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 age3Delta 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 age3Delta cells shows a wild-type-like polarization pattern of the actin cytoskeleton and lipid rafts. Moreover, age3Delta 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 age3Delta 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 age3Delta cells to polarize the actin cytoskeleton and possibly of inefficient endocytosis. The high susceptibility of age3Delta cells to azoles is not caused by inefficient transport of efflux pumps to the cell membrane. A possible role of a vacuolar defect of age3Delta 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

Somatostatin Inhibits Cell Migration and Reduces Cell Counts of Human Keratinocytes and Delays Epidermal Wound Healing in an Ex Vivo Wound Model

The peptide hormone somatostatin (SST) and its five G protein-coupled receptors (SSTR1-5) were described to be present in the skin, but their cutaneous function(s) and skin-specific signalling mechanisms are widely unknown. By using receptor specific agonists we show here that the SSTRs expressed in keratinocytes are functionally coupled to the inhibition of adenylate cyclase. In addition, treatment with SSTR4 and SSTR5/1 specific agonists significantly influences the MAP kinase signalling pathway. As epidermal hormone receptors in general are known to regulate re-epithelialization following skin injury, we investigated the effect of SST on cell counts and migration of human keratinocytes. Our results demonstrate a significant inhibition of cell migration and reduction of cell counts by SST. We do not observe an effect on apoptosis and necrosis. Analysis of signalling pathways showed that somatostatin inhibits cell migration independent of its effect on cAMP. Migrating keratinocytes treated with SST show altered cytoskeleton dynamics with delayed lamellipodia formation. Furthermore, the activity of the small GTPase Rac1 is diminished, providing evidence for the control of the actin cytoskeleton by somatostatin receptors in keratinocytes. While activation of all receptors leads to redundant effects on cell migration, only treatment with a SSTR5/1 specific agonist resulted in decreased cell counts. In accordance with reduced cell counts and impaired migration we observe delayed re-epithelialization in an ex vivo wound healing model. Consequently, our experiments suggest SST as a negative regulator of epidermal wound healing

Inhibition of migration by SST is cAMP-independent.

<p>Quantification of cell migration in scratch assays after treatment with SST (1 µM), FSK (10 µM) or LPA (5 µM) as well as combinations of these substances. Data are presented as percentages of the recovered scratch area relative to untreated control cells (n = 5) Results are shown as means+/−SEM, * P<0.05, compared to controls; * P<0.05 between different treatment groups.</p

SST delays lamellipodium formation in the early phase of keratinocyte migration.

<p>A: Lamellipodium formation in migrating keratinocytes. Cells were scratch wounded, treated as indicated, fixed after 3 h of migration and the actin cytoskeleton was visualized with fluorophor-labelled phalloidin. Lamellipodia are marked by an overlay of red pseudocolor. B: Areas of extending lamellipodia were measured after 3 h for each treatment and compared to control cells (n = 3, total number of analyzed cells is indicated inside bars, means+/−SEM, * P<0.05).</p

Activation of SST receptors results in the inhibition of keratinocyte migration.

<p>A: Keratinocyte monolayers were scratch wounded and the scratched area was examined directly after scratching (0 h) and every 6 hours during an incubation period of 24 h. To prevent proliferative effects, cells were pre-exposed to X-ray irradiation to induce cell cycle arrest. B: Quantification of cell migration in scratch assays after SST stimulation (1 µM SST for 24 h). Data are presented as percentages of the recovered scratch area relative to untreated control cells (n = 7). C: Effect of selective SSTR agonists (1 µM for 24 h) on cell migration compared to untreated cells (n = 5). All subtype-specific agonists tested inhibit keratinocyte migration. Results are shown as means+/−SEM, * P<0.05, ** P<0.01, *** P<0.005.</p

SSTR activation in keratinocytes modulates the MAP kinase pathway.

<p>A: MAP kinase activity assay. Cells were treated with 1 µM SST or 5% FCS (or both) for 5 or 10 min and lysates were analyzed with antibodies against ERK1/2 or phospho-ERK1/2 by Western blotting. B: The relative intensities (control value = 1) of 6 experiments after 5 min treatment were quantified and are shown as means +/− SEM (* P<0.05, *** P<0.005.). C: Effect of different SST receptor agonists on ERK phosphorylation (n = 5; * P<0.05, ** P<0.01), a representative blot is shown below. SST as well as the SSTR4-specific agonist significantly induces ERK phosphorylation, while treatment with agonist sst5/1 results in significantly decreased phospho-ERK levels.</p