Uptake and Metabolism of L‐Serine by Pneumocystis carinii carinii

Serine is an important amino acid that is utilized in the biosyntheses of proteins and lipids. It is directly incorporated into the head group of phosphatidylserine, which in turn can be converted to other phospholipids. Also, it is required for the formation of long chain bases, precursors of sphingolipids. Uptake and incorporation of radiolabeled serine into both lipids and acid-precipitable material were demonstrated in Pneumocystis carinii carinii organism preparations freshly isolated from infected rat lungs. Radioactivity in proteins was about double that observed in lipids. Liquid scintillation spectrometry of metabolically radiolabeled lipids separated by thin-layer chromatography showed 53% of the total radioactivity were in phosphatidylserine, 12% in phosphatidylethanolamine, 24% in ceramides, and 1 1% in long chain bases and other compounds. Four long chain bases were detected by thin-layer chromatography in hydrolyzed P. carinii ceramides metabolically labeled with radioactive serine. Phytosphingosine and dihydrosphingosine were ten- tatively identified by their migrations on thin-layer plates. Radiolabeled ethanolamine was incorporated into P. carinii phosphatidyl- ethanolamine, but relatively low incorporation of radiolabeled choline into phosphatidylcholine occurred. The observations made in this study indicated that P. carinii has the biosynthetic capacity to metabolize phospholipid head groups and to de novo synthesize sphingolipids. L-Cycloserine and @-C1-D-alanine, inhibitors of long chain base synthesis, reduced the incorporation of serine into P. carinii long chain bases and ceramides, which supported the conclusion that the pathogen synthesizes sphingolipids. Supplementary key words. P-Cl-D-Alanine, ceramides, choline, cycloserine, ethanolamine, long chain bases, 2-Br-palmitate, phos- pholipids, sphingolipids. E UMOCYSTIS carinii pneumonitis has emerged in the p" last decade as a serious complication in immunosup- pressed individuals. The pneumonitis resulting from the pro- liferation of this pathogen in the lung is the immediate cause of death of many AIDS patients in North America and Europe. The pathogen proliferates in the lung alveolus in a surfactant milieu comprised of high concentrations of phosphatidylcholine (PC; 78.8% in rat), dominated by palmitic acid (16:O). Other rat lung surfactant phospholipids include phosphatidylglycerol, 9.2%; phosphatidylethanolamine (PE), 4.3%; phosphatidylino- sitol (PI), 3.3%; sphingomyelin (SM), 1.5%; phosphatidylserine (PS), 1.5%; and lyso PC, 1.5% (22). Host lung lipids therefore provide important nutrients and energy sources for the organ- ism. The absence of continuous axenic cultures of P. carinii has been a major hinderance for studying this organism causing a paucity of definitive biochemical data. Direct lipid analyses of the pathogen isolated from infected host lungs are particularly problematic because of potential host surfactant contamination. However, in recent years, protocols for obtaining highly pure and viable organism preparations from infected rat lungs have been developed (lo, 11, 13, 141. Characterizations of these pu- rified preparations according to a number of morphologic and biochemical criteria, including surfactant components, have aided in the interpretation of biochemical data obtained with the isolated organisms ( 141. The amino acid serine is not only a precursor for protein synthesis, but is also used for the formation of various lipids. In lipid biosynthesis, serine is incorporated directly into the head group of PS. Metabolism of phospholipid head groups converts these molecules into different phospholipids. Thus, PE can be formed by decarboxylation of PS, and PC can be formed by methylation of PE. In addition to the formation of phos- pholipid head groups, serine is an important precursor of sphin-