Chromosome-wide histone deacetylation by sirtuins prevents hyperactivation of DNA damage-induced signaling upon replicative stress.

The Saccharomyces cerevisiae genome encodes five sirtuins (Sir2 and Hst1-4), which constitute a conserved family of NAD-dependent histone deacetylases. Cells lacking any individual sirtuin display mild growth and gene silencing defects. However, hst3Δ hst4Δ double mutants are exquisitely sensitive to genotoxins, and hst3Δ hst4Δ sir2Δmutants are inviable. Our published data also indicate that pharmacological inhibition of sirtuins prevents growth of several fungal pathogens, although the biological basis is unclear. Here, we present genome-wide fitness assays conducted with nicotinamide (NAM), a pan-sirtuin inhibitor. Our data indicate that NAM treatment causes yeast to solicit specific DNA damage response pathways for survival, and that NAM-induced growth defects are mainly attributable to inhibition of Hst3 and Hst4 and consequent elevation of histone H3 lysine 56 acetylation (H3K56ac). Our results further reveal that in the presence of constitutive H3K56ac, the Slx4 scaffolding protein and PP4 phosphatase complex play essential roles in preventing hyperactivation of the DNA damage-response kinase Rad53 in response to spontaneous DNA damage caused by reactive oxygen species. Overall, our data support the concept that chromosome-wide histone deacetylation by sirtuins is critical to mitigate growth defects caused by endogenous genotoxins.

The RTA3 Gene, Encoding a Putative Lipid Translocase, Influences the Susceptibility of Candida albicans to Fluconazole.

The RTA3 gene, coding for a member of the Rta1p-like lipid-translocating exporter family, is coordinately upregulated with the ATP-binding cassette transporter genes CDR1 and CDR2 in azole-resistant clinical isolates of Candida albicans that carry activating mutations in the transcription factor Tac1p. We show here that deleting RTA3 in an azole-resistant clinical isolate carrying a Tac1p-activating mutation lowered fluconazole resistance by 2-fold, while overexpressing RTA3 in an azole-susceptible clinical isolate resulted in enhanced fluconazole tolerance associated with trailing growth in a liquid microtiter plate assay. We also demonstrate that an Rta3p-green fluorescent protein (GFP) fusion protein localizes predominantly to the plasma membrane, consistent with a putative function for Rta3p as a lipid translocase.

Positive regulation of the Candida albicans multidrug efflux pump Cdr1p function by phosphorylation of its N-terminal extension.

OBJECTIVES: Overexpression of ATP-binding cassette (ABC) transporters is a frequent cause of multidrug resistance in cancer cells and pathogenic microorganisms. One example is the Cdr1p transporter from the human fungal pathogen Candida albicans that belongs to the pleiotropic drug resistance (PDR) subfamily of ABC transporters found in fungi and plants. Cdr1p is overexpressed in several azole-resistant clinical isolates, causing azole efflux and treatment failure. Cdr1p appears as a doublet band in western blot analyses, suggesting that the protein is post-translationally modified. We investigated whether Cdr1p is phosphorylated and the function of this modification. METHODS: Phosphorylated residues were identified by MS. Their function was investigated by site-directed mutagenesis and expression of the mutants in a C. albicans endogenous system that exploits a hyperactive allele of the Tac1p transcription factor to drive high levels of Cdr1p expression. Fluconazole resistance was measured by microtitre plate and spot assays and transport activity by Nile red accumulation. RESULTS: We identified a cluster of seven phosphorylated amino acids in the N-terminal extension (NTE) of Cdr1p. Mutating all seven sites to alanine dramatically diminished the ability of Cdr1p to confer fluconazole resistance and transport Nile red, without affecting Cdr1p localization. Conversely, a Cdr1p mutant in which the seven amino acids were replaced by glutamate was able to confer high levels of fluconazole resistance and to export Nile red. CONCLUSIONS: Our results demonstrate that the NTE of Cdr1p is phosphorylated and that NTE phosphorylation plays a major role in regulating Cdr1p and possibly other PDR transporter function.

The bZIP transcription factor Rca1p is a central regulator of a novel CO2 sensing pathway in yeast.

Like many organisms the fungal pathogen Candida albicans senses changes in the environmental CO(2) concentration. This response involves two major proteins: adenylyl cyclase and carbonic anhydrase (CA). Here, we demonstrate that CA expression is tightly controlled by the availability of CO(2) and identify the bZIP transcription factor Rca1p as the first CO(2) regulator of CA expression in yeast. We show that Rca1p upregulates CA expression during contact with mammalian phagocytes and demonstrate that serine 124 is critical for Rca1p signaling, which occurs independently of adenylyl cyclase. ChIP-chip analysis and the identification of Rca1p orthologs in the model yeast Saccharomyces cerevisiae (Cst6p) point to the broad significance of this novel pathway in fungi. By using advanced microscopy we visualize for the first time the impact of CO(2) build-up on gene expression in entire fungal populations with an exceptional level of detail. Our results present the bZIP protein Rca1p as the first fungal regulator of carbonic anhydrase, and reveal the existence of an adenylyl cyclase independent CO(2) sensing pathway in yeast. Rca1p appears to regulate cellular metabolism in response to CO(2) availability in environments as diverse as the phagosome, yeast communities or liquid culture.

Nystatin nanosizing enhances in vitro and in vivo antifungal activity against Candida albicans.

OBJECTIVES: In this study, we developed a nanoparticulate nystatin formulation and performed a comparative evaluation against a commercial nystatin preparation of its in vitro and in vivo antifungal activities. METHODS: A nystatin nanosuspension was prepared from a commercially available suspension by wet-media milling. The nanosuspension was characterized for particle size by laser diffraction and assayed for content by HPLC. Its in vitro activity was evaluated against Candida albicans strains SC5314 and LAM-1 (12.5-5000 µg/mL) using an agar plate assay and its in vivo efficacy was evaluated using a murine model of oral candidiasis. Briefly, DBA/2 mice were immunosuppressed with cortisone acetate, orally infected with C. albicans strain LAM-1, and treated for 14 days with conventional nystatin suspension, nystatin nanosuspension or saline control. Efficacy endpoints were oral fungal burden, mouse survival and organ histopathology. A single-dose pharmacokinetic study was also performed. RESULTS: The median particle size of the nystatin suspension was reduced from 6577 to 137 nm. The HPLC assay demonstrated a nystatin content of 98.7% ±â€Š0.8% of the label claim. In vitro activity was superior to that of the conventional nystatin suspension at 100-5000 µg/mL concentrations. Beginning on day 3 of treatment, lower oral burdens of C. albicans were found in the nanosuspension group compared with the suspension and control groups. Mouse survival was also superior in the nanosuspension group. No systemic absorption was observed. CONCLUSIONS: Taken together, these data reveal that nanonization of nystatin provides a novel approach to enhancing its efficacy in the treatment of oral candidiasis.

Improved antifungal activity of itraconazole-loaded PEG/PLA nanoparticles.

Poly(ethylene glycol)/polylactic acid (PEG/PLA) nanoparticles (NPs) containing the hydrophobic antifungal itraconazole (ITZ) were developed to provide a controlled release pattern of ITZ as well as to improve its aqueous dispersibility and hence enhance its antifungal action. Two PEG/PLA copolymers (PEGylated PLA polymers) were used in this study; branched PEGylated polymer in which PEG was grafted on PLA backbone at 7% (mol/mol of lactic acid monomer), PEG7%-g-PLA, and multiblock copolymer of PLA and PEG, (PLA-PEG-PLA)n with nearly similar PEG insertion ratio and similar PEG chain length. ITZ-loaded PLA NPs were also prepared and included in this study as a control. ITZ-NPs were prepared from a 1 : 1 w/w blend of PLA and each PEGylated polymer either PEG7%-g-PLA or (PLA-PEG-PLA)n using an oil-in-water emulsion evaporation method. The NPs morphology, size and size distribution, zeta potential, loading efficiency, release profile and antifungal activity were characterized. All ITZ-NPs were nearly spherical with smooth surface and showed less aggregating tendency with a size range of 185-285 nm. All ITZ-NPs measured nearly neutral zeta potential values close to 0 mV. The % LE of ITZ was ∼94% for PEG7%-g-PLA NPs and ∼83% for (PLA-PEG-PLA)n at 15.3% w/w theoretical loading. PEG/PLA NPs were stable over time regarding size and size distribution and % ITZ loading efficiency (% LE). ITZ release showed an initial burst followed by a gradual release profile for ITZ-NPs over 5 days. (PLA-PEG-PLA)n NPs exhibited faster release rates than PEG7%-g-PLA NPs particularly at the last 2 days. Differential scanning calorimetry and powder X-ray diffractometry data confirmed that ITZ exists in an amorphous state or a solid solution state into the NPs matrix. Fourier transform infrared revealed the possibility of chemical interaction between ITZ and the NPs matrix polymer indicating the successful entrapment of ITZ inside the particles. In haemolysis test, ITZ-NPs caused mild haemolysis over the concentration range (5-20 µg/mL) compared to free ITZ, indicating better safety profile of ITZ-NPs. ITZ-loaded PEG/PLA NPs inhibited fungal growth more efficiently than either free ITZ or ITZ-loaded PLA NPs. Our results suggest that PEG/PLA-ITZ could be used efficiently as a nanocarrier to improve the aqueous dispersibility of ITZ, control its release over time and, thereby, enhance its antifungal efficacy.

Forward genetics in Candida albicans that reveals the Arp2/3 complex is required for hyphal formation, but not endocytosis.

Candida albicans is a diploid fungal pathogen lacking a defined complete sexual cycle, and thus has been refractory to standard forward genetic analysis. Instead, transcription profiling and reverse genetic strategies based on Saccharomyces cerevisiae have typically been used to link genes to functions. To overcome restrictions inherent in such indirect approaches, we have investigated a forward genetic mutagenesis strategy based on the UAU1 technology. We screened 4700 random insertion mutants for defects in hyphal development and linked two new genes (ARP2 and VPS52) to hyphal growth. Deleting ARP2 abolished hyphal formation, generated round and swollen yeast phase cells, disrupted cortical actin patches and blocked virulence in mice. The mutants also showed a global lack of induction of hyphae-specific genes upon the yeast-to-hyphae switch. Surprisingly, both arp2 Delta/Delta and arp2 Delta/Delta arp3 Delta/Delta mutants were still able to endocytose FM4-64 and Lucifer Yellow, although as shown by time-lapse movies internalization of FM4-64 was somewhat delayed in mutant cells. Thus the non-essential role of the Arp2/3 complex discovered by forward genetic screening in C. albicans showed that uptake of membrane components from the plasma membrane to vacuolar structures is not dependent on this actin nucleating machinery.

Regulation of efflux pump expression and drug resistance by the transcription factors Mrr1, Upc2, and Cap1 in Candida albicans.

Constitutive overexpression of the Mdr1 efflux pump is an important mechanism of acquired drug resistance in the yeast Candida albicans. The zinc cluster transcription factor Mrr1 is a central regulator of MDR1 expression, but other transcription factors have also been implicated in MDR1 regulation. To better understand how MDR1-mediated drug resistance is achieved in this fungal pathogen, we studied the interdependence of Mrr1 and two other MDR1 regulators, Upc2 and Cap1, in the control of MDR1 expression. A mutated, constitutively active Mrr1 could upregulate MDR1 and confer drug resistance in the absence of Upc2 or Cap1. On the other hand, Upc2 containing a gain-of-function mutation only slightly activated the MDR1 promoter, and this activation depended on the presence of a functional MRR1 gene. In contrast, a C-terminally truncated, activated form of Cap1 could upregulate MDR1 in a partially Mrr1-independent fashion. The induction of MDR1 expression by toxic chemicals occurred independently of Upc2 but required the presence of Mrr1 and also partially depended on Cap1. Transcriptional profiling and in vivo DNA binding studies showed that a constitutively active Mrr1 binds to and upregulates most of its direct target genes in the presence or absence of Cap1. Therefore, Mrr1 and Cap1 cooperate in the environmental induction of MDR1 expression in wild-type C. albicans, but gain-of-function mutations in either of the two transcription factors can independently mediate efflux pump overexpression and drug resistance.

Depletion of the cullin Cdc53p induces morphogenetic changes in Candida albicans.

Candida albicans is an important opportunistic human fungal pathogen that can cause both mucosal and systemic infections in immunocompromised patients. Critical for the virulence of C. albicans is its ability to undergo a morphological transition from yeast to hyphal growth mode. Proper induction of filamentation is dependent on the ubiquitination pathway, which targets proteins for proteasome-mediated protein degradation or activates them for signaling events. In the present study, we evaluated the role of ubiquitination in C. albicans by impairing the function of the major ubiquitin-ligase complex SCF. This was done by depleting its backbone, the cullin Cdc53p (orf19.1674), using a tetracycline downregulatable promoter system. Cdc53p-depleted cells displayed an invasive phenotype and constitutive filamentation under conditions favoring yeast growth mode, both on solid and in liquid media. In addition, these cells exhibited an early onset of cell death, as judged from propidium iodide staining, suggesting that CDC53 is an essential gene in C. albicans. To identify Cdc53p-dependent pathways in C. albicans, a genome-wide expression analysis was carried out that revealed a total of 425 differentially expressed genes (fold change, >or=2; P

Identification of the Candida albicans Cap1p regulon.

Cap1p, a transcription factor of the basic region leucine zipper family, regulates the oxidative stress response (OSR) in Candida albicans. Alteration of its C-terminal cysteine-rich domain (CRD) results in Cap1p nuclear retention and transcriptional activation. To better understand the function of Cap1p in C. albicans, we used genome-wide location profiling (chromatin immunoprecipitation-on-chip) to identify its transcriptional targets in vivo. A triple-hemagglutinin (HA(3)) epitope was introduced at the C terminus of wild-type Cap1p (Cap1p-HA(3)) or hyperactive Cap1p with an altered CRD (Cap1p-CSE-HA(3)). Location profiling using whole-genome oligonucleotide tiling microarrays identified 89 targets bound by Cap1p-HA(3) or Cap1p-CSE-HA(3) (the binding ratio was at least twofold; P < or = 0.01). Strikingly, Cap1p binding was detected not only at the promoter region of its target genes but also at their 3' ends and within their open reading frames, suggesting that Cap1p may associate with the transcriptional or chromatin remodeling machinery to exert its activity. Overrepresented functional groups of the Cap1p targets (P < or = 0.02) included 11 genes involved in the OSR (CAP1, GLR1, TRX1, SOD1, CAT1, and others), 13 genes involved in response to drugs (PDR16, MDR1, FLU1, YCF1, FCR1, and others), 4 genes involved in phospholipid transport (PDR16, GIT1, RTA2, and orf19.932), and 3 genes involved in the regulation of nitrogen utilization (GST3, orf19.2693, and orf19.3121), suggesting that Cap1p has other cellular functions in addition to the OSR. Bioinformatic analyses of the bound sequences suggest that Cap1p recognizes the DNA motif 5'-MTKASTMA. Finally, transcriptome analyses showed that increased expression generally accompanies Cap1p binding at its targets, indicating that Cap1p functions as a transcriptional activator.

Relative contributions of the Candida albicans ABC transporters Cdr1p and Cdr2p to clinical azole resistance.

Candida albicans frequently develops resistance to treatment with azole drugs due to the acquisition of gain-of-function mutations in the transcription factor Tac1p. Tac1p hyperactivation in azole-resistant isolates results in the constitutive overexpression of several genes, including CDR1 and CDR2, which encode two homologous transporters of the ATP-binding cassette family. Functional studies of Cdr1p and Cdr2p have been carried out so far by heterologous expression in the budding yeast Saccharomyces cerevisiae and by gene deletion or overexpression in azole-sensitive C. albicans strains in which CDR1 expression is low and CDR2 expression is undetectable. Thus, the direct demonstration that CDR1 and CDR2 overexpression causes azole resistance in clinical strains is still lacking, as is our knowledge of the relative contribution of each transporter to clinical azole resistance. In the present study, we used the SAT1 flipper system to delete the CDR1 and CDR2 genes from clinical isolate 5674. This strain is resistant to several azole derivatives due to a strong hyperactive mutation in Tac1p and expresses high levels of Cdr1p and Cdr2p. We found that deleting CDR1 had a major effect, reducing resistance to fluconazole (FLC), ketoconazole (KTC), and itraconazole (ITC) by 6-, 4-, and 8-fold, respectively. Deleting CDR2 had a much weaker effect, reducing FLC or KTC resistance by 1.5-fold, and had no effect on ITC resistance. These results demonstrate that Cdr1p is a major determinant of azole resistance in strain 5674 and potentially in other clinical strains overexpressing Cdr1p and Cdr2p, while Cdr2p plays a more minor role.

Genomewide location analysis of Candida albicans Upc2p, a regulator of sterol metabolism and azole drug resistance.

Upc2p, a transcription factor of the zinc cluster family, is an important regulator of sterol biosynthesis and azole drug resistance in Candida albicans. To better understand Upc2p function in C. albicans, we used genomewide location profiling to identify the transcriptional targets of Upc2p in vivo. A triple hemagglutinin epitope, introduced at the C terminus of Upc2p, conferred a gain-of-function effect on the fusion protein. Location profiling identified 202 bound promoters (P < 0.05). Overrepresented functional groups of genes whose promoters were bound by Upc2p included 12 genes involved in ergosterol biosynthesis (NCP1, ERG11, ERG2, and others), 18 genes encoding ribosomal subunits (RPS30, RPL32, RPL12, and others), 3 genes encoding drug transporters (CDR1, MDR1, and YOR1), 4 genes encoding transcription factors (INO2, ACE2, SUT1, and UPC2), and 6 genes involved in sulfur amino acid metabolism (MET6, SAM2, SAH1, and others). Bioinformatic analyses suggested that Upc2p binds to the DNA motif 5'-VNCGBDTR that includes the previously characterized Upc2p binding site 5'-TCGTATA. Northern blot analysis showed that increased binding correlates with increased expression for the analyzed Upc2p targets (ERG11, MDR1, CDR1, YOR1, SUT1, SMF12, and CBP1). The analysis of ERG11, MDR1, and CDR1 transcripts in wild-type and upc2Delta/upc2Delta strains grown under Upc2p-activating conditions (lovastatin treatment and hypoxia) showed that Upc2p regulates its targets in a complex manner, acting as an activator or as a repressor depending upon the target and the activating condition. Taken together, our results indicate that Upc2p is a key regulator of ergosterol metabolism. They also suggest that Upc2p may contribute to azole resistance by regulating the expression of drug efflux pump-encoding genes in addition to ergosterol biosynthesis genes.

Intestinal apolipoprotein A-IV gene transcription is controlled by two hormone-responsive elements: a role for hepatic nuclear factor-4 isoforms.

In the small intestine, the expression of the apolipoprotein (apo) C-III and A-IV genes is restricted to the enterocytes of the villi. We have previously shown that, in transgenic mice, specific expression of the human apo C-III requires a hormone-responsive element (HRE) located in the distal region of the human apoA-IV promoter. This HRE binds the hepatic nuclear factors (HNF)-4alpha and gamma. Here, intraduodenal injections in mice and infections of human enterocytic Caco-2/TC7 cells with an adenovirus expressing a dominant-negative form of HNF-4alpha repress the expression of the apoA-IV gene, demonstrating that HNF-4 controls the apoA-IV gene expression in enterocytes. We show that HNF-4alpha and gamma functionally interact with a second HRE present in the proximal region of the human apoA-IV promoter. New sets of transgenic mice expressing mutated forms of the promoter, combined with the human apo C-III enhancer, demonstrate that, whereas a single HRE is sufficient to reproduce the physiological cephalo-caudal gradient of apoA-IV gene expression, both HREs are required for expression that is restricted to villi. The combination of multiple HREs may specifically recruit regulatory complexes associating HNF-4 and either coactivators in villi or corepressors in crypts.

Econazole imprinted textiles with antifungal activity.

In this work, we propose pharmaceutical textiles imprinted with lipid microparticles of Econazole nitrate (ECN) as a mean to improve patient compliance while maintaining drug activity. Lipid microparticles were prepared and characterized by laser diffraction (3.5±0.1 µm). Using an optimized screen-printing method, microparticles were deposited on textiles, as observed by scanning electron microscopy. The drug content of textiles (97±3 µg/cm(2)) was reproducible and stable up to 4 months storage at 25 °C/65% Relative Humidity. Imprinted textiles exhibited a thermosensitive behavior, as witnessed by a fusion temperature of 34.8 °C, which enabled a larger drug release at 32 °C (temperature of the skin) than at room temperature. In vitro antifungal activity of ECN textiles was compared to commercial 1% (wt/wt) ECN cream Pevaryl®. ECN textiles maintained their antifungal activity against a broad range of Candida species as well as major dermatophyte species. In vivo, ECN textiles also preserved the antifungal efficacy of ECN on cutaneous candidiasis infection in mice. Ex vivo percutaneous absorption studies demonstrated that ECN released from pharmaceutical textiles concentrated more in the upper skin layers, where the fungal infections develop, as compared to dermal absorption of Pevaryl®. Overall, these results showed that this technology is promising to develop pharmaceutical garments textiles for the treatment of superficial fungal infections.

An inter-laboratory study to evaluate the effects of medium composition on the differentiation and barrier function of Caco-2 cell lines.

Differentiated human intestinal Caco-2 cells are frequently used in toxicology and pharmacology as in vitro models for studies on intestinal barrier functions. Since several discrepancies exist among the different lines and clones of Caco-2 cells, comparison of the results obtained and optimisation of models for use for regulatory purposes are particularly difficult, especially with respect to culture conditions and morphological and biochemical parameters. An inter-laboratory study has been performed on the parental cell line and on three clonal Caco-2 cell lines, with the aim of standardising the culture conditions and identifying the best cell line with respect to parameters relevant to barrier integrity, namely, trans-epithelial electrical resistance (TEER) and mannitol passage, and of epithelial differentiation (alkaline phosphatase activity). Comparison of the cell lines maintained in traditional serum-supplemented culture medium or in defined medium, containing insulin, transferrin, selenium and lipids, showed that parameter performance was better and more reproducible with the traditional medium. The maintenance of the cell lines for 15 days in culture was found to be sufficient for the development of barrier properties, but not for full epithelial differentiation. Caco-2/TC7 cells performed better than the other three cell lines, both in terms of reproducibility and performance, exhibiting low TEER and mannitol passage, and high alkaline phosphatase activity.

Comparative xylose metabolism among the Ascomycetes C. albicans, S. stipitis and S. cerevisiae.

The ascomycetes Candida albicans, Saccharomyces cerevisiae and Scheffersomyces stipitis metabolize the pentose sugar xylose very differently. S. cerevisiae fails to grow on xylose, while C. albicans can grow, and S. stipitis can both grow and ferment xylose to ethanol. However, all three species contain highly similar genes that encode potential xylose reductases and xylitol dehydrogenases required to convert xylose to xylulose, and xylulose supports the growth of all three fungi. We have created C. albicans strains deleted for the xylose reductase gene GRE3, the xylitol dehydrogenase gene XYL2, as well as the gre3 xyl2 double mutant. As expected, all the mutant strains cannot grow on xylose, while the single gre3 mutant can grow on xylitol. The gre3 and xyl2 mutants are efficiently complemented by the XYL1 and XYL2 from S. stipitis. Intriguingly, the S. cerevisiae GRE3 gene can complement the Cagre3 mutant, while the ScSOR1 gene can complement the Caxyl2 mutant, showing that S. cerevisiae contains the enzymatic capacity for converting xylose to xylulose. In addition, the gre3 xyl2 double mutant of C. albicans is effectively rescued by the xylose isomerase (XI) gene of either Piromyces or Orpinomyces, suggesting that the XI provides an alternative to the missing oxido-reductase functions in the mutant required for the xylose-xylulose conversion. Overall this work suggests that C. albicans strains engineered to lack essential steps for xylose metabolism can provide a platform for the analysis of xylose metabolism enzymes from a variety of species, and confirms that S. cerevisiae has the genetic potential to convert xylose to xylulose, although non-engineered strains cannot proliferate on xylose as the sole carbon source.

Modulation of histone H3 lysine 56 acetylation as an antifungal therapeutic strategy.

Candida albicans is a major fungal pathogen that causes serious systemic and mucosal infections in immunocompromised individuals. In yeast, histone H3 Lys56 acetylation (H3K56ac) is an abundant modification regulated by enzymes that have fungal-specific properties, making them appealing targets for antifungal therapy. Here we demonstrate that H3K56ac in C. albicans is regulated by the RTT109 and HST3 genes, which respectively encode the H3K56 acetyltransferase (Rtt109p) and deacetylase (Hst3p). We show that reduced levels of H3K56ac sensitize C. albicans to genotoxic and antifungal agents. Inhibition of Hst3p activity by conditional gene repression or nicotinamide treatment results in a loss of cell viability associated with abnormal filamentous growth, histone degradation and gross aberrations in DNA staining. We show that genetic or pharmacological alterations in H3K56ac levels reduce virulence in a mouse model of C. albicans infection. Our results demonstrate that modulation of H3K56ac is a unique strategy for treatment of C. albicans and, possibly, other fungal infections.

Epidermal growth factor receptor is involved in enterocyte anoikis through the dismantling of E-cadherin-mediated junctions.

Enterocytes of the intestinal epithelium are continually regenerated. They arise from precursor cells in crypts, migrate along villi, and finally die, 3-4 days later, when they reach the villus apex. Their death is thought to occur by anoikis, a form of apoptosis induced by cell detachment, but the mechanism of this process remains poorly understood. We have previously shown that a key event in the onset of anoikis in normal enterocytes detached from the basal lamina is the disruption of adherens junctions mediated by E-cadherin (Fouquet S, Lugo-Martinez VH, Faussat AM, Renaud F, Cardot P, Chambaz J, Pincon-Raymond M, Thenet S. J Biol Chem 279: 43061-43069, 2004). Here we have further investigated the mechanisms underlying this disassembly of the adherens junctions. We show that disruption of the junctions occurs through endocytosis of E-cadherin and that this process depends on the tyrosine-kinase activity of the epidermal growth factor receptor (EGFR). Activation of EGFR was detected in detached enterocytes before E-cadherin disappearance. Specific inhibition of EGFR by tyrphostin AG-1478 maintained E-cadherin and its cytoplasmic partners beta- and alpha-catenin at cell-cell contacts and decreased anoikis. Finally, EGFR activation was evidenced in the intestinal epithelium in vivo, in rare individual cells, which were shown to lose their interactions with the basal lamina. We conclude that EGFR is activated as enterocytes become detached from the basal lamina, and that this mechanism contributes to the disruption of E-cadherin-dependent junctions leading to anoikis. This suggests that EGFR participates in the physiological elimination of the enterocytes.

Hepatocyte nuclear factor 4alpha, a key factor for homeostasis, cell architecture, and barrier function of the adult intestinal epithelium.

Hepatocyte nuclear factor 4alpha (HNF-4alpha) is a transcription factor which is highly expressed in the intestinal epithelium from duodenum to colon and from crypt to villus. The homeostasis of this constantly renewing epithelium relies on an integrated control of proliferation, differentiation, and apoptosis, as well as on the functional architecture of the epithelial cells. In order to determine the consequences of HNF-4alpha loss in the adult intestinal epithelium, we used a tamoxifen-inducible Cre-loxP system to inactivate the Hnf-4a gene. In the intestines of adult mice, loss of HNF-4alpha led to an increased proliferation in crypts and to an increased expression of several genes controlled by the Wnt/beta-catenin system. This control of the Wnt/beta-catenin signaling pathway by HNF-4alpha was confirmed in vitro. Cell lineage was affected, as indicated by an increased number of goblet cells and an impairment of enterocyte and enteroendocrine cell maturation. In the absence of HNF-4alpha, cell-cell junctions were destabilized and paracellular intestinal permeability increased. Our results showed that HNF-4alpha modulates Wnt/beta-catenin signaling and controls intestinal epithelium homeostasis, cell function, and cell architecture. This study indicates that HNF-4alpha regulates the intestinal balance between proliferation and differentiation, and we hypothesize that it might act as a tumor suppressor.

The cellular prion protein PrP(c) is involved in the proliferation of epithelial cells and in the distribution of junction-associated proteins.

BACKGROUND: The physiological function of the ubiquitous cellular prion protein, PrP(c), is still under debate. It was essentially studied in nervous system, but poorly investigated in epithelial cells. We previously reported that PrP(c) is targeted to cell-cell junctions of polarized epithelial cells, where it interacts with c-Src. METHODOLOGY/FINDINGS: We show here that, in cultured human enterocytes and in intestine in vivo, the mature PrP(c) is differentially targeted either to the nucleus in dividing cells or to cell-cell contacts in polarized/differentiated cells. By proteomic analysis, we demonstrate that the junctional PrP(c) interacts with cytoskeleton-associated proteins, such as gamma- and beta-actin, alpha-spectrin, annexin A2, and with the desmosome-associated proteins desmoglein, plakoglobin and desmoplakin. In addition, co-immunoprecipitation experiments revealed complexes associating PrP(c), desmoglein and c-Src in raft domains. Through siRNA strategy, we show that PrP(c) is necessary to complete the process of epithelial cell proliferation and for the sub-cellular distribution of proteins involved in cell architecture and junctions. Moreover, analysis of the architecture of the intestinal epithelium of PrP(c) knock-out mice revealed a net decrease in the size of desmosomal junctions and, without change in the amount of BrdU incorporation, a shortening of the length of intestinal villi. CONCLUSIONS/SIGNIFICANCE: From these results, PrP(c) could be considered as a new partner involved in the balance between proliferation and polarization/differentiation in epithelial cells.