The high mortality rate of immunocompromised patients with fungal infections and the limited availability of highly efficacious and safe agents demand the development of new antifungal therapeutics. To rapidly discover such agents, we developed a high-throughput synergy screening (HTSS) strategy for novel microbial natural products. Specifically, a microbial natural product library was screened for hits that synergize the effect of a low dosage of ketoconazole (KTC) that alone shows little detectable fungicidal activity. Through screening of Ϸ20,000 microbial extracts, 12 hits were identified with broadspectrum antifungal activity. Seven of them showed little cytotoxicity against human hepatoma cells. Fractionation of the active extracts revealed beauvericin (BEA) as the most potent component, because it dramatically synergized KTC activity against diverse fungal pathogens by a checkerboard assay. Significantly, in our immunocompromised mouse model, combinations of BEA (0.5 mg/kg) and KTC (0.5 mg/kg) prolonged survival of the host infected with Candida parapsilosis and reduced fungal colony counts in animal organs including kidneys, lungs, and brains. Such an effect was not achieved even with the high dose of 50 mg/kg KTC. These data support synergism between BEA and KTC and thereby a prospective strategy for antifungal therapy.antifungal ͉ beauvericin ͉ ketoconazole

Candida albicans biofilms are structured microbial communities with high levels of drug resistance. Farnesol, a quorum-sensing molecule that inhibits hyphal formation in C. albicans, has been found to prevent biofilm formation by C. albicans. There is limited information, however, about the molecular mechanism of farnesol against biofilm formation. We used cDNA microarray analysis to identify the changes in the gene expression profile of a C. albicans biofilm inhibited by farnesol. Confocal scanning laser microscopy was used to visualize and confirm normal and farnesol-inhibited biofilms. A total of 274 genes were identified as responsive, with 104 genes up-regulated and 170 genes down-regulated. Independent reverse transcription-PCR analysis was used to confirm the important changes detected by microarray analysis. In addition to hyphal formation-associated genes (e.g., TUP1, CRK1, and PDE2), a number of other genes with roles related to drug resistance (e.g., FCR1 and PDR16), cell wall maintenance (e.g., CHT2 and CHT3), and iron transport (e.g., FTR2) were responsive, as were several genes encoding heat shock proteins (e.g., HSP70, HSP90, HSP104, CaMSI3, and SSA2). Further study of these differentially regulated genes is warranted to evaluate how they may be involved in C. albicans biofilm formation. Consistent with the down-regulation of the cell surface hydrophobicity-associated gene (CSH1), the water-hydrocarbon two-phase assay showed a decrease in cell surface hydrophobicity in the farnesol-treated group compared to that in the control group. Our data provide new insight into the molecular mechanism of farnesol against C. albicans biofilm formation.Candida albicans is a pleiomorphic fungus that can exist as either a commensal or an opportunistic pathogen and is capable of causing superficial to life-threatening infections. Predisposing factors for C. albicans infections include immunosuppressive therapy, antibiotic therapy, human immunodeficiency virus infection, diabetes, and old age. In addition, structured microbial communities attached to surfaces, commonly referred to as biofilms (29), have increasingly been found to be the sources of C. albicans infections. Biomaterials such as stents, shunts, prostheses (voice, heart valve, and knee prostheses), implants (lens and breast implants and dentures), endotracheal tubes, pacemakers, and various types of catheters have all been shown to facilitate C. albicans colonization and biofilm formation (1,20,27,28). It is estimated that biofilms might be involved in 65% of infections (30).Biofilms are spatially organized heterogeneous communities of cells embedded within an extrapolymeric matrix. In comparison with planktonic cells, biofilm cells display unique phenotypic traits (10, 11), the most outstanding of which is that they are notoriously resistant to both antimicrobial agents and host immune factors. Biofilm-associated infections are therefore difficult to treat because of their decreased susceptibilities to antimicrobial therapy. It is reported that C. alb...

Abstract-Endothelial inflammation plays a critical role in the development and progression of cardiovascular disease, albeit the mechanisms need to be fully elucidated. Nur77 is highly expressed in vascular endothelial cells (ECs) and plays a role in the regulation of cell proliferation and angiogenesis; its role in vascular inflammation, however, remains unknown. Treatment of human umbilical vein ECs (HUVECs) with tumor necrosis factor (TNF)-␣ substantially increased the transcription and protein expression of Nur77 in a dose and time-dependent manner, as determined by Northern blot and Western blot analysis. Adenovirus mediated overexpression of Nur77 markedly increased the intracellular levels of IB␣ by approximately 4-fold, whereas overexpression of dominant negative Nur77 (DN-Nur77), which lacks its transactivation domain, had no effect on IB␣ expression, suggesting that Nur77 is an important transcriptional factor in controlling IB␣ expression in ECs. Furthermore, overexpression of Nur77 significantly increased IB␣ promoter activity via directly binding to a Nur77 response element in the IB␣ promoter. Importantly, overexpression of Nur77, but not DN-Nur77, protected ECs against the TNF-␣-and interleukin-1␤-induced endothelial activation, as characterized by attenuation in the nuclear factor B activation, expression of adhesion molecules ICAM-1 and VCAM-1, and monocytic adherence to ECs.

In vitro interaction of fluconazole and berberine chloride was investigated against 40 fluconazole-resistant clinical isolates of Candida albicans. Synergism in fungistatic activity was found with the checkerboard microdilution assay. The findings of agar diffusion tests and time-kill curves confirmed the synergistic interaction, but no antagonistic action was observed.Candida albicans is the most common candidal pathogen, causing mucosal and invasive infections (7,9,23,29). With the increasing clinical use of fluconazole, a choice for the treatment of C. albicans infections (3, 5, 12), fluconazole-resistant isolates are occurring more frequently (2,19,28). Attempts have been made to cope with treatment failures by using combination therapy (14). However, contradictory results of either synergic or antagonistic actions in various antifungal combinations have been reported (8,11,16,27). As for fluconazoleresistant C. albicans, few data are available about the synergism of fluconazole with other antifungal agents (1,4,25).Berberine, a bioactive herbal ingredient, was demonstrated to have weak activity against C. albicans and C. glabrata (17,21,26). Recently, berberine was combined with amphotericin B to treat disseminated candidiasis in mice (10). To seek a novel combination therapy, we investigated the in vitro interaction of fluconazole and berberine chloride (BBR) against fluconazoleresistant clinical isolates of C. albicans.Strains and agents. Forty clinical isolates of fluconazoleresistant C. albicans were used in this study, and C. albicans ATCC 90028 was used as a quality control. Drugs prepared in dimethyl sulfoxide (DMSO) included fluconazole (PfizerRoerig Pharmaceuticals, New York, NY) and BBR (SigmaAldrich, St. Louis, MO).Checkerboard microdilution assay. Assays were performed on all 40 isolates according to methods of the CLSI (formerly NCCLS) (M27-A) (6, 18). The initial concentration of fungal suspension in RPMI 1640 medium was 10 3 CFU/ml, and the final concentrations ranged from 0.125 to 64 g/ml for fluconazole and 1 to 32 g/ml for BBR. Plates were incubated at 35°C for 24 h. Optical density was measured at 630 nm, and background optical densities were subtracted from that of each well. Each isolate was tested in triplicate. MIC 80 and MIC 50 were determined as the lowest concentration of the drugs (alone or in combination) that inhibited growth by 80% or 50%, respectively, compared with that of drug-free wells. The fractional inhibitory concentration (FIC) index is defined as the sum of the MIC of each drug when used in combination divided by the MIC of the drug used alone. Synergy and antagonism were defined by FIC indices of Յ0.5 and Ͼ4, respectively. An FIC index result of Ͼ0.5 but Յ4 was considered indifferent (20).Agar diffusion test. C. albicans 0304103 (one fluconazole-resistant isolate with a MIC of Ͼ32 g/ml for BBR) was tested by agar diffusion assay. A 100-l aliquot of 10 6 -CFU/ml suspension was spread uniformly onto the yeast extract-peptone-dextrose agar plate with or without 64 g/ml ...

CYP51 (Erg11) belongs to the cytochrome P450 monooxygenase (CYP) superfamily and mediates a crucial step of the synthesis of ergosterol, which is a fungal-specific sterol. It is also the target of azole drugs in clinical practice. In recent years, researches on fungal CYP51 have stepped into a new stage attributing to the discovery of crystal structures of the homologs in Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus . This review summarizes the functions, structures of fungal CYP51 proteins, and the inhibitors targeting these homologs. In particular, several drug-resistant mechanisms associated with the fungal CYP51s are introduced. The sequences and crystal structures of CYP51 proteins in different fungal species are also compared. These will provide new insights for the advancement of research on antifungal agents.