“Targeting Phosphoglucomutase in Pneumocystis Pneumonia”

About This Grant

Summary/Abstract. Pneumocystis pneumonia remains a significant cause of morbidity and mortality in AIDS1,2. The glucan-rich cell wall of Pneumocystis is a protective structure, absent from mammals, making it an attractive target for treatment3-6. Cell wall β-glucans are synthesized from donor uridine diphosphate glucose (UDP), requiring the interconversion of glucose-6-phosphate (G-6-P) and glucose-1-phosphate (G-1-P) by phosphoglucomutase (PGM)7-10. Current understanding of Pneumocystis PGM is limited. The overall goal of this proposal is to better understand the role of PGM in Pneumocystis cell wall synthesis, viability and pathogenesis. Prior studies demonstrate the importance of PGM for cell wall integrity and viability in Aspergillus fumigatus, and have shown that a small molecule - ISFP10, can selectively reduce fungal PGM activity with minimal effect on the human isoform11,12. We next identified a functional Pneumocystis PGM enzyme capable of complementing pgm2Δ (deficient) S. cerevisiae, restoring growth and cell wall formation13,14. No studies have yet explored the specific contributions of PGM to Pneumocystis cell wall synthesis, growth, or its relevance to PJP pathogenesis. To address these gaps, we present compelling new data demonstrating a critical role of Pneumocystis PGM in the organism’s life cycle and infection. We hypothesize that Pneumocystis phosphoglucomutase (PGM) plays critical roles in Pneumocystis cell wall synthesis, integrity, viability, growth, and pathogenesis. Aim 1: We will evaluate activity of Pneumocystis PGM and assess the impact of PGM inhibition on Pneumocystis cell wall synthesis, integrity, viability, and growth. Pneumocystis PGM cDNA will be expressed in S. cerevisiae pgm2Δ yeast incubated with sublethal doses of ISFP10. The effects of Pneumocystis PGM complementation in pgm2Δ yeast will be analyzed for global transcriptional responses, total cell wall carbohydrates and b-glucan. Similar assays will be performed on fresh Pneumocystis treated with ISFP10 in vitro. We will assess cell wall integrity in both S. cerevisiae pgm2Δ complemented with Pneumocystis PGM and in Pneumocystis treated with ISFP10. Lastly, we will measure the impact of ISFP10 on growth of S. cerevisiae pgm2Δ complemented with Pneumocystis PGM and assess impact of ISFP10 on Pneumocystis viability in vitro. Aim 2: We will further define the role of Pneumocystis PGM using the AIDS-like PCP immunosuppressed mouse model. Our preliminary studies using both precision-cut lung slices (PCLS) from PCP-infected mice, as well as pilot ISFP10 treated CD4-depleted mice with PCP, demonstrate significant reduction of organism burden. Recent data on maximum tolerated dose (MTD) and pharmacokinetics (PK) of ISFP10 will now guide our next formal studies of higher dose drug administration in mice with PCP. Organism numbers and lung inflammatory burdens will be determined. We anticipate these studies will define the role of PGM in Pneumocystis cell wall synthesis, viability, and pathogenicity, while assessing the therapeutic potential of PGM inhibition during PCP.