Transcriptomic and Proteomic Approaches to Finding Novel Diagnostic and Immunogenic Candidates in <i>Pneumocystis</i>

Abstract

<i>Pneumocystis</i> pneumonia is the most common serious opportunistic infection in patients with HIV/AIDS. Furthermore, <i>Pneumocystis</i> pneumonia is a feared complication of the immunosuppressive drug regimens used to treat autoimmunity, malignancy, and posttransplantation rejection. With an increasing at-risk population, there is a strong need for novel approaches to discover diagnostic and vaccine targets. There are multiple challenges to finding these targets, however. First, <i>Pneumocystis</i> has a largely unannotated genome. To address this, we evaluated each protein encoded within the <i>Pneumocystis</i> genome by comparisons to proteins encoded within the genomes of other fungi using NCBI BLAST. Second, <i>Pneumocystis</i> relies on a multiphasic life cycle, as both the transmissible form (the ascus) and the replicative form (the trophozoite [troph]) reside within the alveolar space of the host. To that end, we purified asci and trophs from <i>Pneumocystis murina</i> and utilized transcriptomics to identify differentially regulated genes. Two such genes, <i>Arp9</i> and <i>Sp</i>, are differentially regulated in the ascus and the troph, respectively, and can be utilized to characterize the state of the <i>Pneumocystis</i> life cycle <i>in vivo</i><i>Gsc1</i>, encoding a β-1,3-glucan synthase with a large extracellular domain previously identified using surface proteomics, was more highly expressed on the ascus form of <i>Pneumocystis</i> GSC-1 ectodomain immunization generated a strong antibody response that demonstrated the ability to recognize the surface of the <i>Pneumocystis</i> asci. GSC-1 ectodomain immunization was also capable of reducing ascus burden following primary challenge with <i>Pneumocystis murina</i> Finally, mice immunized with the GSC-1 ectodomain had limited fungal burden following natural transmission of <i>Pneumocystis</i> using a cohousing model.<b>IMPORTANCE</b> The current report enhances our understanding of <i>Pneumocystis</i> biology in a number of ways. First, the current study provided a preliminary annotation of the <i>Pneumocystis murina</i> genome, addressing a long-standing issue in the field. Second, this study validated two novel transcripts enriched in the two predominant life forms of <i>Pneumocystis</i> These findings allow better characterization of the <i>Pneumocystis</i> life cycle <i>in vivo</i> and could be valuable diagnostic tools. Furthermore, this study outlined a novel pipeline of -omics techniques capable of revealing novel antigens (e.g., GSC-1) for the development of vaccines against <i>Pneumocystis</i>.