Thomas Lettner

UME6 is a crucial downstream target of other transcriptional regulators of true hyphal development in Candida albicans.

True hyphal growth of Candida albicans can be induced by several environmental conditions and contributes significantly to the high virulence of this pathogenic fungus. The transcriptional network that governs hyphal morphogenesis is complex, depends on several regulators and is not completely understood. Recently, CaUME6, a homolog of the Saccharomyces cerevisiae UME6 gene, has been shown to be required for hyphal elongation. In the present study, the C. albicans ume6Delta strain showed a complete defect in hyphae formation under all the growth conditions tested. UME6 was repressed by the Nrg1-Tup1 repressor in yeast-form cells but NRG1 was not repressed by Ume6p under hyphal growth conditions. Wild-type UME6 expression depended on each hyphal regulator tested, and ectopic UME6 expression in efg1Delta, cph1Delta and ras1Delta cells rescued the hyphal defects of these mutants under some hyphal growth conditions. Thus, UME6 is a common downstream target of regulators promoting hyphal development. Ectopic UME6 expression promoted both germ tube formation and hyphal elongation. The expression of all hyphae-specific genes investigated depended on UME6 expression. A model for transcriptional regulation of hyphal development and the role of Ume6p is proposed.

K14 mRNA reprogramming for dominant epidermolysis bullosa simplex

The major challenge to a successful gene therapy of autosomal dominant genetic diseases is a highly efficient and specific knock-down or repair of the disease-causing allele. In epidermolysis bullosa simplex-type Dowling-Meara (EBS-DM), a single amino acid exchange in exon 1 of the keratin 14 gene (K14) triggers a severe skin phenotype, characterized by blistering of the skin and mucous membranes after minor trauma. We chose spliceosome-mediated RNA trans-splicing to specifically replace exons 1-7 of the K14 gene. In this approach, the mutated coding region is replaced by an RNA-trans-splicing molecule (RTM) that incorporates a binding domain (BD) and the wild-type sequence of K14. Since the BD is crucial for the trans-splicing functionality, we developed a fluorescence-based RTM screen consisting of an RTM library containing random BDs. Co-transfection of the library with a target molecule enabled us to identify highly functional RTMs. The best RTMs were adapted for endogenous trans-splicing in an EBS-DM patient cell line. In this cell line, we were able to detect functional, efficient and correct trans-splicing on RNA and protein levels. Scratch assays confirmed phenotypic reversion in vitro. Owing to concomitant knock-down and repair of the mutated allele, we assume that trans-splicing is a promising tool for the treatment of autosomal dominant genetic disease.

The pathogenetic role of IL-1β in severe epidermolysis bullosa simplex.

TO THE EDITOR Dominantly inherited epidermolysis bullosa simplex type Dowling-Meara (EBS-DM) is caused by mutations in either the keratin 5 (K5) or the keratin 14 (K14) gene, which encode the major components of the intermediate filament (IF) network in basal keratinocytes. In healthy keratinocytes, heterodimers of K5 and K14 polymerize to form a cytoplasmic network extending to the cellular periphery and imparting stability to the basal layer of the stratified epithelia. In EBS-DM, causative mutations lead to disintegration and collapse of the IF network, which manifests cytologically as electron-dense aggregates at the periphery of the keratinocyte cytoplasm (Morley et al., 2003). EBS-DM cells exhibit increased fragility to mechanical stress as well as to heat and osmotic shock, making DowlingMeara the most severe subtype of EBS. As a consequence, EBS-DM patients suffer from blistering and wounding of the skin and mucous membranes after even minor trauma (Stephens LETTERS TO THE EDITOR

Cut and Paste: Efficient Homology-Directed Repair of a Dominant Negative KRT14 Mutation via CRISPR/Cas9 Nickases

With the ability to induce rapid and efficient repair of disease-causing mutations, CRISPR/Cas9 technology is ideally suited for gene therapy approaches for recessively and dominantly inherited monogenic disorders. In this study, we have corrected a causal hotspot mutation in exon 6 of the keratin 14 gene (KRT14) that results in generalized severe epidermolysis bullosa simplex (EBS-gen sev), using a double-nicking strategy targeting intron 7, followed by homology-directed repair (HDR). Co-delivery into EBS keratinocytes of a Cas9 D10A nickase (Cas9n), a predicted single guide RNA pair specific for intron 7, and a minicircle donor vector harboring the homology donor template resulted in a recombination efficiency of >30% and correction of the mutant KRT14 allele. Phenotypic correction of EBS-gen sev keratinocytes was demonstrated by immunofluorescence analysis, revealing the absence of disease-associated K14 aggregates within the cytoplasm. We achieved a promising safety profile for the CRISPR/Cas9 double-nicking approach, with no detectable off-target activity for a set of predicted off-target genes as confirmed by next generation sequencing. In conclusion, we demonstrate a highly efficient and specific gene-editing approach for KRT14, offering a causal treatment option for EBS.

Candida albicans AGE3, the Ortholog of the S. cerevisiae ARF-GAP-Encoding Gene GCS1, Is Required for Hyphal Growth and Drug Resistance

Background Hyphal growth and multidrug resistance of C. albicans are important features for virulence and antifungal therapy of this pathogenic fungus. Methodology/Principal Findings Here we show by phenotypic complementation analysis that the C. albicans gene AGE3 is the functional ortholog of the yeast ARF-GAP-encoding gene GCS1. The finding that the gene is required for efficient endocytosis points to an important functional role of Age3p in endosomal compartments. Most C. albicans age3Δ mutant cells which grew as cell clusters under yeast growth conditions showed defects in filamentation under different hyphal growth conditions and were almost completely disabled for invasive filamentous growth. Under hyphal growth conditions only a fraction of age3Δ cells shows a wild-type-like polarization pattern of the actin cytoskeleton and lipid rafts. Moreover, age3Δ cells were highly susceptible to several unrelated toxic compounds including antifungal azole drugs. Irrespective of the AGE3 genotype, C-terminal fusions of GFP to the drug efflux pumps Cdr1p and Mdr1p were predominantly localized in the plasma membrane. Moreover, the plasma membranes of wild-type and age3Δ mutant cells contained similar amounts of Cdr1p, Cdr2p and Mdr1p. Conclusions/Significance The results indicate that the defect in sustaining filament elongation is probably caused by the failure of age3Δ cells to polarize the actin cytoskeleton and possibly of inefficient endocytosis. The high susceptibility of age3Δ cells to azoles is not caused by inefficient transport of efflux pumps to the cell membrane. A possible role of a vacuolar defect of age3Δ cells in drug susceptibility is proposed and discussed. In conclusion, our study shows that the ARF-GAP Age3p is required for hyphal growth which is an important virulence factor of C. albicans and essential for detoxification of azole drugs which are routinely used for antifungal therapy. Thus, it represents a promising antifungal drug target.

MMP-9 and CXCL8/IL-8 Are Potential Therapeutic Targets in Epidermolysis Bullosa Simplex

Epidermolysis bullosa refers to a group of genodermatoses that affects the integrity of epithelial layers, phenotypically resulting in severe skin blistering. Dowling-Meara, the major subtype of epidermolysis bullosa simplex, is inherited in an autosomal dominant manner and can be caused by mutations in either the keratin-5 (K5) or the keratin-14 (K14) gene. Currently, no therapeutic approach is known, and the main objective of this study was to identify novel therapeutic targets. We used microarray analysis, semi-quantitative real-time PCR, western blot and ELISA to identify differentially regulated genes in two K14 mutant cell lines carrying the mutations K14 R125P and K14 R125H, respectively. We found kallikrein-related peptidases and matrix metalloproteinases to be upregulated. We also found elevated expression of chemokines, and we observed deregulation of the Cdc42 pathway as well as aberrant expression of cytokeratins and junction proteins. We further demonstrated, that expression of these genes is dependent on interleukin-1 β signaling. To evaluate these data in vivo we analysed the blister fluids of epidermolysis bullosa simplex patients vs. healthy controls and identified matrix metalloproteinase-9 and the chemokine CXCL8/IL-8 as potential therapeutic targets.

Threonine 150 Phosphorylation of Keratin 5 Is Linked to Epidermolysis Bullosa Simplex and Regulates Filament Assembly and Cell Viability

A characteristic feature of the skin blistering disease epidermolysis bullosa simplex is keratin filament (KF) network collapse caused by aggregation of the basal epidermal keratin type II (KtyII) K5 and its type I partner keratin 14 (K14). Here, we examine the role of keratin phosphorylation in KF network rearrangement and cellular functions. We detect phosphorylation of the K5 head domain residue T150 in cytoplasmic epidermolysis bullosa simplex granules containing R125C K14 mutants. Expression of phosphomimetic T150D K5 mutants results in impaired KF formation in keratinocytes. The phenotype is enhanced upon combination with other phosphomimetic K5 head domain mutations. Remarkably, introduction of T150D K5 mutants into KtyII-lacking (KtyII-/-) keratinocytes prevents keratin network formation altogether. In contrast, phosphorylation-deficient T150A K5 leads to KFs with reduced branching and turnover. Assembly of T150D K5 is arrested at the heterotetramer stage coinciding with increased heat shock protein association. Finally, reduced cell viability and elevated response to stressors is noted in T150 mutant cells. Taken together, our findings identify T150 K5 phosphorylation as an important determinant of KF network formation and function with a possible role in epidermolysis bullosa simplex pathogenesis.A characteristic feature of the skin blistering disease epidermolysis bullosa simplex is keratin filament (KF) network collapse caused by aggregation of the basal epidermal keratin type II (KtyII) K5 and its type I partner keratin 14 (K14). Here, we examine the role of keratin phosphorylation in KF network rearrangement and cellular functions. We detect phosphorylation of the K5 head domain residue T150 in cytoplasmic epidermolysis bullosa simplex granules containing R125C K14 mutants. Expression of phosphomimetic T150D K5 mutants results in impaired KF formation in keratinocytes. The phenotype is enhanced upon combination with other phosphomimetic K5 head domain mutations. Remarkably, introduction of T150D K5 mutants into KtyII-lacking (KtyII–/–) keratinocytes prevents keratin network formation altogether. In contrast, phosphorylation-deficient T150A K5 leads to KFs with reduced branching and turnover. Assembly of T150D K5 is arrested at the heterotetramer stage coinciding with increased heat shock protein association. Finally, reduced cell viability and elevated response to stressors is noted in T150 mutant cells. Taken together, our findings identify T150 K5 phosphorylation as an important determinant of KF network formation and function with a possible role in epidermolysis bullosa simplex pathogenesis.

Increased levels of matrix metalloproteinase‐9 and interleukin‐8 in blister fluids of dystrophic and junctional epidermolysis bullosa patients

ing a Ca pump, cause Darier disease.Nat Genet 1999; 21: 271–277. 9 Ruiz-Perez VL, Carter SA, Healy E et al. ATP2A2 mutations in Darier’s disease: variant cutaneous phenotypes are associated with missense mutations, but neuropsychiatric features are independent of mutation class. Hum Mol Genet 1999; 8: 1621–1630. 10 Ikeda S, Mayuzumi N, Shigihara T, Epstein EH Jr, Goldsmith LA, Ogawa H. Mutations in ATP2A2 in patients with Darier’s disease. J Invest Dermatol 2003; 121: 475–477.

Diacerein orphan drug development for epidermolysis bullosa simplex: A phase 2/3 randomized, placebo-controlled, double-blind clinical trial

Background: Epidermolysis bullosa simplex (EBS) is a rare genetic, blistering skin disease for which there is no cure. Treatments that address the pathophysiology of EBS are needed. Objective: Compare the impact of 1% diacerein cream with placebo in reducing the number of blisters in EBS. Methods: In a randomized, placebo‐controlled, phase 2/3 trial we used a 1% diacerein topical formulation to treat defined skin areas in 17 patients. In a 2‐period crossover trial, patients were randomized to either placebo or diacerein for a 4‐week treatment and a 3‐month follow‐up in period 1. After a washout, patients were crossed over during period 2. The prespecified primary end point was the proportion of patients with a reduction of number of blisters by more than 40% from baseline in selected areas over the treatment episode. Results: Of the patients receiving diacerein, 86% in episode 1 and 37.5% in episode 2 met the primary end point (vs 14% and 17% with placebo, respectively). This effect was still significant after the follow‐up. Changes in absolute blister numbers were significant for the diacerein group only. No adverse effects were observed. Limitations: Low patient numbers and no invasive data acquisition because of clinical burden in children. Conclusion: This trial provides evidence of the impact of 1% diacerein cream in the treatment of EBS.

Threonine 150 phosphorylation of keratin 5 is linked to EBS and regulates filament assembly, cell cycle and oxidative stress response.

A characteristic feature of the skin blistering disease epidermolysis bullosa simplex is keratin filament (KF) network collapse caused by aggregation of the basal epidermal keratin type II (KtyII) K5 and its type I partner keratin 14 (K14). Here, we examine the role of keratin phosphorylation in KF network rearrangement and cellular functions. We detect phosphorylation of the K5 head domain residue T150 in cytoplasmic epidermolysis bullosa simplex granules containing R125C K14 mutants. Expression of phosphomimetic T150D K5 mutants results in impaired KF formation in keratinocytes. The phenotype is enhanced upon combination with other phosphomimetic K5 head domain mutations. Remarkably, introduction of T150D K5 mutants into KtyII-lacking (KtyII-/-) keratinocytes prevents keratin network formation altogether. In contrast, phosphorylation-deficient T150A K5 leads to KFs with reduced branching and turnover. Assembly of T150D K5 is arrested at the heterotetramer stage coinciding with increased heat shock protein association. Finally, reduced cell viability and elevated response to stressors is noted in T150 mutant cells. Taken together, our findings identify T150 K5 phosphorylation as an important determinant of KF network formation and function with a possible role in epidermolysis bullosa simplex pathogenesis.A characteristic feature of the skin blistering disease epidermolysis bullosa simplex is keratin filament (KF) network collapse caused by aggregation of the basal epidermal keratin type II (KtyII) K5 and its type I partner keratin 14 (K14). Here, we examine the role of keratin phosphorylation in KF network rearrangement and cellular functions. We detect phosphorylation of the K5 head domain residue T150 in cytoplasmic epidermolysis bullosa simplex granules containing R125C K14 mutants. Expression of phosphomimetic T150D K5 mutants results in impaired KF formation in keratinocytes. The phenotype is enhanced upon combination with other phosphomimetic K5 head domain mutations. Remarkably, introduction of T150D K5 mutants into KtyII-lacking (KtyII–/–) keratinocytes prevents keratin network formation altogether. In contrast, phosphorylation-deficient T150A K5 leads to KFs with reduced branching and turnover. Assembly of T150D K5 is arrested at the heterotetramer stage coinciding with increased heat shock protein association. Finally, reduced cell viability and elevated response to stressors is noted in T150 mutant cells. Taken together, our findings identify T150 K5 phosphorylation as an important determinant of KF network formation and function with a possible role in epidermolysis bullosa simplex pathogenesis.