Fructose Induces Fluconazole Resistance in Candida albicans through Activation of Mdr1 and Cdr1 Transporters

Candida albicans is a pathogenic fungus that is increasingly developing multidrug resistance (MDR), including resistance to azole drugs such as fluconazole (FLC). This is partially a result of the increased synthesis of membrane efflux transporters Cdr1p, Cdr2p, and Mdr1p. Although all these proteins can export FLC, only Cdr1p is expressed constitutively. In this study, the effect of elevated fructose, as a carbon source, on the MDR was evaluated. It was shown that fructose, elevated in the serum of diabetics, promotes FLC resistance. Using C. albicans strains with green fluorescent protein (GFP) tagged MDR transporters, it was determined that the FLC-resistance phenotype occurs as a result of Mdr1p activation and via the increased induction of higher Cdr1p levels. It was observed that fructose-grown C. albicans cells displayed a high efflux activity of both transporters as opposed to glucose-grown cells, which synthesize Cdr1p but not Mdr1p. Additionally, it was concluded that elevated fructose serum levels induce the de novo production of Mdr1p after 60 min. In combination with glucose, however, fructose induces Mdr1p production as soon as after 30 min. It is proposed that fructose may be one of the biochemical factors responsible for Mdr1p production in C. albicans cells.     

K143R Amino Acid Substitution in 14-α-Demethylase (Erg11p) Changes Plasma Membrane and Cell Wall Structure of Candida albicans

The opportunistic pathogen Candida albicans is responsible for life-threating infections in immunocompromised individuals. Azoles and polyenes are two of the most commonly used antifungals and target the ergosterol biosynthesis pathway or ergosterol itself. A limited number of clinically employed antifungals correspond to the development of resistance mechanisms. One resistance mechanism observed in clinical isolates of azole-resistant C. albicans is the introduction of point mutations in the ERG11 gene, which encodes a key enzyme (lanosterol 14-α-demethylase) on the ergosterol biosynthesis pathway. Here, we demonstrate that a point mutation K143R in ERG11 (C. albicans ERG11^K143R/K143R) contributes not only to azole resistance, but causes increased gene expression. Overexpression of ERG11 results in increased ergosterol content and a significant reduction in plasma membrane fluidity. Simultaneously, the same point mutation caused cell wall remodeling. This could be facilitated by the unmasking of chitin and β-glucan on the fungal cell surface, which can lead to recognition of the highly immunogenic β-glucan, triggering a stronger immunological reaction. For the first time, we report that a frequently occurring azole-resistance strategy makes C. albicans less susceptible to azole treatment while, at the same time, affects its cell wall architecture, potentially leading to exposure of the pathogen to a more effective host immune response.     

Disruption of ergosterol biosynthesis,growth, and the morphological transition in <Emphasis Type="Italic">Candida albicans</Emphasis> by sterol methyltransferase inhibitors containing sulfur at C-25 in the sterol side chain

The sterol substrate analog 25-thialanosterol and its corresponding sulfonium salt were evaluated for their ability to serve as antifungal agents and to inhibit sterol methyltransferase (SMT) activity in Candida albicans. Both compounds inhibited cell proliferation, were fungistatic, interrupted the yeastlike-form to germ-tube-form transition, and resulted in the accumulation of zymosterol and related Δ²⁴-sterols concurrent with a decrease in ergosterol, as was expected for the specific inhibition of SMT activity. Feedback on sterol synthesis was evidenced by elevated levels of cellular sterols in treated vs. control cultures. However, neither farnesol nor squalene accumulated in significant amounts in treated cultures, suggesting that carbon flux is channeled from the isoprenoid pathway to the sterol pathway with minor interruption or redirection until blockage at the C-methylation step. Activity assays using solubilized C. albicans SMT confirmed the inhibitors impair SMT action. Kinetic analysis indicated that 25-thialanosterol inhibited SMT with the properties of a time-dependent mechanismbased inactivator K _i of 5 =gmM and apparent k _inact of 0.013 min⁻¹, whereas the corresponding sulfonium salt was a reversible-type transition state analog exhibiting a K _i of 20 nM. The results are interpreted to imply changes in ergosterol homeostasis as influenced by SMT activity can control growth and the morphological transition in C. albicans, possibly affecting disease development.     

Synthesis, Structure Optimization and Antifungal Screening of Novel Tetrazole Ring Bearing Acyl-Hydrazones

Azoles are generally fungistatic, and resistance to fluconazole is emerging in several fungal pathogens. In an attempt to find novel azole antifungal agents with improved activity, a series of tetrazole ring bearing acylhydrazone derivatives were synthesized and screened for their in vitro antifungal activity. The mechanism of their antifungal activity was assessed by studying their effect on the plasma membrane using flow cytometry and determination of the levels of ergosterol, a fungal-specific sterol. Propidium iodide rapidly penetrated a majority of yeast cells when they were treated with the synthesized compounds at concentrations just above MIC, implying that fungicidal activity resulted from extensive lesions of the plasma membrane. Target compounds also caused a considerable reduction in the amount of ergosterol. The results also showed that the presence and position of different substituents on the phenyl ring of the acylhydrazone pendant seem to play a role on the antifungal activity as well as in deciding the fungistatic and fungicidal nature of the compounds.     

Role of Candida albicans-Secreted Aspartyl Proteinases (Saps) in Severe Early Childhood Caries

Candida albicans is strongly associated with severe early childhood caries (S-ECC). However, the roles of secreted aspartyl proteinases (Saps), an important virulence factor of C. albicans, in the progress of S-ECC are not clear. In our study, the Saps activities were evaluated by the yeast nitrogen base–bovine serum albumi (YNB–BSA) agar plate method and by the MTT method with bovine serum albumin (BSA) as the substrate. Genotypes of C. albicans and gene expression of Sap1–5 were evaluated. The relationships of Saps activities and genotypes with S-ECC were analyzed. The results showed that enzyme activities of Saps in the S-ECC group were significantly higher than those in the caries free (CF) group (p < 0.05). Genotypes A, B and C were detected in the S-ECC group, and genotypes A and C were detected in the CF group. In the genotype A group, Saps activity in the S-ECC group was significantly different from that in the CF group (p < 0.05). The gene expression level of Sap1 in the S-ECC group was significantly higher than that in the CF group (p = 0.001), while Sap4 expression was significantly lower than that in the CF group (p = 0.029). It can be concluded that Sap1–5 are the predominant proteinase genes expressed in C. albicans from dental biofilm and Sap1 may play an important role in the development of S-ECC.     

Evidence for the Role of CYP51A and Xenobiotic Detoxification in Differential Sensitivity to Azole Fungicides in Boxwood Blight Pathogens

Boxwood blight, a fungal disease of ornamental plants (Buxus spp.), is caused by two sister species, Calonectria pseudonaviculata (Cps) and C. henricotiae (Che). Compared to Cps, Che is documented to display reduced sensitivity to fungicides, including the azole class of antifungals, which block synthesis of a key fungal membrane component, ergosterol. A previous study reported an ergosterol biosynthesis gene in Cps, CYP51A, to be a pseudogene, and RNA-Seq data confirm that a functional CYP51A is expressed only in Che. The lack of additional ergosterol biosynthesis genes showing significant differential expression suggests that the functional CYP51A in Che could contribute to reduced azole sensitivity when compared to Cps. RNA-Seq and bioinformatic analyses found that following azole treatment, 55 genes in Cps, belonging to diverse pathways, displayed a significant decrease in expression. Putative xenobiotic detoxification genes overexpressed in tetraconazole-treated Che encoded predicted monooxygenase and oxidoreductase enzymes. In summary, expression of a functional CYP51A gene and overexpression of predicted xenobiotic detoxification genes appear likely to contribute to differential fungicide sensitivity in these two sister taxa.     

Selective Antifungal Activity and Fungal Biofilm Inhibition of Tryptophan Center Symmetrical Short Peptide

Candida albicans, an opportunistic fungus, causes dental caries and contributes to mucosal bacterial dysbiosis leading to a second infection. Furthermore, C.albicans forms biofilms that are resistant to medicinal treatment. To make matters worse, antifungal resistance has spread (albeit slowly) in this species. Thus, it has been imperative to develop novel, antifungal drug compounds. Herein, a peptide was engineered with the sequence of RRFSFWFSFRR-NH2; this was named P19. This novel peptide has been observed to exert disruptive effects on fungal cell membrane physiology. Our results showed that P19 displayed high binding affinity to lipopolysaccharides (LPS), lipoteichoic acids (LTA) and the plasma membrane phosphatidylinositol (PI), phosphatidylserine (PS), cardiolipin, and phosphatidylglycerol (PG), further indicating that the molecular mechanism of P19 was not associated with the receptor recognition, but rather related to competitive interaction with the plasma membrane. In addition, compared with fluconazole and amphotericin B, P19 has been shown to have a lower potential for resistance selection than established antifungal agents.     

Quest for the Molecular Basis of Improved Selective Toxicity of All-Trans Isomers of Aromatic Heptaene Macrolide Antifungal Antibiotics

Three aromatic heptaene macrolide antifungal antibiotics, Candicidin D, Partricin A (Gedamycin) and Partricin B (Vacidin) were subjected to controlled cis-trans → all trans photochemical isomerization. The obtained all-trans isomers demonstrated substantially improved in vitro selective toxicity in the Candida albicans cells: human erythrocytes model. This effect was mainly due to the diminished hemotoxicity. The molecular modeling studies on interactions between original antibiotics and their photoisomers with ergosterol and cholesterol revealed some difference in free energy profiles of formation of binary antibiotic/sterol complexes in respective membrane environments. Moreover, different geometries of heptaene: sterol complexes and variations in polyene macrolide molecule alignment in cholesterol-and ergosterol-containing membranes were found. None of these effects are of the crucial importance for the observed improvement of selective toxicity of aromatic heptaene antifungals but each seems to provide a partial contribution.     

Antifungal Activity and DNA Topoisomerase Inhibition of Hydrolysable Tannins from Punica granatum L.

Punica granatum L. (pomegranate) fruit is known to be an important source of bioactive phenolic compounds belonging to hydrolysable tannins. Pomegranate extracts have shown antifungal activity, but the compounds responsible for this activity and their mechanism/s of action have not been completely elucidated up to now. The aim of the present study was the investigation of the inhibition ability of a selection of pomegranate phenolic compounds (i.e., punicalagin, punicalin, ellagic acid, gallic acid) on both plant and human fungal pathogens. In addition, the biological target of punicalagin was identified here for the first time. The antifungal activity of pomegranate phenolics was evaluated by means of Agar Disk Diffusion Assay and minimum inhibitory concentration (MIC) evaluation. A chemoinformatic analysis predicted for the first time topoisomerases I and II as potential biological targets of punicalagin, and this prediction was confirmed by in vitro inhibition assays. Concerning phytopathogens, all the tested compounds were effective, often similarly to the fungicide imazalil at the label dose. Particularly, punicalagin showed the lowest MIC for Alternaria alternata and Botrytis cinerea, whereas punicalin was the most active compound in terms of growth control extent. As for human pathogens, punicalagin was the most active compound among the tested ones against Candida albicans reference strains, as well as against the clinically isolates. UHPLC coupled with HRMS indicated that C. albicans, similarly to the phytopathogen Coniella granati, is able to hydrolyze both punicalagin and punicalin as a response to the fungal attack. Punicalagin showed a strong inhibitory activity, with IC₅₀ values of 9.0 and 4.6 µM against C. albicans topoisomerases I and II, respectively. Altogether, the results provide evidence that punicalagin is a valuable candidate to be further exploited as an antifungal agent in particular against human fungal infections.     

WMR Peptide as Antifungal and Antibiofilm against Albicans and Non-Albicans Candida Species: Shreds of Evidence on the Mechanism of Action

Candida species are the most common fungal pathogens infecting humans and can cause severe illnesses in immunocompromised individuals. The increased resistance of Candida to traditional antifungal drugs represents a great challenge in clinical settings. Therefore, novel approaches to overcome antifungal resistance are desired. Here, we investigated the use of an antimicrobial peptide WMR against Candida albicans and non-albicans Candida species in vitro and in vivo. Results showed a WMR antifungal activity on all Candida planktonic cells at concentrations between 25 μM to >50 μM and exhibited activity at sub-MIC concentrations to inhibit biofilm formation and eradicate mature biofilm. Furthermore, in vitro antifungal effects of WMR were confirmed in vivo as demonstrated by a prolonged survival rate of larvae infected by Candida species when the peptide was administered before or after infection. Additional experiments to unravel the antifungal mechanism were performed on C. albicans and C. parapsilosis. The time-killing curves showed their antifungal activity, which was further confirmed by the induced intracellular and mitochondrial reactive oxygen species accumulation; WMR significantly suppressed drug efflux, down-regulating the drug transporter encoding genes CDR1. Moreover, the ability of WMR to penetrate within the cells was demonstrated by confocal laser scanning microscopy. These findings provide novel insights for the antifungal mechanism of WMR against Candida albicans and non-albicans, providing fascinating scenarios for the identification of new potential antifungal targets.     

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.     

The Catestatin-Derived Peptides Are New Actors to Fight the Development of Oral Candidosis

Resistance to antifungal therapy of Candida albicans and non-albicans Candida strains, frequently associated with oral candidosis, is on the rise. In this context, host-defense peptides have emerged as new promising candidates to overcome antifungal resistance. Thus, the aim of this study was to assess the effectiveness against Candida species of different Catestatin-derived peptides, as well as the combined effect with serum albumin. Among Catestatin-derived peptides, the most active against sensitive and resistant strains of C. albicans, C. tropicalis and C. glabrata was the D-isomer of Cateslytin (D-bCtl) whereas the efficiency of the L-isomer (L-bCtl) significantly decreases against C. glabrata strains. Images obtained by transmission electron microscopy clearly demonstrated fungal membrane lysis and the leakage of the intracellular material induced by the L-bCtl and D-bCtl peptides. The possible synergistic effect of albumin on Catestatin-derived peptides activity was investigated too. Our finding showed that bovine serum albumin (BSA) when combined with the L- isomer of Catestatin (L-bCts) had a synergistic effect against Candida albicans especially at low concentrations of BSA; however, no synergistic effect was detected when BSA interacted with L-bCtl, suggesting the importance of the C-terminal end of L-bCts (GPGLQL) for the interaction with BSA. In this context in vitro D-bCtl, as well as the combination of BSA with L-bCts are potential candidates for the development of new antifungal drugs for the treatment of oral candidosis due to Candida and non-Candida albicans, without detrimental side effects.     

Antiadhesive Properties of Imidazolium Ionic Liquids Based on (−)-Menthol Against Candida spp.

Infections with Candida spp. are commonly found in long-time denture wearers, and when under immunosuppression can lead to stomatitis. Imidazolium ionic liquids with an alkyl or alkyloxymethyl chain and a natural (1R,2S,5R)-(−)-menthol substituent possess high antifungal and antiadhesive properties towards C. albicans, C. parapsilosis, C. glabrata and C. krusei. We tested three compounds and found they disturbed fungal plasma membranes, with no significant hemolytic properties. In the smallest hemolytic concentrations, all compounds inhibited C. albicans biofilm formation on acrylic, and partially on porcelain and alloy dentures. Biofilm eradication may result from hyphae inhibition (for alkyl derivatives) or cell wall lysis and reduction of adhesins level (for alkyloxymethyl derivative). Thus, we propose the compounds presented herein as potential anti-fungal denture cleaners or denture fixatives, especially due to their low toxicity towards mammalian erythrocytes after short-term exposure.     

Design and Characterization of Myristoylated and Non-Myristoylated Peptides Effective against Candida spp. Clinical Isolates

The increasing resistance of fungi to antibiotics is a severe challenge in public health, and newly effective drugs are required. Promising potential medications are lipopeptides, linear antimicrobial peptides (AMPs) conjugated to a lipid tail, usually at the N-terminus. In this paper, we investigated the in vitro and in vivo antifungal activity of three short myristoylated and non-myristoylated peptides derived from a mutant of the AMP Chionodracine. We determined their interaction with anionic and zwitterionic membrane-mimicking vesicles and their structure during this interaction. We then investigated their cytotoxic and hemolytic activity against mammalian cells. Lipidated peptides showed a broad spectrum of activity against a relevant panel of pathogen fungi belonging to Candida spp., including the multidrug-resistant C. auris. The antifungal activity was also observed vs. biofilms of C. albicans, C. tropicalis, and C. auris. Finally, a pilot efficacy study was conducted on the in vivo model consisting of Galleria mellonella larvae. Treatment with the most-promising myristoylated peptide was effective in counteracting the infection from C. auris and C. albicans and the death of the larvae. Therefore, this myristoylated peptide is a potential candidate to develop antifungal agents against human fungal pathogens.     

General resistance to sterol biosynthesis inhibitors inSaccharomyces cerevisiae

Screening for resistance to fenpropimorph was undertaken in order to isolate yeast mutants affected in the regulation of the ergosterol pathway. Among the mutants isolated, one bearing the recessivefen1-1 mutation was characterized by a 1.5-fold increase in the ergosterol level and a general resistance to sterol biosynthesis inhibitors. Thefen1-1 mutation was linked toMAT locus on chromosome III. The measurement of enzyme activities involved in the ergosterol pathway revealed that isopentenyl diphosphate (IPP) isomerase activity was specifically increased 1.5-fold as compared to the wild type strain. However, overexpression of IPP isomerase in the wild type strain was not by itself sufficient to lead to sterol increase or resistance to sterol biosynthesis inhibitors, showing that IPP isomerase is not a limiting step in the pathway. Thefen1-1 mutation permits viability in aerobiosis of yeast disrupted for sterol-14 reductase in absence of exogenous ergosterol supplementation, whereas the corresponding strain bearing the wild typeFEN1 allele grows only in anaerobiosis. This result shows that ignosterol is able to efficiently replace ergosterol as bulk membrane component and that thefen1-1 mutation eliminates the specific ergosterol requirement in yeast.     

Camphor and Eucalyptol—Anticandidal Spectrum,Antivirulence Effect,Efflux Pumps Interference and Cytotoxicity

Candidaalbicans represents one of the most common fungal pathogens. Due to its increasing incidence and the poor efficacy of available antifungals, finding novel antifungal molecules is of great importance. Camphor and eucalyptol are bioactive terpenoid plant constituents and their antifungal properties have been explored previously. In this study, we examined their ability to inhibit the growth of different Candida species in suspension and biofilm, to block hyphal transition along with their impact on genes encoding for efflux pumps (CDR1 and CDR2), ergosterol biosynthesis (ERG11), and cytotoxicity to primary liver cells. Camphor showed excellent antifungal activity with a minimal inhibitory concentration of 0.125–0.35 mg/mL while eucalyptol was active in the range of 2–23 mg/mL. The results showed camphor’s potential to reduce fungal virulence traits, that is, biofilm establishment and hyphae formation. On the other hand, camphor and eucalyptol treatments upregulated CDR1; CDR2 was positively regulated after eucalyptol application while camphor downregulated it. Neither had an impact on ERG11 expression. The beneficial antifungal activities of camphor were achieved with an amount that was non-toxic to porcine liver cells, making it a promising antifungal compound for future development. The antifungal concentration of eucalyptol caused cytotoxic effects and increased expression of efflux pump genes, which suggests that it is an unsuitable antifungal candidate.     

A Thioether‐Stabilized d‐Proline–l‐Proline‐Induced β‐Hairpin Peptide of Defensin Segment Increases Its Anti‐Candida albicans Ability

We report a β‐hairpin dual stabilizing strategy: a d ‐proline‐l ‐proline (d ‐Pro‐l ‐Pro) dipeptide as the nucleating turn, and a thioether tether as a side‐chain linkage at a precisely designed position to stabilize the β‐hairpin. This method was used to modify the C‐terminal β‐hairpin moiety of the plant defensin, pv‐defensin, in order to obtain a stabilized peptide with enhanced anti‐Candida albicans activity (MIC 84–3.0 μm ), high serum stability (50 % remaining after 48 h) and low hemolysis (<10 % at 152 μm ). This modified peptide penetrated the C. albicans cell membrane within 5 min and showed high activity against clinically isolated antibiotic‐resistant C. albicans and Candida glabrata strains.     

GC-MS-Based Metabolomics Study of Single- and Dual-Species Biofilms of Candida albicans and Klebsiella pneumoniae

Candida albicans and Klebsiella pneumoniae frequently co-exist within the human host as a complex biofilm community. These pathogens are of interest because their association is also related to significantly increased morbidity and mortality in hospitalized patients. With the aim of highlighting metabolic shifts occurring in the dual-species biofilm, an untargeted GC-MS-based metabolomics approach was applied to single and mixed biofilms of C. albicans and K. pneumoniae. Metabolomic results showed that among the extracellular metabolites identified, approximately 40 compounds had significantly changed relative abundance, mainly involving central carbon, amino acid, vitamin, and secondary metabolisms, such as serine, leucine, arabitol, phosphate, vitamin B6, cyclo-(Phe-Pro), trehalose, and nicotinic acid. The results were related to the strict interactions between the two species and the different microbial composition in the early and mature biofilms.     

Network structures of fullerene-like carbon core/nano-crystalline silicon shell nanofibers as anode material for lithium-ion batteries

Fullerene-like carbon (FLC) nanoparticles were prepared by depositing soot of burning castor oil. FLC core/nano-crystalline silicon (nc-Si) shell nanofibers with network structures have been fabricated by electrospinning and plasma enhanced chemical vapor deposition techniques. The morphologies and structures of the materials were characterized using scanning electron microscopy, transmission electron microscopy, and micro-Raman spectroscopy. It was demonstrated that the FLC core/nc-Si shell nanofibers on nickel foam can be used as electrode for lithium-ion batteries without adding any binding or conducting additives. High reversible specific capacity of 1164 mA h g⁻¹ is retained after 50 discharge/charge cycles at a constant current density of 100 mA g⁻¹. The electrode delivers prolonged cycle life and enhanced rate capability compared to pristine nc-Si film. The improved electrochemical performance could be attributed to that the FLC core provides facile strain relaxation to accommodate the large Si volume expansion and shrinkage during lithium-ion insertion and extraction.