LOS ANGELES BIOMEDICAL RESEARCH INSTITUTE AT HARBOR-UCLA MEDICAL CENTER
Grant: $70,460 - National Institutes of Health - Jul. 15, 2009
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Award Description: 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.
Project Description: We have demonstrated in our preliminary study that both Hyr1 and Bcr1 encode fungal neutrophil killing resistance activity in test tube. Since Bcr1 is a transcription factor that positively regulates Hyr1 expression. Our central hypothesis in this proposal is that Hyr1 is a downstream effector of Bcr1 that facilitates fungal survival during hematogenous dissemination candidiasis. To test this hypothesis, we expressed Hyr1 in a bcr1 null mutant. Our very first goal is to test whether the expression of Hyr1, in a bcr1 null mutant could complement the lack of neutrophil resistant activity of the bcr1 null mutant in test tube. We compared neutrophil killing of the bcr1 null mutant strain (CJN702), a BCR1 complemented strain in the bcr1 null mutant background (CJN698), and a wild-type C. albicans strain (DAY185). The bcr1 null mutant was hyper-susceptible to neutrophil-mediated killing compared to the BCR1-complemented and wild-type control strains (Fig 1A). Furthermore, the hyper-susceptibility to killing of bcr1-deficient C. albicans was fully complemented by autonomous expression of HYR1 in the bcr1 mutant background, but not by other cell surface encoding genes regulated by Bcr1p (Fig 1B). These data demonstrated that the hyper-susceptibility to phagocyte killing of the bcr1 null mutant was fully complemented by autonomously expressed HYR1, but not other genes which encode GPI-proteins positively regulated by Bcr1p. Hence, HYR1 is a downstream gene of BCR1 in a phagocyte killing resistance pathway at least in test tube.
Jobs Summary: Through funding of this application, we have been able to retain Guanpingsheng Luo that has expertise in neutrophil killing assay and animal handling needed to complete the studies proposed in this application. Furthermore, Dr. Spellberg provides his immunology expertise (cytokines) and help in conducting and designing the experiments. Dr. Ibrahim works closely with Dr. Fu to coordinate the animal experiments in a strategic and efficient manner. All these efforts directly contribute to achieving the goal in both Specific Aims in this proposal. (Total jobs reported: 0)
Project Status: Less Than 50% Completed
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