Evaluation of in silico designed inhibitors targeting MelF (Rv1936) against Mycobacterium marinum within macrophages.

We recently identified inhibitors targeting Mycobacterium marinum MelF (Rv1936) by in silico analysis, which exhibited bacteriostatic/bactericidal activity against M. marinum and M. tuberculosis in vitro. Herein, we evaluated the effect of best four inhibitors (# 5175552, # 6513745, # 5255829, # 9125618) obtained from the ChemBridge compound libraries, on intracellular replication and persistence of bacteria within IFN-γ activated murine RAW264.7 and human THP-1 macrophages infected with M. marinum. Inhibitors # 5175552 and # 6513745 significantly reduced (p < 0.05) the intracellular replication of bacilli during day 7 post-infection (p.i.) within RAW264.7 and THP-1 macrophages infected at multiplicity of infection (MOI) of ~1.0. These observations were substantiated by electron microscopy, which revealed the protective effect of # 5175552 in clearing the bacilli inside murine macrophages. Strikingly, # 6513745 displayed synergism with isoniazid against M. marinum in murine macrophages, whereas # 5175552 significantly suppressed (p < 0.05) the persistent bacilli during day 10-14 p.i. in infected RAW264.7 and THP-1 macrophages (MOI of ~ 0.1). Moreover, # 5175552 and # 6513745 were non-cytotoxic to host macrophages at both 1X and 5X MIC. Further validation of these inhibitors against M. tuberculosis-infected macrophages and animal models has potential for development as novel anti-tubercular agents.

Bandgap Tunable AgInS based Quantum Dots for High Contrast Cell Imaging with Enhanced Photodynamic and Antifungal Applications.

Herein, we report a facile microwave-assisted synthesis of cadmium-free water-soluble silver indium sulfide (AgInS2 or AIS) and AgInS@ZnS (or AIS@ZnS) core-shell quantum dots (QDs) using glutathione (GSH) as stabilizer. The core and core-shell nanocrystals exhibit tunable bandgap ranging of 2.3-3.1 and 2.4-3.5 eV, mean particle size of 2.5 and 3.25 nm, quantum yield of 26% and 49%, and fluorescence lifetimes of 326 and 438 ns, respectively. The core-shell QDs exhibit color-tunable emission in the visible region (500 to 600 nm), where the tunability was achieved by varying the molar ratio of Ag:In in the precursors. In vitro evaluation of antifungal activity of these water/ buffer stable QDs against the fungal pathogen, Candida albicans demonstrated that these were not toxic to the fungal cells upto a concentration of 100 µg/ml for 16 hours of incubation. Confocal imaging and spectrofluorometric studies showed enhanced fluorescence inside the microbial cells suggesting that AIS@ZnS particles had the capability to easily penetrate the cells. The increased generation of reactive oxygen species was evaluated for the core-shell QDs (photosensitizers) by using 9, 10-anthracenediyl-bis(methylene)dimalonic acid (ABMDMA) as singlet oxygen (1O2) scavenger molecule. These QDs have the potential for use as high contrast cell imaging, photodynamic and antifungal agents.

Curcumin modulates efflux mediated by yeast ABC multidrug transporters and is synergistic with antifungals.

Curcumin (CUR), a natural product of turmeric, from rhizomes of Curcuma longa, is a known agent of reversal of drug resistance phenotypes in cancer cells overexpressing ATP-binding cassette (ABC) transporters, viz., ABCB1, ABCG2, and ABCC1. In the present study, we evaluated whether CUR could also modulate multidrug transporters of yeasts that belong either to the ABC family or to the major facilitator superfamily (MFS). The effect of CUR on multidrug transporter proteins was demonstrated by examining rhodamine 6G (R6G) efflux in Saccharomyces cerevisiae cells overexpressing the Candida albicans ABC transporters Cdr1p and Cdr2p (CaCdr1p and CaCdr2p, respectively) and the MFS transporters CaMdr1p and S. cerevisiae Pdr5p. CUR decreased the extracellular concentration of R6G in ABC transporter-expressing cells but had no effect on methotrexate efflux mediated through the MFS transporter CaMdr1p. CUR competitively inhibited R6G efflux and the photolabeling of CaCdr1p by [(125)I]iodoarylazidoprazosin, a drug analogue of the substrate prazosin (50% inhibitory concentration, 14.2 microM). Notably, the mutant variants of CaCdr1p that displayed abrogated efflux of R6G also showed reduced modulation by CUR. Drug susceptibility testing of ABC protein-expressing cells by spot assays and checkerboard tests revealed that CUR was selectively synergistic with drug substrates such as R6G, ketoconazole, itraconazole, and miconazole but not with fluconazole, voriconazole, anisomycin, cycloheximide, or FK520. Taken together, our results provide the first evidence that CUR modulates only ABC multidrug transporters and could be exploited in combination with certain conventional antifungal drugs to reverse multidrug resistance in Candida cells.

Iron deprivation induces EFG1-mediated hyphal development in Candida albicans without affecting biofilm formation.

In this study, we investigated the role of cellular iron status in hyphae and biofilm formation in Candida albicans. Iron deprivation by a chelator, bathophenanthrolene disulfonic acid, promoted hyphal development even in nonhyphal-inducing media without affecting growth of C. albicans cells. Iron-acquisition defective mutants, Deltaftr1 and Deltaccc2, also showed hyphal formation, which was prevented by iron supplementation. Notably, most of the tested morphological mutants Deltacph1, Deltaefh1 and Deltatpk1 continued to form hyphae under iron-deprived conditions, except the Deltaefg1 null mutant, which showed a complete block in hyphae formation. The role of EFG1 in filamentation under iron-deprived conditions was further confirmed by Northern analysis, which showed a considerable upregulation of the EFG1 transcript. Of notable importance, all the morphological mutants including Deltaefg1 mutant possessed enhanced membrane fluidity under iron-deprived conditions; however, this did not appear to contribute to hyphal development. Interestingly, iron deprivation did not affect the ability of C. albicans to form biofilms on the catheter surface and led to no gross defects in azole resistance phenotype of these biofilms of C. albicans cells. Our study, for the first time, establishes a link between cellular iron, Efg1p and hyphal development of C. albicans cells that is independent of biofilm formation.

Membrane fluidity and lipid composition of fluconazole resistant and susceptible strains of Candida albicans isolated from diabetic patients / Fluidez e composição lipídica da membrana de cepas de Candida albicans resistentes e sensíveis ao fluconazol, isoladas de pacientes diabéticos

Ten clinical isolates of Candida albicans, five strains belonging to each of fluconazole resistant and susceptible groups isolated from diabetic patients, were studied for the membrane fluidity and lipid composition. Compared to fluconazole susceptible strains, fluconazole resistant ones exhibited enhanced membrane fluidity as measured by fluorescence polarization technique. The increased membrane fluidity was reflected in the decreased p-values exhibited by the resistant strains. On the other hand, susceptible isolates contained higher amount of ergosterol, almost twice as compared to resistant isolates which might have contributed to their lower membrane fluidity. However, no significant alteration was observed in the phospholipid and fatty acid composition of these isolates. Labeling experiments with fluorescamine dye revealed that the percentage of the exposed aminophospholipid, phosphatidylethanolamine was highest in the resistant strains as compared to the susceptible strains, indicating a possible overexpression of CDR1 and CDR2 genes in resistant strains. The results presented here suggest that the changes in the ergosterol content and overexpression of ABC transporter genes CDR1 and CDR2 could contributeto fluconazole resistance in C. albicans isolated from diabetic patients.

Dez isolados clínicos, sendo cinco resistentes e cinco sensíveis ao fluconazol, obtidos de pacientes diabéticos, foram estudados quanto à fluidez e composição química da membrana. Quando comparados aos isolados sensíveis ao fluconazol, os isolados resistentes apresentaram fluidez de membrana aumentada, conforme mensurado pela técnica de polarização fluorescente. A fluidez de membrana aumentada refletiu-se pelos valores mais baixos de p. Por outro lado, os isolados sensíveis continham quantidades mais elevadas de ergosterol, quase o dobro dos isolados resistentes, o que pode ter contribuído para a fluidez de membrana mais baixa. Entretanto, não se observou alteração significativa na composição fosfolipídica e de ácidos graxos nesses isolados. Experimentos de marcação com corante fluorescamina indicaram que a porcentagem de aminofosfolípides e fosfatidiletanolamina expostos foi mais elevada nos isolados resistentes do que nos sensíveis, indicando uma possível superexpressão dos genes CDR1 e CDR2 nos isolados resistentes. Os resultados aqui apresentados sugerem que alterações no teor de ergosterol e superexpressão dos genes ABC transportadores CDR1 e CDR2 podem contribuir na resistência ao fluconazol em isolados de C. albicans de pacientes diabéticos.

Unexpected link between iron and drug resistance of Candida spp.: iron depletion enhances membrane fluidity and drug diffusion, leading to drug-susceptible cells.

Inthis study, we show that iron depletion in Candida albicans with bathophenanthrolene disulfonic acid and ferrozine as chelators enhanced its sensitivity to several drugs, including the most common antifungal, fluconazole (FLC). Several other species of Candida also displayed increased sensitivity to FLC because of iron restriction. Iron uptake mutations, namely, Deltaftr1 and Deltaftr2, as well as the copper transporter mutation Deltaccc2, which affects high-affinity iron uptake in Candida, produced increased sensitivity to FLC compared to that of the wild type. The effect of iron depletion on drug sensitivity appeared to be independent of the efflux pump proteins Cdr1p and Cdr2p. We found that iron deprivation led to lowering of membrane ergosterol by 15 to 30%. Subsequently, fluorescence polarization measurements also revealed that iron-restricted Candida cells displayed a 29 to 40% increase in membrane fluidity, resulting in enhanced passive diffusion of the drugs. Northern blot assays revealed that the ERG11 gene was considerably down regulated in iron-deprived cells, which might account for the lowered ergosterol content. Our results show a close relationship between cellular iron and drug susceptibilities of C. albicans. Considering that multidrug resistance is a manifestation of multifactorial phenomena, the influence of cellular iron on the drug susceptibilities of Candida suggests iron as yet another novel determinant of multidrug resistance.

Squalene epoxidase encoded by ERG1 affects morphogenesis and drug susceptibilities of Candida albicans.

OBJECTIVES: Functional characterization of the erg1 mutant of ergosterol biosynthesis of Candida albicans. METHODS: We disrupted the ERG1 gene of C. albicans, which encodes squalene epoxidase (EC 1.14.99.7). Since the disruption of both alleles of ERG1 was lethal, the second allele of a heterozygous disruptant was placed under the control of a regulable promoter, MET3p, which is repressed by methionine and cysteine. RESULTS: The reverse-phase HPLC analysis of sterol, extracted from the conditional mutant strain, showed a total lack of ergosterol and instead accumulation of squalene. This imbalance in sterol composition led to defects in growth and increased susceptibilities to drugs including fluconazole, ketoconazole, cycloheximide, nystatin, amphotericin B and terbinafine. Reduced drug efflux activity of the erg1 mutant was associated with poor surface localization of Cdr1p, suggesting that enhanced passive diffusion and reduced efflux mediated by the ABC (ATP binding cassette) transporter Cdr1p increases drug susceptibility. Additionally, conditional erg1 mutant strains were unable to form hyphae in various media. CONCLUSIONS: Taken together, our results demonstrate that the absence of ergosterol, which is one of the constituents of membrane microdomains (rafts), has a direct effect on drug susceptibility and morphogenesis of C. albicans.