Pneumocystis melanins confer enhanced organism viability

Abstract

Pneumocystis continues 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 in Pneumocystis. In the present study, we sought to further characterize these components and define the function for these melanins. Melanins extracted from Pneumocystis and melanized Pneumocystis cells 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 across Pneumocystis host species, including mouse-, ferret-, and human-derived Pneumocystis organisms, as well as Pneumocystis carinii derived from rat. Using immunoelectron microscopy, melanins were found to localize to the cell wall and cytoplasm of P. carinii cysts, as well as to intracystic bodies within mature cysts. Next, the role of melanins on the maintenance of Pneumocystis viability 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 of Pneumocystis, we observed that strongly melanized Pneumocystis retained viability to a greater degree when exposed to UV irradiation or desiccation compared to less-pigmented organisms. These studies support our previous identification of Pneumocystis melanins across the genus, further characterize these Pneumocystis components, and demonstrate that melanins protect Pneumocystis from environmental stressors.

FIG. 1.

l-DOPA enhances melanization of P. carinii. Panels A and B depict Pneumocystis incubated in the presence or absence of l-DOPA (melanin substrate). Panel A shows the dramatic difference between natively melanized Pneumocystis (left tube) and highly melanized Pneumocystis (driven by l-DOPA [right tube]). Panel B depicts transmission electron micrographs of native melanized compared to panel C showing the highly melanized Pneumocystis resulting from incubation with l-DOPA. Panel D shows that glyphosate inhibition of Pneumocystis phenoloxidase activity impairs the l-DOPA-driven generation of Pneumocystis melanin pigment. The first bar represents P. carinii (Pc) alone, the second bar represents l-DOPA alone, and bars 3 through 10 show results for P. carinii incubated with l-DOPA in the presence of the phenoloxidase inhibitor glyphosate at the indicated concentrations. Error bars represent the standard deviations.

FIG. 2.

ESR spectra of Pneumocystis melanins. Panel A depicts the ESR spectra for P. carinii melanin ghosts, panel B depicts the spectra for highly melanized P. carinii whole cells, and panel C depicts the spectra for highly melanized P. murina whole cells.

FIG. 3.

Immunofluorescence visualization of Pneumocystis melanins. (A) P. carinii (rat); (B) P. jirovecii (human); (C) P. murina (mouse); (D) ferret-derived Pneumocystis. For each set of images, panel 1 represents staining with the anti-melanin MAb 6D2 and viewed under fluorescence microscopy, pPanel 2 is the phase-contrast image of the identical field shown in panel 1, panel 3 represents staining with nonimmune isotype control antibody and viewed under fluorescence microscopy, and panel 4 is the phase-contrast image of the identical field shown in panel 3.

FIG. 4.

Immunoelectron microscopic localization of Pneumocystis melanins. Melanins were visualized by using MAb 6D2 antibody and immunogold particle detection. Panel A demonstrates a Pneumocystis cyst containing four visible intracystic bodies, and panel B is a magnified portion of panel A as indicated by the inset box in panel A.

FIG. 5.

Pneumocystis melanins protect organisms viability. Shown are the Pneumocystis heat shock protein 70 content (in arbitrary units) as a means to assess the viability of native melanized and highly melanized (l-DOPA driven) Pneumocystis after exposure to environmental stressors. The left bars depict the relative amounts of Pneumocystis heat shock protein 70 in highly melanized and native melanized Pneumocystis after treatment with UV irradiation. Heat shock protein 70 measurements were collected 48 h after UV irradiation treatment. The right bars depict the relative amounts of Pneumocystis heat shock protein 70 in highly melanized and native melanized Pneumocystis after desiccation for 48 h. ★, P < 0.004; ★★, P < 0.008 (comparing native melanized to highly melanized Pneumocystis).