a synergistic interaction between compound MMV688768 and fluconazole against preformed biofilms. Overall, our results indicate that this compound may constitute a potential candidate for further clinical development.

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Iron Chelator Deferasirox Reduces Candida albicans Invasion of Oral Epithelial Cells and Infection Levels in Murine Oropharyngeal Candidiasis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02152-18 ◽

2019 ◽

Vol 63 (4) ◽

Cited By ~ 6

Author(s):

Sumant Puri ◽

Rohitashw Kumar ◽

Isolde G. Rojas ◽

Ornella Salvatori ◽

Mira Edgerton

Keyword(s):

Candida Albicans ◽

Treatment Options ◽

Fungal Growth ◽

Bloodstream Infections ◽

Preventive Treatment ◽

Iron Chelator ◽

Oropharyngeal Candidiasis ◽

Content Type ◽

Oral Epithelial

ABSTRACT Candida albicans, the causative agent of mucosal infections, including oropharyngeal candidiasis (OPC), as well as bloodstream infections, is becoming increasingly resistant to existing treatment options. In the absence of novel drug candidates, drug repurposing aimed at using existing drugs to treat off-label diseases is a promising strategy. C. albicans requires environmental iron for survival and virulence, while host nutritional immunity deploys iron-binding proteins to sequester iron and reduce fungal growth. Here we evaluated the role of iron limitation using deferasirox (an FDA-approved iron chelator for the treatment of patients with iron overload) during murine OPC and assessed deferasirox-treated C. albicans for its interaction with human oral epithelial (OE) cells, neutrophils, and antimicrobial peptides. Therapeutic deferasirox treatment significantly reduced salivary iron levels, while a nonsignificant reduction in the fungal burden was observed. Preventive treatment that allowed for two additional days of drug administration in our murine model resulted in a significant reduction in the number of C. albicans CFU per gram of tongue tissue, a significant reduction in salivary iron levels, and significantly reduced neutrophil-mediated inflammation. C. albicans cells harvested from the tongues of animals undergoing preventive treatment had the differential expression of 106 genes, including those involved in iron metabolism, adhesion, and the response to host innate immunity. Moreover, deferasirox-treated C. albicans cells had a 2-fold reduction in survival in neutrophil phagosomes (with greater susceptibility to oxidative stress) and reduced adhesion to and invasion of OE cells in vitro. Thus, deferasirox treatment has the potential to alleviate OPC by affecting C. albicans gene expression and reducing virulence.

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Candida albicans-Induced Epithelial Damage Mediates Translocation through Intestinal Barriers

mBio ◽

10.1128/mbio.00915-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 47

Author(s):

Stefanie Allert ◽

Toni M. Förster ◽

Carl-Magnus Svensson ◽

Jonathan P. Richardson ◽

Tony Pawlik ◽

...

Keyword(s):

Candida Albicans ◽

Bloodstream Infections ◽

Cellular Damage ◽

Intestinal Damage ◽

Epithelial Integrity ◽

Content Type ◽

Epithelial Damage ◽

Peptide Toxin ◽

Fungal Genes

ABSTRACTLife-threatening systemic infections often occur due to the translocation of pathogens across the gut barrier and into the bloodstream. While the microbial and host mechanisms permitting bacterial gut translocation are well characterized, these mechanisms are still unclear for fungal pathogens such asCandida albicans, a leading cause of nosocomial fungal bloodstream infections. In this study, we dissected the cellular mechanisms of translocation ofC. albicansacross intestinal epitheliain vitroand identified fungal genes associated with this process. We show that fungal translocation is a dynamic process initiated by invasion and followed by cellular damage and loss of epithelial integrity. A screen of >2,000C. albicansdeletion mutants identified genes required for cellular damage of and translocation across enterocytes. Correlation analysis suggests that hypha formation, barrier damage above a minimum threshold level, and a decreased epithelial integrity are required for efficient fungal translocation. Translocation occurs predominantly via a transcellular route, which is associated with fungus-induced necrotic epithelial damage, but not apoptotic cell death. The cytolytic peptide toxin ofC. albicans, candidalysin, was found to be essential for damage of enterocytes and was a key factor in subsequent fungal translocation, suggesting that transcellular translocation ofC. albicansthrough intestinal layers is mediated by candidalysin. However, fungal invasion and low-level translocation can also occur via non-transcellular routes in a candidalysin-independent manner. This is the first study showing translocation of a human-pathogenic fungus across the intestinal barrier being mediated by a peptide toxin.IMPORTANCECandida albicans, usually a harmless fungus colonizing human mucosae, can cause lethal bloodstream infections when it manages to translocate across the intestinal epithelium. This can result from antibiotic treatment, immune dysfunction, or intestinal damage (e.g., during surgery). However, fungal processes may also contribute. In this study, we investigated the translocation process ofC. albicansusingin vitrocell culture models. Translocation occurs as a stepwise process starting with invasion, followed by epithelial damage and loss of epithelial integrity. The ability to secrete candidalysin, a peptide toxin deriving from the hyphal protein Ece1, is key:C. albicanshyphae, secreting candidalysin, take advantage of a necrotic weakened epithelium to translocate through the intestinal layer.

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Effect of Streptococcus salivarius K12 on the In Vitro Growth of Candida albicans and Its Protective Effect in an Oral Candidiasis Model

Applied and Environmental Microbiology ◽

10.1128/aem.07055-11 ◽

2012 ◽

Vol 78 (7) ◽

pp. 2190-2199 ◽

Cited By ~ 48

Author(s):

Sanae A. Ishijima ◽

Kazumi Hayama ◽

Jeremy P. Burton ◽

Gregor Reid ◽

Masashi Okada ◽

...

Keyword(s):

Candida Albicans ◽

Therapeutic Option ◽

Oral Treatment ◽

Oral Candidiasis ◽

Oral Care ◽

Petri Dish ◽

Dependent Manner ◽

Streptococcus Salivarius ◽

Content Type

ABSTRACTOral candidiasis is often accompanied by severe inflammation, resulting in a decline in the quality of life of immunosuppressed individuals and elderly people. To develop a new oral therapeutic option for candidiasis, a nonpathogenic commensal oral probiotic microorganism,Streptococcus salivariusK12, was evaluated for its ability to modulateCandida albicansgrowthin vitro, and its therapeutic activity in an experimental oral candidiasis model was tested.In vitroinhibition of mycelial growth ofC. albicanswas determined by plate assay and fluorescence microscopy. Addition ofS. salivariusK12 to modified RPMI 1640 culture medium inhibited the adherence ofC. albicansto the plastic petri dish in a dose-dependent manner. Preculture ofS. salivariusK12 potentiated its inhibitory activity for adherence ofC. albicans. Interestingly,S. salivariusK12 was not directly fungicidal but appeared to inhibitCandidaadhesion to the substratum by preferentially binding to hyphae rather than yeast. To determine the potentially anti-infective attributes ofS. salivariusK12 in oral candidiasis, the probiotic was administered to mice with orally induced candidiasis. Oral treatment withS. salivariusK12 significantly protected the mice from severe candidiasis. These findings suggest thatS. salivariusK12 may inhibit the process of invasion ofC. albicansinto mucous surfaces or its adhesion to denture acrylic resins by mechanisms not associated with the antimicrobial activity of the bacteriocin.S. salivariusK12 may be useful as a probiotic as a protective tool for oral care, especially with regard to candidiasis.

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