Pneumocystis pneumonia remains the number one cause of fungal pneumonia in infancy and is a common infection in immunocompromised individuals. Research in the field has been hampered by the inability to culture this fungus leading to a delay in obtaining genomic/transcriptomic information on fungal life cycle, mechanisms of fungal attachment as well as a lack of in vitro antibiotic susceptibility. When placed in media, Pneumocystis murina dies within hours. However we have preliminary data that with P. murina is placed in culture lung tissue form precision cut lung slices (PCLS) that both the troph and ascus can survive up to 14 days. Moreover, we have observed evidence of fungal biofilms in this model. Additional preliminary data shows that this model can be used for in vitro antibiotic susceptibility. Thus the goal of this proposal is to refine this model and test submerged versus air liquid interface model to optimize fungal attachment, survival, and growth. Additionally we will investigate if this model can be used for antibiotic susceptibility testing with both folate metabolism antagonists as well as echinocandins. Furthermore, we will investigate of this model can be used to assess macrophage mediated killing as well as the role of both mannan and β-glucan recognition receptors in this process.