Targeting the Sexual Cycle of Pneumocystis

Cincinnati Va Medical Center Research, Cincinnati OH

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Abstract

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, β -1,3 -D-glucan synthase inhibitors, are a newer family of antifungal agents. Reports of the efficacy of echinocandins for PjP have been contradictory, due in large part to the anecdotal nature of the reports and combinations with other agents. However, new data from meta-analyses indicate that a combination of TMP-SMX with echinocandins can lead to a reduction in mortality and improved treatment responses. This new finding together with the novel insights on the effects of echinocandins on Pneumocystis pneumonia we accrued, position us to take the next steps in optimizing and understanding this synergy. Mouse and rat models of Pneumocystis pneumonia are surrogates for P. jirovecii infection, since no species of Pneumocystis can be cultivated outside the lung. Fortunately, rodent models are predictive of drug efficacy in humans. During the tenure of the previous VA Award, we have made several significant discoveries that we will use to our advantage for the proposed studies, most notably the fact that Pneumocystis spp. are obligated to undergo sexual replication to survive, grow, and transmit the infection. Treatment with echinocandins interrupt the sexual cycle, leading to its eventual elimination. We will concentrate on the effects of anidulafungin monotherapy (AnM) and combination therapy with TMP- SMX (AnTS), on the P. carinii sexual cycle and the ability of the host to transmit the infection. We posit that as sexual obligates, a better understanding of these treatments on the sexual cycle will lead to more efficient regimens and identify at what point hosts are unable to transmit the infection. Our approaches will employ single cell RNA-seq and in vivo animal strategies. Typically used to interrogate mammalian cells, application of sc- RNA-seq to a human fungal pathogen is highly innovative. The aims of this proposal are to: Aim 1. Define the perturbations by anidulafungin monotherapy (AnM) and anidulafungin/TMP-SMX combination therapy (AnTS) on the sexual cycle of Pneumocystis carinii using single cell profiling (scRNA-seq). We have shown that scRNA-seq can be used to identify clusters of life cycle stages from BALF of P. carinii-infected rats (Preliminary Data). In the present aim, three groups of P. carinii-infected rats will be employed to achieve our goals: (i) Untreated, infected, and immunosuppressed rats (Control); (ii) infected, immunosuppressed treated with AnM; and (iii) infected, immunosuppressed, treated with AnTS. BALF will be used for scRNA-seq and harvested at 3 time points during treatment. Data from these studies will vastly improve our understanding of the effects of the treatments on the infection and potential synergy. Aim 2. Temporally assess the recovery of the sexual cycle of P. carinii after release from echinocandins. We have shown that asci return 6-7 days after cessation of anidulafungin treatment. Here we will withdraw AnM and AnTS treatment after the 3-week period while the rats will continue on immunosuppression to allow repopulation of infection. We will use scRNA-seq to determine the life cycle stages that repopulate and at what point the rats can transmit the infection. While we anticipate the AnTS may eradicate the infection, we know that An does not. Concomitant with the weekly scRNA-seq data, we will evaluate when the rats are infectious to immunosuppressed recipient rats. These studies will allow us to examine the early events of the sexual cycle and determine at what point the fungi can transmit the infection and what genes are associated with the infective phase.