Metal complexes of 1,10-phenanthroline-5,6-dione alter the susceptibility of the yeast Candida albicans to Amphotericin B and Miconazole

Growth of the pathogenic yeast Candida albicans in sub-MIC (minimum inhibitory concentration) levels of Cu(ClO4)2 6H2O and [Cu(phendio)3](ClO4)2 4H2O (phendio = 1,10-phenanthroline-5,6-dione) increased the concentration of miconazole and amphotericin B required to achieve the MIC90 whereas pre-growth in AgClO4 and [Ag(phendio)2]ClO4 resulted in a small decrease in the relevant MIC90 values. The copper complexes reduce the oxygen consumption of C. albicans while the silver complexes increase oxygen consumption. In addition, pre-growth of cells in the copper complexes resulted in a lower ergosterol content while the silver complexes induced an elevation in ergosterol synthesis. The ability of copper and silver complexes to alter the susceptibility of C. albicans to miconazole and amphotericin B may be influenced by their action on respiration, since reduced respiration rates correlate with reduced cellular ergosterol which is the target for amphotericin B. Lower levels of ergosterol have previously been associated with elevated tolerance to this drug. In the case of reduced sensitivity to miconazole, tolerance may be mediated by lower ergosterol synthesis giving rise to fewer toxic side products once biosynthesis is inhibited by miconazole.     

Effects of amphotericin B and its methyl ester on plasma membranes of Candida albicans and erythrocytes as examined by freeze-fracture electron microscopy

Freeze-fracture electron microscopy of the plasma membranes of Candida albicans yeast cells and red blood cells treated with amphotericin methyl ester and amphotericin B showed that amphotericin B (50 micrograms ml-1) caused extreme aggregation of intramembranous particles on the protoplasmic fracture face of the C. albicans membrane, and a marked reduction of the density of intramembranous particles. On the other hand, the rearrangement of intramembranous particles induced by amphotericin methyl ester (50 micrograms ml-1) produced elevations of the particle-free membrane domains toward the outside of the cells, so that the particles were aggregated in linear furrows surrounding these elevations on the protoplasmic fracture face, and the corresponding ridges on the exoplasmic fracture face. The density of intramembranous particles was greatly reduced on the protoplasmic fracture face. Both polyenes produced only small changes in the erythrocyte membranes at the same concentration. These results suggest that amphotericin methyl ester affects the ergosterol-containing membranes more than amphotericin B, and that ergosterol has a higher sensitivity for these two polyene antibiotics than cholesterol.     

Amphotericin B resistance leads to enhanced proteinase and phospholipase activity and reduced germ tube formation in Candida albicans

Resistance to amphotericin B is an emerging phenomenon in Candida albicans. Amphotericin B-resistant strain of C. albicans was developed under laboratory conditions and the stability of acquired resistance was confirmed in vitro as well as in vivo. This AMB-resistant strain exhibited reduced germ tube formation as compared to parent strain of C. albicans (ATCC10231). Enzymatic activity of virulence factors like secreted aspartyl proteinase and phospholipase were found to be significantly high in AMB-R as compared to parent strain whereas ergosterol content of AMB-R was drastically reduced. The behavior of AMB-R strain is an interesting phenomenon and opens up a wide area of research regarding pathways and mechanisms.     

Mitochondrial sorting and assembly machinery subunit Sam37 in Candida albicans: insight into the roles of mitochondria in fitness, cell wall integrity, and virulence

Recent studies indicate that mitochondrial functions impinge on cell wall integrity, drug tolerance, and virulence of human fungal pathogens. However, the mechanistic aspects of these processes are poorly understood. We focused on the mitochondrial outer membrane SAM (Sorting and Assembly Machinery) complex subunit Sam37 in Candida albicans. Inactivation of SAM37 in C. albicans leads to a large reduction in fitness, a phenotype not conserved with the model yeast Saccharomyces cerevisiae. Our data indicate that slow growth of the sam37ΔΔ mutant results from mitochondrial DNA loss, a new function for Sam37 in C. albicans, and from reduced activity of the essential SAM complex subunit Sam35. The sam37ΔΔ mutant was hypersensitive to drugs that target the cell wall and displayed altered cell wall structure, supporting a role for Sam37 in cell wall integrity in C. albicans. The sensitivity of the mutant to membrane-targeting antifungals was not significantly altered. The sam37ΔΔ mutant was avirulent in the mouse model, and bioinformatics showed that the fungal Sam37 proteins are distant from their animal counterparts and could thus represent potential drug targets. Our study provides the first direct evidence for a link between mitochondrial function and cell wall integrity in C. albicans and is further relevant for understanding mitochondrial function in fitness, antifungal drug tolerance, and virulence of this major pathogen. Beyond the relevance to fungal pathogenesis, this work also provides new insight into the mitochondrial and cellular roles of the SAM complex in fungi.     

Proteomic analysis of Rta2p-dependent raft-association of detergent-resistant membranes in Candida albicans

In Candida albicans, lipid rafts (also called detergent-resistant membranes, DRMs) are involved in many cellular processes and contain many important proteins. In our previous study, we demonstrated that Rta2p was required for calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Here, we found that Rta2p was co-localized with raft-constituted ergosterol on the plasma membrane of C. albicans. Furthermore, this membrane expression pattern was totally disturbed by inhibitors of either ergosterol or sphingolipid synthesis. Biochemical fractionation of DRMs together with immunoblot uncovered that Rta2p, along with well-known DRM-associated proteins (Pma1p and Gas1p homologue), was associated with DRMs and their associations were blocked by inhibitors of either ergosterol or sphingolipid synthesis. Finally, we used the proteomic analysis together with immunoblot and identified that Rta2p was required for the association of 10 proteins with DRMs. These 5 proteins (Pma1p, Gas1p homologue, Erg11p, Pmt2p and Ali1p) have been reported to be DRM-associated and also that Erg11p is a well-known target of azoles in C. albicans. In conclusion, our results showed that Rta2p was predominantly localized in lipid rafts and was required for the association of certain membrane proteins with lipid rafts in C. albicans.     

Mitochondrial quality control: Cell-type-dependent responses to pathological mutant mitochondrial DNA

Pathological mutations in the mitochondrial DNA (mtDNA) produce a diverse range of tissue-specific diseases and the proportion of mutant mitochondrial DNA can increase or decrease with time via segregation, dependent on the cell or tissue type. Previously we found that adenocarcinoma (A549.B2) cells favored wild-type (WT) mtDNA, whereas rhabdomyosarcoma (RD.Myo) cells favored mutant (m3243G) mtDNA. Mitochondrial quality control (mtQC) can purge the cells of dysfunctional mitochondria via mitochondrial dynamics and mitophagy and appears to offer the perfect solution to the human diseases caused by mutant mtDNA. In A549.B2 and RD.Myo cybrids, with various mutant mtDNA levels, mtQC was explored together with macroautophagy/autophagy and bioenergetic profile. The 2 types of tumor-derived cell lines differed in bioenergetic profile and mitophagy, but not in autophagy. A549.B2 cybrids displayed upregulation of mitophagy, increased mtDNA removal, mitochondrial fragmentation and mitochondrial depolarization on incubation with oligomycin, parameters that correlated with mutant load. Conversely, heteroplasmic RD.Myo lines had lower mitophagic markers that negatively correlated with mutant load, combined with a fully polarized and highly fused mitochondrial network. These findings indicate that pathological mutant mitochondrial DNA can modulate mitochondrial dynamics and mitophagy in a cell-type dependent manner and thereby offer an explanation for the persistence and accumulation of deleterious variants.     

Effect of the aggregation state of amphotericin B on its interaction with ergosterol

The interaction of amphotericin B with ergosterol was studied in aqueous solutions of propanol. The mode of the interaction was found to be related to the aggregation state of amphotericin B. Ergosterol does not react (or reacts extremely slowly) with monomeric amphotericin B. Traces of a small aggregate, probably a dimer, enable a cooperative reaction. At high concentrations of the dimer, the reaction is immediate and the concentration of amphotericin B complexed with ergosterol is twice as high as the amount of added sterol. The interaction with ergosterol is hindered when the antibiotic is in micellar form. The pharmaceutical form, Fungizone, behaves similarly to the pure amphotericin B. Fungizone's greater solubility in water does not modify either the extent or the mode of interaction with ergosterol.     

High-dose methylprednisolone influences the physiology and virulence of Candida albicans ambiguously and enhances the candidacidal activity of the polyene antibiotic amphotericin B and the superoxide-generating agent menadione

Although exposure of Candida albicans cells to high-dose (4 mM) methylprednisolone stimulated microbial growth, germination rate in serum and phospholipase release, it also promoted the recognition of C. albicans cells by polymorphonuclear leukocytes. Pretreatment of C. albicans cells with methylprednisolone did not result in any increase in the pathogenicity of the fungus in intraperitoneal and intravenous mouse assays. Therefore, the virulence of C. albicans is unlikely to increase in patients treated with comparably high-dose methylprednisolone on skin and mucosal membranes. Methylprednisolone treatments also increased the production of conjugated dienes and thiobarbituric acid-reactive substances, and the menadione sensitivity of C. albicans cells, which can be explained by a significant decrease in the specific activities of several antioxidant enzymes. The combination of methylprednisolone with oxidants, e.g. in topical applications, may be of clinical importance when the predisposition to candidiasis is high. Methylprednisolone treatments negatively affected membrane fluidity and decreased the antifungal effects of both the polyene antibiotic nystatin and the ergosterol biosynthesis inhibitor lovastatin, and also enhanced the deleterious effects of the polyene antimycotic amphotericin B on C. albicans cells. These corticosteroid-polyene drug interactions should be considered in the treatment of C. albicans infections in patients with prolonged topical application of corticosteroids.     

Association of polyene antibiotics with sterols

Molecular interaction between amphotericin B and sterols in non-aqueous solutions was examined quantitatively by spectroscopic methods in order to support the view point that selectivity of amphotericin B is more pronounced in the presence of ergosterol than of cholesterol. The most likely association complexes between ergosterol and amphotericin B are 4:1, 6:1 stoichiometric complex when the concentrations of amphotericin B are 3.93 x 10(-4) M, 1.94 x 10(-4) M respectively. The presence of 3 beta-OH group is necessary but not enough for the association with amphotericin B. It appears that the extent of spectral change of amphotericin B induced by complexing sterol is greater for ergosterol than cholesterol.     

Characteristics of a strain of C. albicans resistant to polyene antibiotics]

It was found that a resistant strain R2 of C. albicans obtained as a result of passages on media containing increasing concentrations of amphotericin B differed from the initial strain by its lower pathogenicity. Treatment of the infection caused by the resistant strain on modeling of candidiasis in mice was not successful. The decrease in the average life span of the mice infected with the resistant strain R2 and treated with amphotericin B was lower than that in the control animals and such indices of the disease as the levels of the kidney dissemination and the cell vegetation even increased under the effect of amphotericin B. The results of the study suggest that the resistant strain R2 of C. albicans depend on amphotericin B in the host. The data obtained emphasize the necessity of determinining the antibiotic sensitivity of C. albicans strains isolated from patients.     

Candida albicans VPS1 contributes to protease secretion, filamentation, and biofilm formation

To investigate the pre-vacuolar secretory pathway in Candida albicans, we cloned and analyzed the C. albicans homolog of the Saccharomyces cerevisiae vacuolar protein sorting gene VPS1. C. albicans VPS1 encodes a predicted 694-aa dynamin-like GTPase that is 73.3% similar to S. cerevisiae Vps1p. Plasmids bearing C. albicans VPS1 complemented the temperature-sensitive growth, abnormal class F vacuolar morphology, and carboxypeptidase missorting of a S. cerevisiae vps1 null mutant. To study VPS1 function in C. albicans, a conditional mutant strain (tetR-VPS1) was generated by deleting the first allele of VPS1 and placing the second allele under control of a tetracycline-regulatable promoter. With doxycycline, the tetR-VPS1 mutant was hyper-susceptible to sub-inhibitory concentrations of fluconazole, but not amphotericin B, 5-fluorocytosine, or non-specific osmotic stresses. The repressed tetR-VPS1 mutant was defective in filamentation and secreted less extracellular protease activity. Biofilm production and filamentation within the biofilm were markedly reduced. These results suggest that C. albicans VPS1 has a key role in several important virulence-related phenotypes.     

Nitroxide spin-probe study of amphotericin B-ergosterol interaction in egg phosphatidylcholine multilayers

The interaction between the polyene macrolide antibiotic, amphotericin B, and ergosterol in egg phosphatidylcholine multilayers was investigated using head group and acyl chain nitroxide spin-labelled phosphatidylcholine as probes. At physiological concentrations of less than 15 mol% sterol in egg phosphatidylcholine multilayers amphotericin B accumulates near the head group region until an amphotericin B : ergosterol ratio of approximately 0.7 is achieved. As the proportion of amphotericin B is increased above this value, formation of an acyl chain disordering complex occurs which has an approximate antibiotic:sterol ratio of unity. Dicetyl phosphate was used to increase the solubility of ergosterol past its normal limit in pure egg phosphatidylcholine (approximately 15 mol%). At concentrations of ergosterol higher than 15 mol% a complex of two ergosterol molecules and one amphotericin B was postulated when there was insufficient antibiotic to form a 1:1 complex.     

Disruption of the human COQ5-containing protein complex is associated with diminished coenzyme Q10 levels under two different conditions of mitochondrial energy deficiency

BackgroundThe Coq protein complex assembled from several Coq proteins is critical for coenzyme Q₆ (CoQ₆) biosynthesis in yeast. Secondary CoQ₁₀ deficiency is associated with mitochondrial DNA (mtDNA) mutations in patients. We previously demonstrated that carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) suppressed CoQ₁₀ levels and COQ5 protein maturation in human 143B cells.MethodsThis study explored the putative COQ protein complex in human cells through two-dimensional blue native-polyacrylamide gel electrophoresis and Western blotting to investigate its status in 143B cells after FCCP treatment and in cybrids harboring the mtDNA mutation that caused myoclonic epilepsy with ragged-red fibers (MERRF) syndrome. Ubiquinol-10 and ubiquinone-10 levels were detected by high-performance liquid chromatography. Mitochondrial energy status, mRNA levels of various PDSS and COQ genes, and protein levels of COQ5 and COQ9 in cybrids were examined.ResultsA high-molecular-weight protein complex containing COQ5, but not COQ9, in the mitochondria was identified and its level was suppressed by FCCP and in cybrids with MERRF mutation. That was associated with decreased mitochondrial membrane potential and mitochondrial ATP production. Total CoQ₁₀ levels were decreased under both conditions, but the ubiquinol-10:ubiquinone-10 ratio was increased in mutant cybrids. The expression of COQ5 was increased but COQ5 protein maturation was suppressed in the mutant cybrids.ConclusionsA novel COQ5-containing protein complex was discovered in human cells. Its destabilization was associated with reduced CoQ₁₀ levels and mitochondrial energy deficiency in human cells treated with FCCP or exhibiting MERRF mutation.General significanceThe findings elucidate a possible mechanism for mitochondrial dysfunction-induced CoQ₁₀ deficiency in human cells.     

A deafness-associated tRNAHis mutation alters the mitochondrial function,ROS production and membrane potential

In this report, we investigated the molecular genetic mechanism underlying the deafness-associated mitochondrial tRNA^His 12201T>C mutation. The destabilization of a highly conserved base-pairing (5A-68U) by the m.12201T>C mutation alters structure and function of tRNA^His. Using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mtDNA-less (ρ^o) cells, we showed ∼70% decrease in the steady-state level of tRNA^His in mutant cybrids, compared with control cybrids. The mutation changed the conformation of tRNA^His, as suggested by slower electrophoretic mobility of mutated tRNA with respect to the wild-type molecule. However, ∼60% increase in aminoacylated level of tRNA^His was observed in mutant cells. The failure in tRNA^His metabolism was responsible for the variable reductions in seven mtDNA-encoded polypeptides in mutant cells, ranging from 37 to 81%, with the average of ∼46% reduction, as compared with those of control cells. The impaired mitochondrial translation caused defects in respiratory capacity in mutant cells. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These mitochondrial dysfunctions caused an increase in the production of reactive oxygen species in the mutant cells. The data provide the evidence for a mitochondrial tRNA^His mutation leading to deafness.     

Quantitative and qualitative analyses of the cell death process in Candida albicans treated by antifungal agents

The death process of Candida albicans was investigated after treatment with the antifungal agents flucytosine and amphotericin B by assessing morphological and biophysical properties associated with cell death. C. albicans was treated varying time periods (from 6 to 48 hours) and examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM). SEM and AFM images clearly showed changes in morphology and biophysical properties. After drug treatment, the membrane of C. albicans was perforated, deformed, and shrunken. Compared to the control, C. albicans treated with flucytosine was softer and initially showed a greater adhesive force. Conversely, C. albicans treated with amphotericin B was harder and had a lower adhesive force. In both cases, the surface roughness increased as the treatment time increased. The relationships between morphological changes and the drugs were observed by AFM clearly; the surface of C. albicans treated with flucytosine underwent membrane collapse, expansion of holes, and shrinkage, while the membranes of cells treated with amphotericin B peeled off. According to these observations, the death process of C. albicans was divided into 4 phases, CDP(0), CDP(1), CDP(2), and CDP(4), which were determined based on morphological changes. Our results could be employed to further investigate the antifungal activity of compounds derived from natural sources.     

In vitro activity of amphotericin B and anidulafungin against Candida spp. biofilms

Invasive infections caused by Candida spp. are increasing worldwide and are becoming an important cause of morbidity and mortality in immunocompromised patients. A large number of manifestations of candidiasis are associated with the formation of biofilms on inert or biological surfaces. Candida spp. biofilms are recalcitrant to treatment with conventional antifungal therapies. The aim of this study was dual 1) to determine the prevalence of biofilm producers among clinical isolates from catheter (16 C. albicans ) and blood culture (2 C. albicans and 30 C. tropicalis), and 2) to determine the activity of amphotericin B and anidulafungin against C. albicans and C. tropicalis biofilms of 24 and 48 hours of maturation. Biofilms were developed using a 96-well microtitre plate model and production and activity of antifungal agents against biofilms were determined by the tetrazolium (XTT) reduction assay. Of catheter and blood isolates, 62.5 and 56.25%, respectively, produced biofilms. By species, 68.42% of C. albicans and 53.33% of C. tropicalis were biofilm producers. C. albicans biofilms showed more resistance to amphotericin B and anidulafungin than their planktonic counterparts. Complete killing of biofilms was never achieved, even at the highest concentrations of the drugs tested. Anidulafungin displayed more activity than amphotericin B against C. albicans biofilms of 24 hours of maturation (GM MIC 0.354 vs. 0.686 microg/ml), but against C. tropicalis biofilms amphotericin B was more active (GM MIC 11.285 vs. 0.476 microg/ml). In contrast, against biofilms with 48 hours maturation, amphotericin B was more active against both species.     

Comparative in vitro studies on liposomal formulations of amphotericin B and its derivative,N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME)

N-Methyl-N-D-fructosyl amphotericin B methyl ester (MFAME) is a semisynthetic derivative of the antifungal antibiotic amphotericin B (AMB). In contrast to the parent antibiotic, the derivative is characterised by low toxicity to mammalian cells and good solubility in water of its salts. Comparative studies on biological properties of free MFAME, AMB and their liposomal formulations were performed. To obtain liposomal forms, the antibiotics were incorporated into small unilamellar vesicles composed of dimyristoyl phosphatidylcholine (DMPC) and DMPC:cholesterol or ergosterol, 8:2 molar ratio. The effectivity of the liposomal and free forms of AMB and MFAME were compared by determination of fungistatic and fungicidal activity against Candida albicans ATCC 10261, potassium release from erythrocytes, and haemolysis. The results obtained indicate that in contrast to AMB, incorporation of MFAME into liposomes did not further improve its selective toxicity. Studies on the antagonistic effect of ergosterol and cholesterol on the antifungal activity of the antibiotics indicated that sterol interference was definitely less pronounced in the case of MFAME than in the case of AMB.     

Aminoglycoside Stress Together with the 12S rRNA 1494C>T Mutation Leads to Mitophagy

Aminoglycosides as modifying factors modulated the phenotypic manifestation of mitochondrial rRNA mutations and the incomplete penetrance of hearing loss. In this report, using cybrids harboring the m.1494C>T mutation, we showed that gentamycin aggravated mitochondrial dysfunction in a combination of the m.1494C>T mutation. The m.1494C>T mutation was responsible for the dramatic reduction in three mtDNA-encoded proteins of H-strand, with the average of 39% reduction, except of the MT-ND6 protein, accompanied with 21% reduction of ATP production and increase in mitochondrial reactive oxygen species, compared with those of control cybrids. After exposure to gentamycin, 35% reduction of mitochondrial ATP production was observed in mutant cybrids with a marked decrease of the mitochondrial membrane potential. More excessive cellular reactive oxygen species was detected with stimulus of gentamycin than those in mutant cells. Under gentamycin and m.1494C>T stress together, more dysfunctional mitochondria were forced to fuse and exhibited mitophagy via up-regulated LC3-B, as a compensatory protective response to try to optimize mitochondrial function, rather than undergo apoptosis. These findings may provide valuable information to further understand of mechanistic link between mitochondrial rRNA mutation, toxicity of AGs and hearing loss.