Elucidating the Sexual Cycle of HIV/AIDS-Related Pneumocystis Pneumonia - Pneumocystis jirovecii is a fungal pathogen that causes pneumonia (PjP) in immunocompromised patients. Trimethoprim-sulfamethoxazole (TMP-SMX) is the first line drug for the treatment and prophylaxis of PjP. However, many patients cannot tolerate TMP-SMX due to severe allergic reactions. Secondary treatments such as pentamidine are either toxic or not as effective as TMP-SMX. PjP remains refractory to most common antifungal drugs necessitating novel approaches for new agent discovery. Echinocandins are anti-fungal drugs that target the synthesis of β -1,3 -D-glucan (BG), found in fungal cell walls. Treatment with echinocandins in rodent models of PjP resulted in disappearance of asci and inability to transmit infection. Bulk RNA-Seq studies of anidulafungin treated mice showed an apparent dysregulation in mating type receptor expression and other genes associated with sexual reproduction in addition to BG inhibition. These findings led to the concept that Pneumocystis spp. are obligated to undergo sexual reproduction to survive. We showed that 8 weeks of treatment with the echinocandins, rezafungin and anidulafungin, could eradicate the pneumonia in mice. Since there was no “salvage” asexual replication, this finding provides further evidence that these fungi must sexually replicate to survive and that the sexual cycle presents a novel drug target. Based on the critical role of asci production via the sexual process we propose to define the sexual cycle of these fungi, then use the defined sexual stages to identify potential drug targets that could be used in combination therapy with echinocandins to eradicate/prevent the infection. As a test of concept, we will evaluate antibody-directed therapy to the mating receptor proteins as the initiators of the sexual cycle, alone and in combination with anidulafungin. Our aims are to: Aim 1. Define the stages of the sexual cycle of Pneumocystis species using single cell profiling (scRNA-seq). Two surrogate rat models, corticosteroid immunosuppressed and CD4+ depletion, will be used to define the sexual cycle and determine whether the host immune milieu might affect the life cycle. Male and female sources of P. carinii will identify any sex-related variances. We will then determine whether the life cycle of P. jirovecii corresponds to that of P. carinii. Aim 2. Identify reconstituted sexual cycle genes after release from anidulafungin treatment. T h e goal of this aim will be to identify the genes temporally expressed during reconstitution of the sexual cycle after anidulafungin treatment as a strategy to prioritize potential drug targets. Along with the scRNA-seq, fluorescent microscopy using the antibodies to the mating receptors, the anti-BG mAb, and DAPI staining will reveal the kinetics of repopulation of the sexual life cycle stages. Aim 3. Treatment with antibodies to Mam2p and Map3p of P. carinii with and without anidulafungin: a test-of-concept study. We posit that identification of genes upstream of the BG target, will provide candidate drug targets that will act in synergy with anidulafungin to eradicate the infection. As a test of concept for combination therapy, we will treat PjP in the rat model with antibodies to Mam2p andMap3p and anidulafungin.
