Pneumocystis spp. are 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. Typically, PCP has been associated with patients infected with HIV, however, the fulminate pneumonia, PCP, and colonization with Pneumocystis jirovecii (the species infecting humans) are emerging clinical problems in newly susceptible populations in the general and veterans? populations including bone marrow recipients; patients receiving chronic immunotherapy for rheumatoid arthritis and other chronic inflammatory diseases; and cancer chemo- and immunotherapies. The life cycle of Pneumocystis is suggested to contain both an asexual replication cycle and a sexual cycle involving mating with subsequent formation of asci containing 8 ascospores (1). During the previous Merit Review, we showed that echinocandin treatment of rodents infected with P. murina and P. carinii, which target ?-1,3-D-glucan synthesis (BG), depleted the asci which contain BG but large numbers of non-BG expressing life cycle stages remained in the lungs and were unable to proliferate. We further demonstrated that anidulafungin and caspofungin could prevent infection in a prophylactic model, suggesting that formation of asci via the sexual cycle may be required for a productive infection (2). Analysis of gene expression profiles of P. murina in mice treated with anidulafungin, showed strong upregulation of genes associated with sexual replication, though the resulting infections were devoid of asci, the product of sexual reproduction, 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 to facilitate progression through the life cycle leading to a productive infection. We further posit that presence of asci is required for transmission of Pneumocystis infection. In the present proposal, we will explore 2 critical, but unanswered questions that will lead to a deeper knowledge of the life cycle of Pneumocystis, and also suggest potential vulnerabilities for targeted treatment concomitant with anidulafungin therapy: (1) Is sexual replication required for completion of the life cycle of Pneumocystis? Tracking of the replication status of P. murina during prolonged treatment with anidulafungin by global gene analysis, BG content, and microscopic methods will reveal whether the non-BG expressing forms numbers remain: 1) static over time, 2) increase, or 3) decrease; suggesting: 1) the lack of BG blocks replication; 2) that an asexual or alternative replication phase permits survival of the fungi; or 3) the lack of sexual replication results in elimination of the infection. (2) Can sexual replication rebound after cessation of prolonged anidulafungin treatment? Mice will be treated with anidulafungin for up to 8 weeks, with 2 cessation time points. Mice in the cessation groups will be tracked for microscopic, BG content, and gene expression evidence of asci formation and return of the pneumonia while remaining under immunosuppression. Mice in the treated and cessation groups will be evaluated for their ability to transmit the infection and the critical number of asci needed for transmission. All studies will be conducted in male and female mice, recognizing sex as a biological variable. The echinocandins are clinically available in the United States. Current monotherapy with any echinocandin for PCP is not warranted as withdrawal can result in return of the pneumonia. The results of the proposed studies will have immediate clinical relevance by determining the length of time viable Pneumocystis can remain in the lungs with concurrent anidulafungin treatment, providing a rationale for duration of therapy with eradication as the goal. They will also identify whether immunosuppressed mice can transmit the infection after withdrawal of anidulafungin and if there is a critical number of asci needed. The studies will also elaborate the life cycle of Pneumocystis and suggest new target strategies.
Pneumocystis causes an oftentimes lethal pneumonia (PCP) in humans with compromised immune status. Today, this includes not only HIV (+) patients, but also those on a variety of immune- and chemo-therapies. Over 28,000 HIV (+) US veterans receive care from the VA; over 10,000 HIV (-) veterans take anti-TNF-alpha inhibitors for management of chronic arthritides; 40,000 have cancer diagnoses; and 42,000 have COPD in the context of HIV. All are at high risk for PCP. Previous studies supported by the VA allowed us to report several properties unique to echinocandin-treated Pneumocystis, including the depletion of asci, leading to the inability to transmit the infection while under treatment and when given prophylactically, could prevent the infection. We posit that the sexual cycle resulting in asci formation, is necessary for a productive infection. In this proposal, we will explore the mechanisms how an echinocandin, anudulafungin, suppresses the sexual cycle of these fungi and whether prolonged treatment will lead to clearance of the infection.
