Pneumocystis spp. are host obligate fungal pathogens that cause a fatal pneumonia (PCP) in immunocompromised hosts. Few drugs are effective against PCP and there have been no new therapies for its treatment in decades. PCP remains a problem in HIV-infected patients and is emerging as a clinical concern in newly susceptible populations including patients receiving immunotherapy for chronic conditions. We recently reported that treatment of PCP in murine models with the echinocandins, anti-fungal agents that target ?-1,3-D-glucan synthesis (BG), depleted the asci which contain BG, but large numbers of forms that do not express BG remained in the infected lungs and were apparently unable to proliferate. Notably, the treated mice could not transmit the infection without asci. The echinocandins prevented PCP when administered prophylactically at higher doses, suggesting that formation of asci via the sexual cycle may be required for a productive infection. Genes associated with sexual replication and cell cycle perturbation comprised the strongest up-regulated signals in P. murina from anidulafungin treated mice. These infections were devoid of asci, suggesting that P. murina attempted to undergo sexual replication, but could not due to a lack of BG. Based on these data, we posit that asci, and thus sexual replication, is required for progression through the Pneumocystis life cycle. The importance of the sexual cycle for survival of Pneumocystis will be investigated using the anidulafungin treatment and prophylactic models.
The specific aims address 2 major questions we propose to pursue: Question 1: Is sexual replication required for completion of the life cycle of Pneumocystis? 3 aims will be used to address this question. The first will be to assess the replication competency of P. murina treated with anidulafungin by transferring them to new hosts. Replication will be determined by a combination of microscopic methods, BG measurement, and quantitative PCR.
The second aim will use the prophylactic model to allow low level replication to ask whether replication occurs via the sexual cycle, using the same methods.
The third aim will determine whether P. murina can survive during prolonged anidulafungin treatment in the same hosts without proceeding through the sexual cycle or if it is eventually cleared. Question 2: At what point in the Pneumocystis life cycle can the infection be transmitted? The sexual cycle returns after withdrawal of anidulafungin. During 10 days to 4 weeks post-cessation, we will track the emergence of the sexual cycle and identify gene signatures, biomarkers and numbers of asci prior to exposing these mice to nave recipients. Using these data, we will determine at what point in the life cycle transmission takes place and the thresholds necessary for transmission. Selected proteins based on gene expression data will be validated by proteomics as potential biomarkers associated with return of the sexual cycle and transmission. These studies will identify potential vulnerabilities in the Pneumocystis life cycle that could be exploited for interdiction and add to our understanding of its life cycle and transmission.
Few drugs are available to treat the pneumonia caused by Pneumocystis, PCP. Using a novel echinocandin treated model of PCP, we determined that the sexual cycle may be necessary for its survival, offering a potential therapeutic target. Using this model we will ascertain whether these fungi truly require the sexual cycle to replicate and transmit the infection and in doing so will further define their life cycle.