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

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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.