In order to proliferate within host cells and subsequently promote disease, fungal pathogens require an active calcium-calmodulin-dependent signaling cascade. The molecular mechanisms that determine how the calcium response is initiated and propagated in fungal pathogens remain largely unknown. A possible working model would state that calcineurin is activated by an increase in cytosolic calcium levels and calmodulin in response to signals specific to the host environment. Activated calcineurin would then subsequently dephosphorylate specific proteins required for fungal pathogenesis. We propose that calcium channels in pathogenic fungal cells initiate calcium signaling by responding to particular stimuli that are specific to the host environment (i.e. alkaline pH, 5% CO2, iron levels etc). Signal-specificity is achieved by the association of the calcium channel with key signaling proteins that could be recruited by calmodulin's interaction with the C-terminus of the calcium channel. The overall aim of the proposed research is to characterize the cellular and molecular mechanism by which pathogenic fungi use calcium channels to couple host-specific signals to a calcium/calmodulin-mediated signaling cascade that is required for colonization of the host environment. In order to elucidate the molecular mechanism of calcium channel function and regulation, structure-function studies using conventional patch clamp techniques in conditions that mimic the host environment will be performed. The calcium channel mutants generated for the structure-function studies, will be tested for virulence in an animal model of cryptococcal meningitis. Channel activation and regulation will be examined in cells that lack key signaling molecules in order to determine whether these signaling proteins regulate channel function as a means to impart signal specificity. A detailed study of calcium channel function and regulation is imperative not only for a clear understanding of the mechanism(s) underlying the signal-response coupling in the pathogenic fungal-host relationship but also for the potential development of small molecules that could function to prevent fungal proliferation in the host. For example, occlusion of the channel pore, or a change in channel voltage-sensitivity or the prevention of regulatory proteins from interacting with the channel could represent viable means by which small molecules may function to perturb channel activity, inhibit fungal cell proliferation within the host and ultimately prevent disease.
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