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The Role of MAPK Signal Transduction Pathways in the Response to Oxidative Stress in the Fungal Pathogen Candida albicans: Implications in Virulence
Abstract
In recent years, Mitogen-Activated Protein Kinase (MAPK) pathways have emerged as major regulators of cellular physiology. In the fungal pathogen Candida albicans, three different MAPK pathways have been characterized in the last years. The HOG pathway is mainly a stress response pathway that is activated in response to osmotic and oxidative stress and also participates regulating other pathways. The SVG pathway (or mediated by the Cek1 MAPK) is involved in cell wall formation under vegetative and filamentous growth, while the Mkc1-mediated pathway is involved in cell wall integrity. Oxidative stress is one of the types of stress that every fungal cell has to face during colonization of the host, where the cell encounters both hypoxia niches (i.e. gut) and high concentrations of reactive oxygen species (upon challenge with immune cells). Two pathways have been shown to be activated in response to oxidative stress: the HOG pathway and the MKC1-mediated pathway while the third, the Cek1 pathway is deactivated. The timing, kinetics, stimuli and functional responses generated upon oxidative stress differ among them; however, they have essential functional consequences that severely influence pathogenesis. MAPK pathways are, therefore, valuable targets to be explored in antifungal research.
... The pathogenicity of C. albicans mainly depends on its virulence factors, such as adherence and invasion, hyphal and biofilm formation, cell wall integrity, and hydrolase secretion (Staniszewska, 2020). The cyclic AMP (cAMP) and mitogenactivated protein kinase (MAPK) signaling pathways has further been found to play significant roles in regulating the expression of various virulence factors (de Dios et al., 2010;Huang et al., 2019). The cAMP signaling pathway is crucial in regulating C. albicans morphogenesis and environmental sensing . ...
... In terms of signaling pathways, studies have shown that both cAMP and MAPK signaling pathways could play essential roles in the pathogenesis of C. albicans (de Dios et al., 2010;Huang et al., 2019). The cAMP signaling pathway is crucial in regulating C. albicans morphogenesis and environmental sensing . ...
Magnolol, a lignin compound extracted from Magnolia officinalis Cortex, has been found to have prominent antifungal effects against Candida albicans. However, the specific mechanism still remains unclear. Therefore, this study aimed to further explore the inhibition mechanism of magnolol against Candida albicans virulence factors and the related signaling pathways. By an XTT reduction assay, a hyphal formation assay, confocal laser scanning microscopy, transmission electron microscopy, a calcofluor white staining assay, and a cell wall β-glucan quantitative detection assay, we evaluated the inhibitory effects of magnolol against the adhesion, hyphal formation, biofilm viability, biofilm spatial structure, and cell wall ultrastructure of Candida albicans. Moreover, by RNA sequencing and qRT-PCR, we confirmed the effects of magnolol in inhibiting the gene expression of Candida albicans virulence factors and the related signaling pathways. The results revealed that the adhesion and hyphal formation of Candida albicans were inhibited significantly by magnolol. The viability and spatial structures of Candida albicans biofilms were further weakened. Candida albicans ultrastructure showed partial thinning of cell walls and even rupture, with cytoplasmic leakage. The cell wall intergrity and β-glucan content were also radically reduced. Moreover, magnolol caused significant inhibition of the expression of Candida albicans adhesion, invasion, hyphal formation, biofilm formation, β-1,3-glucan synthesis, and hydrolase secretion-related genes, including ALS1, ALS3, EFG1, EAP1, FKS1, FKS2, PLB2, and SAP2. Furthermore, the PKC pathway-related genes (RHO1, PKC1, BCK1, MKK2, MKC1) and Cek1 pathway-related genes (CDC42, CST20, STE11, HST7, CEK1) were also significantly downregulated, indicating that the inhibition of magnolol against Candida albicans virulence factors might be related to PKC and Cek1 MAPK signaling pathways. In conclusion, the findings of this study confirmed the inhibition mechanism of magnolol against Candida albicans virulence factors, which might be related to PKC and Cek1 MAPK pathways, thus laying the theoretical foundation for its clinical antifungal applications.
... Thus far several partially redundant signaling pathways have been shown to contribute to or regulate fungal oxidative / ROS stress responses [13,14]. These include the high osmolarity glycerol (Hog1) mitogen-activated protein (MAP) kinase, the protein kinase C-like, PKC1-MAP kinase, and cell wall integrity (CWI) pathways [15][16][17][18]. In addition, the cytokinesis-required Cdc14 phosphatase and a P-type calcium ATPase have been shown to affect these signaling pathways in response to oxidative stress [19,20]. ...
Background
Response to oxidative stress is universal in almost all organisms and the mitochondrial membrane protein, BbOhmm, negatively affects oxidative stress responses and virulence in the insect fungal pathogen, Beauveria bassiana. Nothing further, however, is known concerning how BbOhmm and this phenomenon is regulated.
Results
Three oxidative stress response regulating Zn2Cys6 transcription factors (BbOsrR1, 2, and 3) were identified and verified via chromatin immunoprecipitation (ChIP)-qPCR analysis as binding to the BbOhmm promoter region, with BbOsrR2 showing the strongest binding. Targeted gene knockout of BbOsrR1 or BbOsrR3 led to decreased BbOhmm expression and consequently increased tolerances to free radical generating compounds (H2O2 and menadione), whereas the ΔBbOsrR2 strain showed increased BbOhmm expression with concomitant decreased tolerances to these compounds. RNA and ChIP sequencing analysis revealed that BbOsrR1 directly regulated a wide range of antioxidation and transcription-associated genes, negatively affecting the expression of the BbClp1 cyclin and BbOsrR2. BbClp1 was shown to localize to the cell nucleus and negatively mediate oxidative stress responses. BbOsrR2 and BbOsrR3 were shown to feed into the Fus3-MAPK pathway in addition to regulating antioxidation and detoxification genes. Binding motifs for the three transcription factors were found to partially overlap in the promoter region of BbOhmm and other target genes. Whereas BbOsrR1 appeared to function independently, co-immunoprecipitation revealed complex formation between BbClp1, BbOsrR2, and BbOsrR3, with BbClp1 partially regulating BbOsrR2 phosphorylation.
Conclusions
These findings reveal a regulatory network mediated by BbOsrR1 and the formation of a BbClp1-BbOsrR2-BbOsrR3 complex that orchestrates fungal oxidative stress responses.
... In addition to the problem of carbon sources and trace elements, C. albicans faces pressure from the guts' physical and chemical environment, but C. albicans can resist them through specific signaling pathways [50,51]. For example, resistance to the osmotic pressure and oxidative stress can be promoted through the Hog1-mediated MAP kinase pathway [52][53][54]; the gene CAP1, which codes for a bZip transcription factor of the AP-1 family, drives transcriptional responses to oxidative stress [55][56][57]; Mkc1-and Cek1-mediated MAP kinase pathways promote C. albicans resistance to cell wall stress [51,58,59]; transcription factors Cta4 and Hsf1 respond to the intestinal nitrosation stress and heat shock [60][61][62]. ...
Background
The gut microbiota plays an important role in human health, as it can affect host immunity and susceptibility to infectious diseases. Invasive intestinal candidiasis is strongly associated with gut microbiota homeostasis. However, the nature of the interaction between Candida albicans and gut bacteria remains unclear.
Objective
This review aimed to determine the nature of interaction and the effects of gut bacteria on C. albicans so as to comprehend an approach to reducing intestinal invasive infection by C. albicans.
Methods
This review examined 11 common gut bacteria’s interactions with C. albicans, including Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus faecalis, Staphylococcus aureus, Salmonella spp., Helicobacter pylori, Lactobacillus spp., Bacteroides spp., Clostridium difficile, and Streptococcus spp.
Results
Most of the studied bacteria demonstrated both synergistic and antagonistic effects with C. albicans, and just a few bacteria such as P. aeruginosa, Salmonella spp., and Lactobacillus spp. demonstrated only antagonism against C. albicans.
Conclusions
Based on the nature of interactions reported so far by the literature between gut bacteria and C. albicans, it is expected to provide new ideas for the prevention and treatment of invasive intestinal candidiasis.
... Environmental cues and QS molecules modulate the morphological transition of C. albicans by affecting a set of transcription factors through various upstream pathways, including cyclic adenosine monophosphate (cAMP)-dependent pathways and mitogen-activated protein kinase (MAPK) pathways (Biswas et al., 2007;de Dios et al., 2010;Leberer et al., 2001;Martin et al., 2005;Ramage et al., 2002;Staib, 2002). In view of the important role of hyphal formation and biofilm formation in the pathogenic process of C. albicans, it is a general trend to prevent and treat C. albicans infection by inhibiting the pathogenic factors of C. albicans without affecting its growth. ...
Candida albicans is an important human fungal pathogen. Our previous study disclosed that aryloxy‐phenylpiperazine skeleton was a promising molecule to suppress C. albicans virulence by inhibiting hypha formation and biofilm formation. In order to deeply understand the efficacy and mechanism of action of phenylpiperazine compounds, and obtain new derivatives with excellent activity against C. albicans, hence, we synthesized three series of (1‐heteroaryloxy‐2‐hydroxypropyl)‐phenylpiperazines and evaluated their inhibitory activity against C. albicans both in vitro and in vivo in this study. Compared with previously reported aryloxy‐phenylpiperazines, part of these heteroaryloxy derivatives improved their activities by strongly suppressing hypha formation and biofilm formation in C. albicans SC5314. Especially, (9H‐carbazol‐4‐yl)oxy derivatives 25, 26, 27 and 28 exhibited strong activity in reducing C. albicans virulence in both human cell lines in vitro and mouse infection models in vivo. The compound 27 attenuated the virulence of various clinical C. albicans strains, including clinical drug‐resistant C. albicans strains. Moreover, additive effects of the compound 27 with antifungal drugs against drug‐resistant C. albicans strains were also discussed. Furthermore, the compound 27 significantly improved the composition and richness of the faecal microbiota in mice infected by C. albicans. These findings indicate that these piperazine compounds have great potential to be developed as new therapeutic drugs against C. albicans infection.
... Nevertheless, KEGG enrichment showed that a large number of genes in the MAPK pathway were altered greatly in response to AMP-17 ( Figure S4). Previous reports suggested that MAPK participates in cell wall forming under vegetative and filamentous growth, playing an important role in morphogenesis [25,46]. The gene expression level of HOG1, CEK1, CCP1, CPH1, and WOR3 was found to be downregulated after AMP-17 treatment. ...
The biofilm formation of C. albicans represents a major virulence factor during candidiasis. Biofilm-mediated drug resistance has necessitated the search for a new antifungal treatment strategy. In our previous study, a novel antimicrobial peptide named AMP-17 derived from Musca domestica was confirmed to have significant antifungal activity and suppress hyphal growth greatly in C. albicans. In the current work, we aimed to investigate the antibiofilm property of AMP-17 in C. albicans and explore the underlying mechanism. An antifungal susceptibility assay showed that AMP-17 exerted a strong inhibitory efficacy on both biofilm formation and preformed biofilms in C. albicans. Furthermore, AMP-17 was found to block the yeast-to-hypha transition and inhibit the adhesion of biofilm cells with a reduction in cellular surface hydrophobicity. A morphological analysis revealed that AMP-17 indeed suppressed typical biofilm formation and damaged the structures of the preformed biofilm. The RNA-seq showed that the MAPK pathway, biosynthesis of antibiotics, and essential components of the cell were mainly enriched in the biofilm-forming stage, while the citrate cycle (TCA cycle), phenylamine metabolism, and propanoate metabolism were enriched after the biofilm matured. Moreover, the co-expressed DEGs in the two pairwise comparisons highlighted the terms of transmembrane transporter activity, regulation of filamentation, and biofilm formation as important roles in the antibiofilm effect of AMP-17. Additionally, qRT-PCR confirmed that the level of the genes involved in cell adhesion, filamentous growth, MAPK, biofilm matrix, and cell dispersal was correspondingly altered after AMP-17 treatment. Overall, our findings reveal the underlying antibiofilm mechanisms of AMPs in C. albicans, providing an interesting perspective for the development of effective antifungal agents with antibiofilm efficacy in Candida spp.
... They comprise a conserved module of three protein kinases: the MAP kinase (MAPK), the MAP kinase kinase (MAPKK), and the MAP kinase kinase kinase (MAPKKK) 243-245 , and additional upstream elements such as transmembrane sensors, scaffold proteins and downstream transcription factors.In C. albicans, two MAPKs become activated under oxidative stress conditions: the Hog1 and the Mkc1 pathways246,247 (Figure 18). The Hog1-mediated pathway is considered as a general stress response cascade, involved in morphogenesis and cell wall integrity, and is essential for the response to osmotic and oxidative stress (reviewed in248 ). The Mkc1mediated pathway is mainly implicated in cell wall reconstruction and morphogenesis, but other stimuli have been reported to activate this cascade. ...
Candida albicans is the most prevalent human fungal pathogen. Despite being a harmless commensal organism, it is also an opportunistic fungus which can cause life-threatening infections in immunocompromised people. Although major virulence factors have been characterized, the exact mechanism of Candida pathogenesis still remains unknown. Mechanisms of adaptation under the different environments it faces during body invasion are yet poorly characterized.
In this thesis, functional genomic analyses were utilized to study the global response of C. albicans to two environmental insults it faces during invasion of the human body: oxidative and hypoxic stress. Several genes were found to be involved in the adaptation to these stresses, as reflected by the up or down regulation of their mRNA and protein products. Interestingly, the expression of the genes involved in the response to oxidative stress was found to be regulated mainly at the post-transcriptional level.
To explore the possible implication of non-coding RNAs in the response to these stresses, an online web application was created to enable the detection of ncRNAs which levels change under stress conditions. Two groups of 154 and 159 ncRNAs, including intergenic and antisense to ORFs, were found to be involved in the responses to hypoxia and oxidative stress respectively.
To study changes in nascent transcription under these stresses, Genomic run-on (GRO), a well-established technique used for the study of the nascentome in Saccharomyces cerevisiae, was implemented for its use in C. albicans in its most up-to-date version, BioGROseq. The GRO technique was later used for the study of changes in gene expression parameters at different growth temperatures, such as RNApol II density or translation rate, using the model yeast S. cerevisiae as a tool. The results showed that RNA pol II density decreases as growth temperature increases, and that this decrease in density is compensated with an increase in RNA pol II speed to maintain mRNA homeostatic levels, compensation that is regulated at the RNA pol II initiation level. Within the optimal range of growth temperatures in S. cerevisiae (26-34 oC) both total RNA and mRNA amounts were found to be kept at homeostatic levels, decreasing markedly at temperatures above and below. Finally, an increase in translation rate was observed with the increase in growth temperatures. Overall, the results presented in this thesis provide clues into the regulatory mechanisms that yeast utilize to grow at different temperatures and contribute to the understanding of how C. albicans thrives during infection of the human body.
Candida albicans, the most prevalent fungal pathogen, exists as a commensal in the human host. It is subjected to myriad physiological stress conditions in different host niches, which jeopardizes its fitness to survive and propagate as an established commensal. C. albicans has highly labile chromatin which gets remodeled in response to the stress conditions to facilitate the expression of several stress-responsive genes. Several epigenetic factors including histone variants, histone modifiers and chromatin remodelers that define the chromatin architecture play crucial roles in the regulation of the stress-responsive genes in this organism. Here we investigated the roles of the ATP-dependent chromatin remodeler RSC (Remodel the Structure of Chromatin) in several stress responses in C. albicans, by targeting the key ATPase component, Sth1, given its profound and similar roles exist in Saccharomyces cerevisiae. We have unraveled the crucial roles of the RSC complex (Sth1) in maintaining cell wall integrity and fighting against osmotic and oxidative stresses. We found that the mutant conditionally depleted of Sth1 was sensitive to the cell wall disrupting agents, and the mutant without exposure to any stressor accumulated higher chitin content in the cell wall as a defense mechanism to restore the cell wall integrity. Further, this was supported by the phosphorylation of MAPK1 protein Mkc1, which happens due to activation of the cell wall integrity pathway PKC1. We also observed the Sth1 mutant to be sensitive to oxidative and osmotic stresses in vitro, which are very important and imparted by the host defense mechanism. This suggests that the mutant could get attenuated and hence become less virulent than the wild-type when loss of function of Sth1 happens. We also found that Sth1 has a crucial role in maintaining genomic integrity as sth1 mutant cells accumulate extensive DNA damages and show the loss in cell viability. Overall this work suggests that Sth1 has an important role in fighting against some of the clinically relevant and physiologically important stresses. It also has a crucial role in fighting against stress to the genomic integrity and hence functions in DNA damage repair.
Commensal fungus-Candida albicans turn pathogenic during the compromised immunity of the host, causing infections ranging from superficial mucosal to dreadful systemic ones. C. albicans has evolved various adaptive measures which collectively contribute towards its enhanced virulence. Among fitness attributes, metabolic flexibility and vigorous stress response are essential for its pathogenicity and virulence. Metabolic flexibility provides a means for nutrient assimilation and growth in diverse host microenvironments and reduces the vulnerability of the pathogen to various antifungals besides evading host immune response(s). Inside the host micro-environments, C. albicans efficiently utilizes the multiple fermentable and non-fermentable carbon sources to sustain and proliferate in glucose deficit conditions. The utilization of alternative carbon sources further highlights the importance of understanding these pathways as the attractive and potential therapeutic target. A thorough understanding of metabolic flexibility and adaptation to environmental stresses is warranted to decipher in-depth insights into virulence and molecular mechanisms of fungal pathogenicity. In this review, we have attempted to provide a detailed and recent understanding of some key aspects of fungal biology. Particular focus will be placed on processes like nutrient assimilation and utilization, metabolic adaptability, virulence factors, and host immune response in C. albicans leading to its enhanced pathogenicity.
Alternaria brassicicola causes black spot disease in Brassicaceae. During host infection, this necrotrophic fungus is exposed to various antimicrobial compounds, such as the phytoalexin brassinin which is produced by many cultivated Brassica species. To investigate the cellular mechanisms by which this compound causes toxicity and the corresponding fungal adaptive strategies, we first analyzed fungal transcriptional responses to short-term exposure to brassinin and then used additional functional approaches. This study supports the hypothesis that indolic phytoalexin primarily targets mitochondrial functions in fungal cells. Indeed, we notably observed that phytoalexin treatment of A. brassicicola disrupted the mitochondrial membrane potential and resulted in a significant and rapid decrease in the oxygen consumption rates. Secondary effects, such as Reactive oxygen species production, changes in lipid and endoplasmic reticulum homeostasis were then found to be induced. Consequently, the fungus has to adapt its metabolism to protect itself against the toxic effects of these molecules, especially via the activation of high osmolarity glycerol and cell wall integrity signaling pathways and by induction of the unfolded protein response.
MAPK pathways regulate different responses yet can share common components. Although core regulators of MAPK pathways are well known, new pathway regulators continue to be identified. Overexpression screens can uncover new roles for genes in biological processes and are well suited to identify essential genes that cannot be evaluated by gene deletion analysis. In this study, a genome-wide screen was performed to identify genes that, when overexpressed, induce a reporter (FUS1-HIS3) that responds to ERK-type pathways (Mating and filamentous growth or fMAPK) but not p38-type pathways (HOG) in yeast. Approximately 4500 plasmids overexpressing individual yeast genes were introduced into strains containing the reporter by high-throughput transformation. Candidate genes were identified by measuring growth as a readout of reporter activity. Fourteen genes were identified and validated by re-testing: two were metabolic controls (HIS3, ATR1), five had established roles in regulating ERK-type pathways (STE4, STE7, BMH1, BMH2, MIG2) and seven represent potentially new regulators of MAPK signaling (RRN6, CIN5, MRS6, KAR2, TFA1, RSC3, RGT2). MRS6 encodes a Rab escort protein and effector of the TOR pathway that plays a role in nutrient signaling. MRS6 overexpression stimulated invasive growth and phosphorylation of the ERK-type fMAPK, Kss1. Overexpression of MRS6 reduced the osmotolerance of cells and phosphorylation of the p38/HOG MAPK, Hog1. Mrs6 interacted with the PAK kinase Ste20 and MAPKK Ste7 by two-hybrid analysis. Based on these results, Mrs6 may selectively propagate an ERK-dependent signal. Identifying new regulators of MAPK pathways may provide new insights into signal integration among core cellular processes and the execution of pathway-specific responses.
The response of the pathogenic yeast Candida albicans to the silver (I) perchlorate salt (AgClO4) was assessed. By employing an anti-phospho-p38 MAPK antibody, dual phosphorylation of Hog1p in C. albicans in the presence of AgClO4 was demonstrated. Phosphorylation of C. albicans Hog1p in response to hydrogen peroxide or AgClO4 resulted in the translocation of this MAP kinase to the nucleus. Nuclear translocation of Cap1p was demonstrated by Western blot analysis and detected using polyclonal anti-Cap1p antibody. Upon AgClO4-induced translocation of Cap1p there was a concomitant activation of genes coding for glutathione reductase-1 and Mn-superoxide dismutase but no increase in the expression of flavin oxidoreductase or mitochondrial processing protease was recorded. In addition, exposure to AgClO4 increased the activity of superoxide dismutase, glutathione reductase and catalase. The activation of C. albicans oxidative stress response genes and enzymes following exposure to AgClO4 is evidence for the generation of oxidative stress within this medically important yeast.
Signal transduction mediated by the mitogen-activated protein kinase (MAPK) Slt2 pathway is essential to maintain the cell wall integrity in Saccharomyces cerevisiae. Stimulation of MAPK pathways results in activation by phosphorylation of conserved threonine and tyrosine residues of MAPKs. We have used an antibody that specifically recognizes dually phosphorylated Slt2 to gain insight into the activation and modulation of signaling through the cell integrity pathway. We show that caffeine and vanadate activate this pathway in the absence of osmotic stabilization. The lack of the putative cell surface sensor Mid2 prevents vanadate- but not caffeine-induced Slt2 phosphorylation. Disruption of the Rho1-GTPase-activating protein genes SAC7 and BEM2 leads to constitutive Slt2 activation, indicating their involvement as negative regulators of the pathway. MAPK kinases also seem to participate in signaling regulation, Mkk1 playing a greater role than Mkk2 in signal transmission to Slt2. Additionally, one of the phosphatases involved in Slt2 dephosphorylation is likely to be the dual specificity phosphatase Msg5, since overexpression of MSG5 in a sac7Delta mutant eliminates the high Slt2 phosphorylation, and disruption of MSG5 in wild type cells results in increased phospho-Slt2 levels. These data present the first evidence for a negative regulation of the cell integrity pathway.
Cells respond to changes in osmotic pressure with compensatory molecular adaptations that allow them to reestablish homeostasis of osmotically disturbed aspects of cell structure and function, In addition, some cell types respond to osmotic stress by changing their phenotype or, if their tolerance threshold is exceeded, by initiating programmed cell death, To understand how cells achieve these different types of adaptive response to osmotic stress, it is necessary to identify the key elements of osmosensory signal transduction and to analyze the complex networks that process osmotic stimuli imposed upon cells by their environment. This review highlights mitogen-activated protein kinase (MAPK) cascades as important intracellular signal-transduction pathways activated in response to changes in osmolality, A unifying theme of osmotic stress signaling via MAPKs seems to be regulation of the cell cycle as part of the cellular stress response. This very important physiological capacity may have been conserved throughout evolution as a major function of MAPKs from many different subfamilies. The evidence for this conjecture is discussed, and our current knowledge about osmotic stress signaling pathways in yeast, animals and plants is briefly reviewed.
Using a DNA fragment derived from the Saccharomyces cerevisiae protein kinase C gene (PKC1) as a probe to screen an ordered array library of genomic DNA from the dimorphic pathogenic fungus Candida albicans, the C. albicans PKC1 gene (CaPKC1) was isolated. The CaPKC1 gene is predicted to encode a protein of 1079 amino acids with 51% sequence identity over the entire length with the S. cerevisiae Pkc1 protein and is capable of functionally complementing the growth defects of a S. cerevisiae pkc1Δ mutant strain on hypo-osmotic medium. Deletion of both endogenous copies of the CaPKC1 gene in diploid C. albicans cells resulted in an osmotically remedial cell lysis defect of both the budding and the hyphal growth form and morphologically aberrant cells of the budding form. Despite these abnormalities, the transition between the two growth forms of C. albicans occurred normally in pkc1/pkc1 double disruptants. Capkc1p was modified at its C-terminus with two repeats of the Staphylococcus aureus protein A IgG-binding fragment (ZZ-sequence tag) and partially purified by chromatography on DEAE–Sepharose and IgG–Sepharose. In vitro, Capkc1p preferably phosphorylated the S. cerevisiae Pkc1p pseudosubstrate peptide and myelin basic protein, but not histones, protamine or dephosphorylated casein, and failed to respond to cofactors known to activate several mammalian PKC isozymes.
We have shown previously that pure polyphenol curcumin I (CUR-I) shows antifungal activity against Candida species. By employing the chequerboard method, filter disc and time-kill assays, in the present study we demonstrate that CUR-I at non-antifungal concentration interacts synergistically with azoles and polyenes. For this, pure polyphenol CUR-I was tested for synergy with five azole and two polyene drugs - fluconazole (FLC), miconazole, ketoconazole (KTC), itraconazole (ITR), voriconazole (VRC), nystatin (NYS) and amphotericin B (AMB) - against 21 clinical isolates of Candida albicans with reduced antifungal sensitivity, as well as a drug-sensitive laboratory strain. Notably, there was a 10-35-fold drop in the MIC(80) values of the drugs when CUR-I was used in combination with azoles and polyenes, with fractional inhibitory concentration index (FICI) values ranging between 0.09 and 0.5. Interestingly, the synergistic effect of CUR-I with FLC and AMB was associated with the accumulation of reactive oxygen species, which could be reversed by the addition of an antioxidant such as ascorbic acid. Furthermore, the combination of CUR-I and FLC/AMB triggered apoptosis that could also be reversed by ascorbic acid. We provide the first evidence that pure CUR-I in combination with azoles and polyenes represents a novel therapeutic strategy to improve the activity of common antifungals.