Yue Fu, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): A number of key virulence factors of Candida albicans are encoded by glycosyl-phosphatidylinositol- anchored proteins (GPI-proteins). Amongst these virulence associated GPI-proteins are those that regulate morphogenesis, adherence to and/or invasion of host cells, and resistance to phagocyte-mediated killing. In the studies proposed herein, we will use an innovative, function-based approach to investigate the pathogenesis of C. albicans by identifying unique, surface GPI-proteins that encode virulence associated phenotypes. We will: 1) over-express putative GPI-proteins identified by the C. albicans sequencing project using the tetracycline regulated promoter; and 2) identify C. albicans GPI-proteins responsible for seven virulence associated phenotypes through "gain/increase-of-function" screening. This new, complementary approach overcomes phenotypic unavailability, functional redundancy, and essential gene problems that plague gene disruption approaches. It also greatly facilitates large-scale functional screening. Because these GPI-proteins are localized on the Candida cell surface, results will yield potential avenues for future research and provide important insights into the pathogenesis of the organism and host-pathogen interaction. Additionally, GPI-proteins are easily accessible to drugs, and therefore may reveal promising new targets for therapeutic interventions against C. albicans infections. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Candida albicans is an opportunistic fungal pathogen that is able to survive as a commensal organism in several anatomically distinct sites. In compromised hosts, the manifestations of invasive infection caused by C. albicans vary based upon the particular anatomical origin of the causative strain. Phagocytes are the central effectors in host defense against hematogenously disseminated candidiasis. Using a new function-based approach, we have successfully identified a Candida albicans putative gene, HYR1, which encodes resistance to neutrophil-killing activity in vitro. In addition, we found that Bcr1p, a transcription factor positively regulating HYR1 expression, is also required for full resistance to neutrophil-killing. Based on these data, we hypothesize that Bcr1p, through the activation of its downstream effector, Hyr1p, contributes to survivability of C. albicans during hematogenous dissemination, increasing severity of infection. Our objective is to determine the impact of BCR1 during hematogenously disseminated candidiasis in mice, and to determine the relationship between BCR1 and HYR1 in regulating these processes. In this application, we will 1) determine the impact of BCR1 disruption on tissue fungal burden, inflammatory response and survivability during hematogenously disseminated murine candidiasis;and 2) determine if HYR1 autonomous expression complements the bcr1 null mutation in vitro and in vivo. While neutrophil-resistance mechanisms are well described for bacteria, the proposed studies will elucidate the first genetic relationship between an upstream regulator and a downstream effector for fungal resistance to neutrophil killing in vitro. Furthermore, the results of the proposed aims will define the in vivo effects of genes which control candidal resistance to phagocytic-killing in vitro. These studies will enable submission of a follow up R01 to identify additional members of the signal transduction pathway and to elucidate the mechanisms by which these genes mediate resistance to neutrophil killing in vitro and in vivo. As well, these putative virulence gene products will become potential targets for development of novel prophylactic and therapeutic strategies. PUBLIC HEALTH RELEVANCE: Candida is a cause of lethal infections in hospitalized patients. It is critical to understand how Candida causes such infections in order to create new ways to prevent and treat these infections. We have identified two genes that help the fungus to resist neutrophil killing in test tube. We will determine how these genes help the fungus to cause disease in mice, which will enable development of treatment strategies targeting these genes.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Candidiasis is a life-threatening infection that occurs in patients whose immune systems are not working properly. Candida albicans is the leading cause of Candida infections originating from the blood stream. Despite antifungal therapy, mortality resulting from these infections due to C. albicans remains unacceptably high and continues to increase. In light of these concerns, it is critical that mechanisms by which C. albicans initiate the infection in the blood stream be identified. C. albicans must bind to and traverse the vascular endothelium (cells that line the blood vessels)to escape from the blood vessel and infect a target organ such as kidneys. Therefore, a critical step in crossing the endothelial barrier involves adherence to and invasion of vascular endothelial cells by C. albicans. Additionally, during these processes C. albicans must evade killing by circulating white blood cells. Glycosyl-phosphatidylinositol-anchored proteins (GPI-proteins)are cell surface proteins of C. albicans and are known to encode key elements that promote infection. Amongst these GPI-proteins are those that regulate adherence to and invasion of endothelial cells, and resistance to white blood cells-mediated killing. In the studies proposed herein, we will use a new, genetic approach that depends on over producing these proteins under specific conditions to investigate the contribution of these proteins to the infectious process. After overproduction of these proteins in C. albicans, the generated organisms will be used to compare their ability to the parent strain (containing regular amount of the protein under study)in adhering adhering to and damaging endothelial cells as well as resistance to white blood cell killing in the test tube. To perform these experiments, it is necessary to grow in the test tube the same type of human cells that the fungus normally interacts with while in the blood stream (i.e. human endothelial cells and white blood cells). The most available, richest source of endothelial cells is the umbilical cord veins. The umbilical cords are anonymously removed from placentas that are to be discarded after delivery. There is no link between the patient and the umbilical cord that would allow the patient?s identity to be known. Given the absence of any link between the umbilical cord, the fact that the umbilical cords are anonymously removed from the placenta after delivery of the child, the fact that the umbilical cords would be destroyed if they were not used for this research, the risk to the patient is minimal. White blood cells (neutrophils)will be harvested from healthy volunteers after signing an informed consent. The volunteers will be from our division which is will diversified in ethnicity and gender, including African Americans, Asians, Hispanics and Caucasians. Approximately 10 ml blood will be harvested per draw. Phlebotomy will not be performed on any given volunteer more frequently than once per month. Phlebotomy is a routine procedure with minimal adverse effects that might include discomfort and some swelling in the arm due to needle insertion. Given the potential for discoveries to be made that could lead to a vaccine to prevent common and life-threatening Candida albicans, the potential benefit of this research is high. Therefore, the risk:benefit ratio is highly favorable. It is improtant to mention that all procedures involving harvesting endothelial cells from umbilical cords and obtaining blood samples are required only for performing the research outlined in the attached grant application.