Candidemia, the fourth most common bloodstream infection in the U.S., and other forms of systemic candidiasis are associated with mortality rates of 40% or more despite treatment with antifungal agents. The Candida albicans cell wall is central to the pathogenesis of candidiasis, but mechanisms that link cell wall regulation and virulence are only beginning to be understood. Recently, we demonstrated that C. albicans rapidly delocalizes phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) and septins as part of the natural response to the cell wall-active antifungal caspofungin. Furthermore, we identified a novel C. albicans PI(4,5)P2-septin pathway that regulates cell wall integrity and virulence among mice with candidiasis. We hypothesize that the ability to activate or down-regulate the PI(4,5)P2-septin pathway as dictated by the environment (i.e., balanced regulation) is necessary for optimal C. albicans responses to cell wall stress during drug exposure or invasive candidiasis. The objectives of this project are to prove our balanced regulation hypothesis, validate the proposed PI(4,5)P2-septin pathway, and identify its outputs. We will pursue three specific aims.
The first aim i s to demonstrate that balanced PI(4,5)P2 regulation correlates with protective responses to caspofungin. Dynamic PI(4,5)P2 responses will be correlated with cellular viability in PI(4,5)P2- regulatory mutants and caspofungin-susceptible and -resistant C. albicans strains.
The second aim i s to establish interactions between PI(4,5)P2 and other PI(4,5)P2-septin pathway components during caspofungin exposure and invasive candidiasis. Interactions will be assessed by tracking components during time-lapse live cell imaging, demonstrating physical interactions, and visualizing interactions within cells by fluorescence resonance energy transfer (FRET). PI(4,5)P2 levels in pathway mutants will be directly correlated with PKC- MAPK cell wall integrity pathway activation.
The third aim i s to link transcription factors to the PI(4,5)P2-septin pathway, and identify transcriptional outputs and pathway targets that contribute to caspofungin responses and pathogenesis. Transcriptional outputs will be defined during intra-abdominal candidiasis of mice by using RNA-Seq, a largely unbiased method that comprehensively quantitates gene expression. The project employs a series of innovative techniques to study a novel pathway that is relevant to antifungal drug resistance and the pathogenesis of candidiasis. Therefore, it is likely to yield clinically useful insights that would not be obtained through other studies. Our findings will be significant because they will explain how the PI(4,5)P2-septin pathway governs cell wall integrity, echinocandin susceptibility and resistance, and pathogenesis, and place the pathway within the context of other regulators of these processes. The project will open new avenues of investigation that will define, in detail, the molecular and cellular mechanisms by which the PI(4,5)P2-septin pathway contributes to diverse types of candidiasis, and the impact of cell wall regulation on interactions with the host.
Candidemia is the fourth most common bloodstream infection in the U.S. Candidemia and other forms of systemic candidiasis are particularly relevant to the V.A. because major risk factors like neutropenia, other immunosuppressed states, receipt of cancer chemotherapy and broad-spectrum antibiotics, presence of intravenous catheters, gastrointestinal surgery and renal failure are so common among our veterans. The cell wall-active echinocandin antifungal agents such as caspofungin have emerged as frontline therapy for systemic candidiasis, but mortality rates remain unacceptable high. In this project, we will characterize a novel cell wall response pathway that contributes to decreased caspofungin susceptibility and pathogenesis of candidiasis. Our long-term goal is to target the pathway and its outputs to develop more effective treatments and diagnostic tests for systemic candidiasis, and thereby improve the health of veterans and other patients.