On-site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans.

Candida albicans is an opportunistic fungal pathogen that colonises the skin as well as genital and intestinal mucosa of most healthy individuals. The ability of C. albicans to switch between different morphological states, for example, from an ellipsoid yeast form to a highly polarised, hyphal form, contributes to its success as a pathogen. In highly polarised tip-growing cells such as neurons, pollen tubes, and filamentous fungi, delivery of membrane and cargo to the filament apex is achieved by long-range delivery of secretory vesicles tethered to motors moving along cytoskeletal cables that extend towards the growing tip. To investigate whether such a mechanism is also critical for C. albicans filamentous growth, we studied the dynamics and organisation of the C. albicans secretory pathway using live cell imaging and three-dimensional electron microscopy. We demonstrate that the secretory pathway is organised in distinct domains, including endoplasmic reticulum membrane sheets that extend along the length of the hyphal filament, a sub-apical zone exhibiting distinct membrane structures and dynamics and a Spitzenkörper comprised of uniformly sized secretory vesicles. Our results indicate that the organisation of the secretory pathway in C. albicans likely facilitates short-range "on-site" secretory vesicle delivery, in contrast to filamentous fungi and many highly polarised cells.

Role of Arf GTPases in fungal morphogenesis and virulence.

Virulence of the human fungal pathogen Candida albicans depends on the switch from budding to filamentous growth, which requires sustained membrane traffic and polarized growth. In many organisms, small GTPases of the Arf (ADP-ribosylation factor) family regulate membrane/protein trafficking, yet little is known about their role in fungal filamentous growth. To investigate these GTPases in C. albicans, we generated loss of function mutants in all 3 Arf proteins, Arf1-Arf3, and 2 Arf-like proteins, Arl1 and Arl3. Our results indicate that of these proteins, Arf2 is required for viability and sensitivity to antifungal drugs. Repressible ARF2 expression results in defects in filamentous growth, cell wall integrity and virulence, likely due to alteration of the Golgi. Arl1 is also required for invasive filamentous growth and, although arl1/arl1 cells can initiate hyphal growth, hyphae are substantially shorter than that of the wild-type, due to the inability of this mutant to maintain hyphal growth at a single site. We show that this defect does not result from an alteration of phospholipid distribution and is unlikely to result from the sole Golgin Imh1 mislocalization, as Imh1 is not required for invasive filamentous growth. Rather, our results suggest that the arl1/arl1 hyphal growth defect results from increased secretion in this mutant. Strikingly, the arl1/arl1 mutant is drastically reduced in virulence during oropharyngeal candidiasis. Together, our results highlight the importance of Arl1 and Arf2 as key regulators of hyphal growth and virulence in C. albicans and identify a unique function of Arl1 in secretion.

Phosphatidylinositol-4-phosphate-dependent membrane traffic is critical for fungal filamentous growth.

The phospholipid phosphatidylinositol-4-phosphate [PI(4)P], generated at the Golgi and plasma membrane, has been implicated in many processes, including membrane traffic, yet its role in cell morphology changes, such as the budding to filamentous growth transition, is unknown. We show that Golgi PI(4)P is required for such a transition in the human pathogenic fungus Candida albicans. Quantitative analyses of membrane traffic revealed that PI(4)P is required for late Golgi and secretory vesicle dynamics and targeting and, as a result, is important for the distribution of a multidrug transporter and hence sensitivity to antifungal drugs. We also observed that plasma membrane PI(4)P, which we show is functionally distinct from Golgi PI(4)P, forms a steep gradient concomitant with filamentous growth, despite uniform plasma membrane PI-4-kinase distribution. Mathematical modeling indicates that local PI(4)P generation and hydrolysis by phosphatases are crucial for this gradient. We conclude that PI(4)P-regulated membrane dynamics are critical for morphology changes.

CWH43 is required for the introduction of ceramides into GPI anchors in Saccharomyces cerevisiae.

After glycosylphosphatidylinositols (GPIs) are added to GPI proteins of Saccharomyces cerevisiae, the fatty acid in sn-2 of the diacylglycerol moiety can be replaced by a C26:0 fatty acid by a deacylation-reacylation cycle catalysed by Per1p and Gup1p. Furthermore the diacylglycerol moiety of the yeast GPI anchor can also be replaced by ceramides. CWH43 of yeast is homologous to PGAP2, a gene that recently was implicated in a similar deacylation reacylation cycle of GPI proteins in mammalian cells, where PGAP2 is required for the reacylation of monoradylglycerol-type GPI anchors. Here we show that mutants lacking CWH43 are unable to synthesize ceramide-containing GPI anchors, while the replacement of C18 by C26 fatty acids on the primary diacylglycerol anchor by Per1p and Gup1p is still intact. CWH43 contains the COG3568 metal hydrolase motif, which is found in many eukaryotic and prokaryotic enzymes. The conserved His 802 residue of this motif was identified as being essential for ceramide remodelling. Ceramide remodelling is not required for the normal integration of GPI proteins into the cell wall. All remodelling reactions are dependent on prior removal of the inositol-linked fatty acid by Bst1p.