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An H(+)-ATPase regulates cytoplasmic pH in Pneumocystis carinii trophozoites
R Docampo1, N M Vanderheyden, M M Shaw
1Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign 61801, USA.
Abstract:
Pneumocystis carinii is an opportunistic fungus which causes interstitial pneumonia in patients with acquired immunodeficiency syndrome (AIDS). Cytoplasmic pH (pHi) regulation in short-term-cultured P. carinii trophozoites was studied using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(-6)-carboxyfluorescein. With an extracellular pH of 7.4, the mean baseline pHi of P. carinii trophozoites was 7.40 +/- 0.10 (n = 8). This steady-state pHi was not significantly affected in the absence of extracellular Na+ or K+. Moreover, steady-state pHi was maintained in the nominal absence of HCO3- and was not affected by the Cl-/HCO(3-)-exchanger inhibitor 4, 4'-di-isothiocyanato-dihydrostilbene-2, 2'-disulphonic acid (100 microM), or the Na+/H(+)-exchanger inhibitor N-ethyl-N-isopropylamiloride (100 microM). In contrast, the general inhibitors of ATPases, N-ethylmaleimide (1 mM), and dicyclohexylcarbodi-imide (100 microM), and the inhibitor of yeast H(+)-ATPase, diethylstilbestrol (12.5-100 microM), decreased pHi, while the K+/H(+)-ATPase inhibitor omeprazole (50-400 microM), and the vacuolar-type H(+)-ATPase inhibitor bafilomycin A1 (1-5 microM) only produced a dose-dependent acidification of the cells when used at high concentrations. In addition, steady-state pHi depended on the availability of cellular ATP, since it was decreased by the ATP synthase inhibitors oligomycin (1 microgram/ml) and sodium azide (1 mM), and by the uncoupler of oxidative phosphorylation carbonyl cyanide p-trifluorophenylhydrazone (1 microM), agents that were able to deplete significantly the intracellular ATP levels. Taken together, these results are consistent with an important role of an H(+)-ATPase similar to those found in other fungi in the regulation of pHi homoeostasis in P. carinii trophozoites.
Insights
Pneumocystis carinii uses an H(+)-ATPase to regulate its internal pH, crucial for survival in AIDS patients. This study highlights the role of ATP-dependent proton pumps in maintaining pH homeostasis in this opportunistic fungus.
Area of Science:
- Medical Mycology
- Cell Physiology
- Biochemistry
Background:
- Pneumocystis carinii causes opportunistic pneumonia in immunocompromised individuals, particularly those with AIDS.
- Understanding the cellular mechanisms of P. carinii, such as pH regulation, is vital for developing targeted therapies.
Purpose of the Study:
- To investigate the mechanisms of cytoplasmic pH (pHi) regulation in Pneumocystis carinii trophozoites.
- To determine the role of ion transporters and ATPases in maintaining pHi homeostasis.
Main Methods:
- Short-term culture of P. carinii trophozoites.
- Measurement of intracellular pH using the fluorescent dye BCECF.
- Treatment with various ion transporter inhibitors (e.g., amiloride, DIDS) and ATPase inhibitors (e.g., NEM, DCCD, diethylstilbestrol, omeprazole, bafilomycin A1).
- Assessment of cellular ATP levels and the impact of ATP depletion on pHi.
Main Results:
- Baseline pHi of P. carinii trophozoites was maintained at 7.40 +/- 0.10.
- pHi regulation was independent of extracellular Na+, K+, and HCO3-.
- Inhibitors of general ATPases and yeast H(+)-ATPase significantly decreased pHi.
- Vacuolar-type H(+)-ATPase inhibitors caused acidification at high concentrations.
- pHi was dependent on cellular ATP levels, decreasing with inhibitors of ATP synthase and oxidative phosphorylation uncouplers.
Conclusions:
- P. carinii trophozoites rely on an H(+)-ATPase, similar to those in other fungi, for pHi homeostasis.
- Cellular ATP availability is critical for maintaining steady-state pHi.
- These findings provide insights into the fundamental physiology of P. carinii and potential therapeutic targets.