Fungi have emerged as the fourth most common pathogens isolated in nosocomial bloodstream infections, and Candida albicans is the most common human fungal pathogen. Only a few antibiotics are effective in the treatment of fungal infections. In addition, the repetition and lengthy duration of fluconazole therapy has led to an increased incidence of azole resistance and treatment failure associated with C. albicans. To investigate the mechanism of drug resistance and explore new targets to treat clinically resistant fungal pathogens, we examined the large-scale gene expression profile of two sets of matched fluconazole-susceptible and -resistant bloodstream C. albicans isolates from bone marrow transplanted (BMT) patients for the first time by microarray analysis. More than 198 differentially expressed genes were identified and they were confirmed and validated by RT-PCR independently. Not surprisingly, the resistant phenotype is associated with increased expression of CDR mRNA, as well as some common genes involved in drug resistance such as CaIFU5, CaRTA2 and CaIFD6. Meanwhile, some special functional groups of genes, including ATP binding cassette (ABC) transporter genes (IPF7530, CaYOR1, CaPXA1), oxidative stress response genes (CaALD5, CaGRP1, CaSOD2, IPF10565), copper transport and iron mobilization-related genes (CaCRD1/2, CaCTR1/2, CaCCC2, CaFET3) were found to be differentially expressed in the resistant isolates. Furthermore, among these differentially expressed genes, some co-regulated with CaCDR1, CaCDR2 and CaIFU5, such as CaPDR16 and CaIFD6, have a DRE-like element and may interact with TAC1 in the promoter region. These findings may shed light on mechanisms of azole resistance in C. albicans and clinical antifungal therapy.

14-3-3 proteins are a family of conserved dimeric molecules that bind to a range of cellular proteins involved in signal transduction and oncogenesis. Our solution of the crystal structure of 14-3-3ζrevealed a conserved amphipathic groove that may allow the association of 14-3-3 with diverse ligands (Liu, D., Bienkowska, J., Petosa, C., Collier, R. J., Fu, H., and Liddington, R. (1995) Nature 376, 191–194). Here, the contributions of three positively charged residues (Lys-49, Arg-56, and Arg-60) that lie in this Raf-binding groove were investigated. Two of the charge-reversal mutations greatly (K49E) or partially (R56E) decreased the interaction of 14-3-3ζwith Raf-1 kinase, whereas R60E showed only subtle effects on the binding. Interestingly, these mutations exhibited similar effects on the functional interaction of 14-3-3ζwith another target protein, exoenzyme S (ExoS), an ADPribosyltransferase from Pseudomonas aeruginosa. The EC50 values of 14-3-3ζrequired for ExoS activation increased by ;110-, 5-, and 2-fold for the K49E, R56E, and R60E mutants, respectively. The drastic reduction of 14-3-3ζ/ligand affinity by the K49E mutation is due to a local electrostatic effect, rather than the result of a gross structural alteration, as evidenced by partial proteolysis and circular dichroism analysis. This work identifies the first point mutation (K49E) that dramatically disrupts 14-3-3ζ/ligand interactions. The parallel effects of this single point mutation on both Raf-1 binding and ExoS activation strongly suggest that diverse associated proteins share a common structural binding determinant on 14-3-3ζ.

Apoptosis signal-regulating kinase 1 (ASK1) is a pivotal component of a signaling pathway induced by many death stimuli, including tumor necrosis factor a, Fas, and the anticancer drugs cisplatin and paclitaxel. Here we report that ASK1 proapoptotic activity is antagonized by association with 14-3-3 proteins. We found that ASK1 specifically bound 14-3-3 proteins via a site involving Ser-967 of ASK1. Interestingly, overexpression of 14-3-3 in HeLa cells blocked ASK1-induced apoptosis whereas disruption of the ASK1/14-3-3 interaction dramatically accelerated ASK1-induced cell death. Targeting of ASK1 by a 14-3-3-mediated survival pathway may provide a novel mechanism for the suppression of apoptosis.

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 ≈20,000 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.

Exoenzyme S (ExoS) is a mono-ADP-ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa. ExoS requires a eukaryotic factor, the 14-3-3 protein, for enzymatic activity. Here, two aspects of the activation of the ADP-ribosyltransferase activity of ExoS by 14-3-3 proteins are examined. Initial studies showed that several isoforms of 14-3-3, including β, ξ, η, σ, and ô, activated ExoS with similar efficiency. This implicates a conserved structure in 14-3-3 that contributes to the interaction between 14-3-3 and ExoS. One candidate structure is the conserved amphipathic groove that mediates the 14-3-3/Raf-1 interaction. The next series of experiments examined the role of individual amino acids of the amphipathic groove of 14-3-3ξ in ExoS activation and showed that ExoS activation required the basic residues lining the amphipathic groove of 14-3-3ξ　 without extensive involvement of the hydrophobic residues. Strikingly, mutations of Val-176 of 14-3-3ξ that disrupted its interaction with Raf-1 did not affect the binding and activation of ExoS by 14-3-3. Thus, ExoS selectively employs residues in the Raf-binding groove for its association with 14-3-3 proteins.