Icenhour, Crystal R.

ABSTRACTPneumocystiscontinues to represent an important opportunistic fungal pathogen of those with compromised immunity. Thus, it is crucial to identify factors that affect its viability and pathogenicity. We previously reported the first identification of melanins inPneumocystis. In the present study, we sought to further characterize these components and define the function for these melanins. Melanins extracted fromPneumocystisand melanizedPneumocystiscells were analyzed by electron spin resonance spectroscopy, revealing spectra consistent with melanins from other fungi. Immunofluorescence assays using anti-melanin monoclonal antibodies showed that melanins are widely present acrossPneumocystishost species, including mouse-, ferret-, and human-derivedPneumocystisorganisms, as well asPneumocystis cariniiderived from rat. Using immunoelectron microscopy, melanins were found to localize to the cell wall and cytoplasm ofP. cariniicysts, as well as to intracystic bodies within mature cysts. Next, the role of melanins on the maintenance ofPneumocystisviability was determined by using quantitative reverse transcription-PCR measurement of the heat shock protein mRNA under adverse environmental conditions. Using a new method to promote the melanization ofPneumocystis, we observed that strongly melanizedPneumocystisretained viability to a greater degree when exposed to UV irradiation or desiccation compared to less-pigmented organisms. These studies support our previous identification ofPneumocystismelanins across the genus, further characterize thesePneumocystiscomponents, and demonstrate that melanins protectPneumocystisfrom environmental stressors.

ABSTRACT Fluorescein isothiocyanate-labeled monoclonal antibodies specific for fungal melanin were used in this study to visualize melanin-like components of the Pneumocystis carinii cell wall. A colorimetric enzyme assay confirmed these findings. This is the first report of melanin-like pigments in Pneumocystis .