The role of the Cdc28 Cbk1 and Tpk1 kinases in the formation of hyphae of the human fungal pathogen Candida albicans

Candida albicans is a fungus that is responsible for causing vaginitis (thrush) in women and oral infections in AIDs patients. In certain groups of vulnerable individuals, C. albicans can enter the blood stream and spread to internal organs. Such bloodstream infections are extremely serious with a mortality rate of up to 50%. Some surveys place Candida infections as the second most common cause of death from infections acquired in hospitals. A particular feature of its biology is its ability to grow as unicellular budding yeasts or in long filaments of tube-like cells called hyphae. This ability to switch growth modes is thought to be important for its pathogenicity. The hyphae allow the fungus to penetrate through mucosal membranes and enter the bloodstream. They also allow the fungus to escape after they have been engulfed by macrophages. Understanding the molecular mechanisms operating when the organisms switches from the yeast to the hyphal growth form is an important first step towards the development of new forms of treatment and prevention. We know the switch from yeast to hyphal growth form involves the production of new types of protein encoded by the activation of genes that are normally silent in yeast cells. One gene that is activated encodes a protein called Hgc1 that partners another protein called Cdc28 which is a type of enzyme called a kinase. Kinases control the activity of other proteins by adding a phosphate group to certain amino acid subunits which make up the proteins and in doing so change their properties in a different ways. We know that kinases play key role orchestrating a wide variety of the complex process in living cells, so it is not surprising that kinases should be important in promoting hyphal growth. Two other kinases, Cbk1 and Tpk1, are known to be required for hyphal growth. The proposed work is designed to identify the proteins that are targeted by Cdc28, Cbk1 and Tpk1. This will tell us which proteins form part of the hyphal growth machinery and allow us to investigate how their properties are altered during hyphal growth. Our existing knowledge of cell growth allows us to guess some of the proteins that might be targeted by the kinases. We have already instigated a program of experiments on this basis and already have some promising results. The first stage of the program aims to finish these studies. Because it would be foolish to suppose that our knowledge is good enough to guess all the proteins involved, the second stage of the investigation will broaden the search for kinase targets based on a procedure to identify which proteins physically associate with each of the kinases. A short stretch of amino acids called an epitope tag will be added to each kinase that is recognized by antibodies attached to magnetic beads which can be collected by a magnet allowing the kinase to isolated from a cell lysate. Any protein to which the kinase physically interacts will also be captured and its identity can be revealed by Mass Spectroscopy. A potential problem is that there may many false positive hits by proteins which attach to the antibody or magnetic beads non-specifically. To recognize these proteins we will mix the lysate culture containing the tagged kinase that recognizes the antibody with an equal amount of lysate from an untagged culture. The tagged culture will be labeled with a heavy isotope. Fragments of protein that bind non-specifically will be present in 1:1 heavy:light ratio because they will be present in equal amounts from the tagged and untagged culture, but proteins that the kinase binds specifically will contain an excess of heavy isotope that can be detected by mass spectroscopy. Despite this modification it will still be necessary to carry out further experiments to verify the hits from the screen. When this has been completed we hope to be able to construct a network showing the proteins modified by the kinases and their role in promoting hyphal growth.

The polymorphic fungus Candida albicans is a major human pathogen. Hyphae, which grow in a highly polarized fashion from their tip, are required for virulence. The project focuses on the role of three kinases, Cdc28, Cbk1 and Tpk1, and two regulatory subunits Mob2 and Hgc1 that are known to be essential for hyphal growth. Low throughput studies in the PI's laboratory has already identified Sec2 as a target of Cdc28. Preliminary data has led to the identification of a number of putative possible targets for Cdc28 and Cbk1 and uncovered a complex pattern of phosphorylation regulating the activity of Cbk1. The first objective is complete these preliminary studies by unambiguously identifying the targeted residues by Mass Spectrometry, and determining the physiological relevance by mutation of the targeted residues. The second objective seeks to extend the search for targets of the three kinases by an unbiased survey of the proteome. This will adopt the approach successfully pioneered in Saccharomyces cerevisiae of using mass spectrometry to identify proteins that co-immuneprecipitate with the kinases. Rapid magnetic bead capture, minimal washes and on-bead protein digestion will be used to address the transient nature of the interaction between kinases and their targets. To eliminate non-specific binders a metabolic labeling approach will be adopted whereby the lysate from a kinase-tagged strain, grown on heavy medium will be mixed with an equal quantity of lysate from a non-tagged strain grown on light medium. Deviation from 1:1 peptide ration will identify proteins that genuinely interact with the kinases. Hits from this screen will be verified by appropriate tests and the targeted residues of selected proteins mutagenised to phosphomimetic and non-phosphorylatable residues to determine the physiological role of the phosphorylation events. Finally, the data will be used to construct a kinase interaction map required for hyphal growth and development.

1) Pharmaceutical companies developing novel antifungal agents and Health professionals preventing and managing C. albicans infections C. albicans is a significant cause of morbidity to an otherwise healthy and sizable fraction of the overall UK population and a common cause of life-threatening bloodstream infections responsible in vulnerable patient groups in hospitals. The research proposed here focusing on hyphal growth mechanisms address a key aspect of its pathogenicity. The benefit here is long term derived from understanding the basic mechanisms of an important pathogen 2) Education of the wider public Due to the high prevalence of vulvovaginal candidiasis VVC in women there is considerable interest in C. albicans among the general population. There is also a good deal of inaccurate and misleading information propagated in the media; often attributing C. albicans as the cause of ill-defined and chronic disorders for which there is no evidence that it is involved. 3) Research and professional skills of the Research assistant. The RA Dr Caballero-Lima is highly skilled in C. albicans genetics and cell biology. The work will require him to be trained in operational aspects of Mass Spectrometry and analysis of the resultant data. This will allow him to develop a completely new skill set and greatly enhance his future career. Furthermore, it add to the limited pool of scientists competent in this important technology.