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%.

The lipids of purified preparations of Pneumocystis carinii carinii freshly isolated from infected rats were analyzed and compared with those of whole lungs from normal and methylprednisolone-immunosuppressed uninfected rats. In this study, the neutral lipid fraction was examined in detail; the relative concentrations of individual classes making up this fraction were quantified. Of particular interest was the nature of the organism's ubiquinone (coenzyme Q, CoQ) fraction because atovaquone, a hydroxynaphtho-quinone (566C80) analog of ubiquinone, is efficacious in the treatment of P. carinii pneumonia. The ubiquinone concentration in both P. carinii and lung tissues was relatively low compared to that present in rat heart and liver tissues. Two homologs were identified in the organism: CoQ10 was the predominant homolog with lesser amounts of CoQ9 present. In contrast, the lungs of normal and immunosuppressed uninfected rats had CoQ9 and lesser amounts of CoQ8, but no detectable CoQ10. Furthermore, radiolabeled mevalonic acid was incorporated in vitro into the ubiquinone fraction of P. carinii indicating that the organism has the de novo branch of the isoprenoid biosynthetic pathway leading to polyprenyl formation. Hence, it was concluded that CoQ10 (if not both CoQ10 and CoQ9) in P. carinii was not scavenged from the host but was synthesized by the organism. Although lung tissues contained substantial free fatty acids, the organism was enriched in these lipids. The high concentration of free fatty acids and relatively low level of triglycerides in P. carinii suggest that fatty acids may represent major carbon sources for ATP production by the organism.

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Natural products remain an important source of new therapeutics for emerging drug-resistant pathogens like Candida albicans, which particularly affects immunocompromised patients. A bioactive 3-decalinoyltetramic acid, pyrrolocin A, was isolated from extracts of a novel Amazonian fungal endophyte, E6927E, of the Diaporthales family. The structure of the natural product was solved using NMR and CD spectroscopy and it is structurally related to the fungal setins, equisetin and phomasetin, which are well-characterized tetramic acid antibiotics specific for Gram-positive organisms. We show that the compound inhibits growth of Staphylococcus aureus and Enterococcus faecalis. It shows selective and potent bioactivity against fungal strains, with an MIC of 4 µg/mL for C. albicans, 100 µg/mL for Aspergillus sp. and greater than 100 µg/mL for Saccharomyces cerevisiae. Further, the compound is less toxic to mammalian cells (IC 50 = 150 µg/mL), with an inhibitory concentration greater than forty times that for C. albicans. Pyrrolocin A retained potent activity against eight out of seventeen strains of clinical Candida sp. isolates tested.