Intracellular pH and pH gradients of Leishmania donovani amastigotes and promastigotes were determined over a broad range of extracellular pH values. Intracellular pH was determined by 31p NMR and by equilibrium distribution studies with 5,5-dimethyloxazolidine-2,4-dione or methylamine. Promastigotes maintain intracellular pH values close to neutral between extracellular pH values of 5.0 and 7.4. Amastigote intracellular pH is maintained close to neutral at external pH values as low as 4.0. Both life stages maintain a positive pH gradient to an extracellular pH of 7.4, which is important for active transport of substrates. Treatment with ionophores, such as nigericin and carbonyl cyanide mchlorophenylhydrazone and the ATPase inhibitor dicyclohexylcarbodiimide, reduced pH gradients in both stages. Maintenance of intracellular pH in the physiologic range is especially relevant for the survival ofthe amastigote in its acidic in vivo environment. where 59 is a factor for the conversion of ApH to mV and is equal to (2.3 RT)/F; R, T, and F are the gas constant, absolute temperature, and the Faraday constant, respectively (4). Knowledge of the pHi enables the calculation of ApH and ultimately the magnitude of the driving force for active transport. Such knowledge also provides insight into the homeostatic mechanisms of these parasites, which encounter substantial changes of pH in their extracellular environments.Here we report pH, values for Leishmania donovani over a broad range of extracellular pH values (pHe). Promastigote pHi was determined by two methods: the equilibrium distribution of the weak acid 5,5-dimethyl-oxazolidine-2,4-dione (DMO) and the chemical shift of intracellular inorganic phosphate as determined by 31P NMR spectrometry. The high density of cells required for NMR prohibited the use of this technique for amastigote pHi determination; therefore, amastigote pH1 values were determined by only the DMO method. MATERIALS AND METHODSMaintenance and Preparation of Amastigotes. Amastigotes of L. donovani Sudan strain IS were maintained by serial passage in female Syrian golden hamsters. The organism was passaged by i.p. injection of spleen homogenates containing 1 x 107 parasites. Hamsters were sacrificed 8 weeks postinfection, and parasites were isolated from liver and spleens by a described method (5).Maintenance and Preparation of Promastigotes. Promastigotes of L. donovani IS were maintained at 260C in medium 199 (GIBCO) supplemented with 15% fetal bovine serum. Cells were harvested during stationary phase growth by centrifugation at 1100 x g for 10 min and washed twice in a basal salts solution (6).Determination of pH, by DMO or Methylamine. The pHi of L. donovani promastigotes and amastigotes was determined from the distribution of the weak acid DMO or the weak base methylamine as described by Rottenberg (7), a method based on the principle that the nondissociated acid is fully permeable to the membrane but is impermeable in its ionic form. Therefore, as long as the pHi is more alkaline relati...

Pneumocysterol [(24Z)-ethylidenelanost-8-en-3␤-ol], a rare sterol detected in the opportunistic pathogenCommunicated by William Trager, The Rockefeller University, New York, NY, November 2, 1998 (received for review February 4, 1998 ABSTRACTPneumocystis carinii pneumonia (PcP) remains among the most prevalent opportunistic infections among AIDS patients. Currently, drugs used clinically for deep mycosis act by binding ergosterol or disrupting its biosynthesis. Although classified as a fungus, P. carinii lacks ergosterol. Instead, the pathogen synthesizes a number of distinct ⌬ 7 , 24-alkylsterols, despite the abundance of cholesterol, which it can scavenge from the lung alveolus. Thus, the pathogen-specific sterols appear vital for organism survival and proliferation. In the present study, high concentrations of a C 32 sterol were found in humanderived P. carinii hominis. The definitive structural identities of two C-24 alkylated lanosterol compounds, previously not reported for rat-derived P. carinii carinii, were determined by using GLC, MS, and NMR spectroscopy together with the chemical syntheses of authentic standards. The C 31 and C 32 sterols were identified as euphorbol (24-methylenelanost-8-en-3␤-ol) and pneumocysterol [(24Z)-ethylidenelanost-8-en-3␤-ol], respectively. The identification of these and other 24-alkylsterols in P. carinii hominis suggests that (i) sterol C-24 methyltransferase activities are extraordinarily high in this organism, (ii) 24-alkylsterols are important components of the pathogen's membranes, because the addition of these side groups onto the sterol side chain requires substantial ATP equivalents, and (iii) the inefficacy of azole drugs against P. carinii can be explained by the ability of this organism to form 24-alkysterols before demethylation of the lanosterol nucleus. Because mammals cannot form 24-alkylsterols, their biosyntheses in P. carinii are attractive targets for the development of chemotherapeutic strategies against this opportunistic infection.Sterols and their biosyntheses are excellent targets for chemotherapeutic attack against infectious microbes, especially the fungi. Polyene antibiotics such as amphotericin B bind avidly to ergosterol in fungal cell membranes. After the sterol-drug complexes aggregate, large pores in the membranes are formed, dissipating ion gradients. Fluconazole and some other compounds routinely used clinically for systemic mycosis target ergosterol biosynthesis at nuclear demethylation steps. Ergosterol was not detected in Pneumocystis carinii carinii that was isolated and purified from the lungs of corticosteroid-immunosuppressed rats. In this respect, the pathogen appears to be unlike higher fungi. However, the organism synthesizes its own distinct sterols, e.g., fungisterol (24-methylcholest-7-en-3␤-ol and 24-ethylcholest-7-en-3␤-ol; refs. 1-4). Parasites generally scavenge sterols (e.g., cholesterol) from the host and utilize them for membrane formation and other cell functions. If host sterols do not fulfill the precise stereochemi...

No abstract