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Characterization of Pneumocystis carinii Preparations Developed for Lipid Analysis
Author(s) -
KANESHIRO EDNA S.,
WYDER MICHAEL A.,
ZHOU LINDA HUA,
ELLIS JAYNE E.,
VOELKER DENNIS R.,
LANGRETH SUSAN G.
Publication year - 1993
Publication title -
journal of eukaryotic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 1066-5234
DOI - 10.1111/j.1550-7408.1993.tb04479.x
Subject(s) - biology , pneumocystis carinii , centrifugation , microbiology and biotechnology , lysis , bacteria , pulmonary surfactant , biochemistry , virology , genetics , human immunodeficiency virus (hiv) , pneumocystis jirovecii
Pneumocystis carinii organisms were isolated from viral antibody‐negative rats that had been infected by intratracheal intubation of organism preparations tested negative for common bacteria and fungi. Infection scores of lungs from infected animals at the time of parasite isolation was > 5 (100‐1,000 organisms/oil immersion field). Electron microscopy of heavily infected lungs revealed that the pathogens adhered to Type I pneumocytes and to each other, resulting in obstructions up to several cell layers thick, which extended into the alveolar lumen. Protocols for purifying the organisms were developed to optimize separation from each other and from host cells, and to optimize preparation purity, recovery efficiency, and organism viability. The study tested mucolytic agents, sieving, various centrifugation speeds, lysis of host cells by osmotic shock and filtration through membranes of different pore diameter. Final preparations contained no intact host cells as determined by light microscopy. Only minor amounts (< 5%) of host debris were detected by electron microscopy. Most organisms and their pellicles were ultrastructurally intact but no longer adhered to one another. The final preparation was characterized biochemically by quantitation of the specific lung surfactant marker surfactant protein A, which indicated > 99.5% purity. The total non‐ P. carinii protein in the final preparation (< 6%, depending on the level of infection) was estimated by the protein content of pelletable material resulting from processing uninfected lungs in an identical manner. Elimination of free cholesterol and phospholipids from host lung tissue was monitored during the purification process. Exogenous stigmasterol, added as an extracellular marker, decreased during the purification process and was undetectable in the final organism preparation. Yields of 10 8 ‐10 9 organisms/rat were routinely obtained. Viability, assessed by the calcein acetoxymethyl ester‐propidium iodide assay, was 80–95%.