Vacuole segregation and mycelial development of Candida albicans

Candida albicans is the most common fungus that is associated with life-threatening infection. It is one of many fungi that can grow either as an ovoid yeast that grows by forming expanding buds or as a mould that elaborates branching tubular cells to form a mycelium. Because infections are associated with transitions between these two forms the process of yeast-to-hypha transition has been heavily studied in recent years. However, most of these investigations have focussed on the signals that stimulate the transitions rather than the growth of the primary germ tube and its associated branches. This is important because mycelial growth may be vital for tissue invasion, and because it has become clear that the process of hyphal C. albicans is unusual and enables novel hypotheses to be tested about the physiological requirements for cell division of eukaryotic cells in general. Consequently we will investigate how mycelial cells grow and divide both from the point of view of its role in fungal disease and the insights it can provide into the cell biology of the cell cycle. We have observed that during the growth of germ tubes, a large vacuole forms behind the growing tip that fills up most of the cell. Although the overall dimensions of this cell are similar to other cells, it has less cytoplasm. We propose that it does not have sufficient cytoplasm to progress through the cell division cycle. At a specific point in the cell cycle called START, cells must achieve a certain minimal cell size. We therefore hypothesise that the large vacuole left behind after cell division prevents these cells from passing through START in the division cycle. We have also shown that the vacuole is not equally divided between the two daughter cells formed after cell division, and that the younger daughter cell acquires most of the cytoplasm and the mother cell retains most of the vacuole. Vacuole division and inheritance is a carefully regulated process about which we have learned much from the related yeast-like fungus Saccharomyces cerevisiae. To test out hypothesis that vacuole inheritance and distribution determines whether growth or growth arrest of cells occurs within the mycelium, we will make defined mutations in genes that regulate vacuole partitioning at cell division. We can predict from studies in S. cerevisiae exactly what mutations we will need to make to be able to alter the normal vacuole pattern in cells. These mutants will enable us not only to find out how vacuoles control cell division, but also to address questions about how the cell cycle in regulated in higher eukaryotic cells. With a range of mutants we will be able to dissect the regulatory pathways through which hyphae and branches of mycelia grow. We will also be able to assess whether normal branching is important for the ability of this fungus to establish diseased lesions in animal tissues. Importantly, many other important fungi, including many plant pathogens, control the type of growth they undergo by forming a greater or lesser amount of vacuole. We will therefore be able to advance the field of fungal physiology, by undertaking the first in- depth study of the genetic links between vacuolation and fungal growth.

This project will test our novel hypothesis linking the cell cycle, vacuole inheritance, mycelial growth and pathogenesis of the human pathogenic fungus Candida albicans. The project is based on the strong platform of two publications from our group that describes a novel pattern of the inheritance of vacuole during filamentous growth of this fungus. During the first cell division cycle of the growth of the true hyphal form, vacuole is partitioned asymmetrically such that the daughter germ tube inherits most cytoplasm and the mother cell inherits most vacuole. As a consequence, the mother cell does not immediately re-enter the cell cycle and secondary germ tube formation is delayed. This pattern of asymmetric vacuole inheritance is reiterated in subsequent cell cycles of the germ tube resulting in the generation of a series of highly vacuolated hyphal compartments behind the apex that are cell cycle arrested. Our interpretation of these events is that cell cycle entry at START, which is thought to be regulated by cell size, is delayed if the cytoplasmic cell volume is significantly less that the total cell volume. We hypothesise that cytoplasmic volume, rather than cell size, influences cell cycle progression, and therefore that asymmetric vacuolar inheritance modulates the growth and branching of C. albicans mycelia. To test this hypothesis we will create isogenic mutants that are blocked at known stages in the normal vacuole inheritance pathway. Low organelle copy number compartments are partitioned at cytokinesis by first fragmenting the organelle into smaller sized organelles that can be more ready segregated equally at cytokinesis. In the budding yeast Saccharomyces cerevisiae an 'inheritance structure' is formed that extrudes vesicles or tubules of vacuoles into the bud. Homotypic vacuole formation occurs that involves the capture of vacuolar protein tags in v-SNARE complexes of integral membrane proteins that achieve docking and subsequent vacuole fusion. The genetic regulation of this process is well understood in yeast, providing a conceptual framework to identify genes that, in C. albicans, would be predicted to be altered in the extension of germ tubes and the formation of hyphal branches and the cell cycle in true hyphae. The specific aims are as follows. (i) To use advanced live-cell imaging techniques to describe the unusable vacuole inheritance pattern in C. albicans to compare the cell biology of vacuole inheritance during growth of yeast cells, pseudohyphae and true hyphae of C. albicans. (ii) To assemble a collection of null or conditional mutants that are altered in the normal pattern of vacuole inheritance. Accordingly we will disrupt VAC7, VAC17 (required for normal vacuole inheritance), YKT6 (v-SNARE component involved in vacuole fusion and ER-Golgi trafficking), NYV1 (V-SNARE acting on vacuole-t-vacuole fusion) and VAM2 (vacuole transport fusion/ fragmentation). We have already created vac8 (Barelle et al., unpublished; Veses et al., 2005. Euk. Cell 4, 1088-1101) and abg1 mutants and have been given access to vps11, vps21 (defective in fusion events at the pre-vacuole compartment) and ypt7 (required for trafficking and tethering between late endosomes and vacuole) mutants of C. albicans created elsewhere (Palmer et al., 2003. Euk. Cell 2, 411-421; Palmer, personal communication). (iii) To measure the growth kinetics of germ tube formation and branch formation and cell cycle timing in these mutants and in cells treated with agents that interfere with vacuole inheritance or vacuole fusion. (iv) To assess the consequences to perturbations of such vacuole inheritance / altered branching mutants in the pathogenesis of C. albicans in ex vivo and in vivo models of infection.