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Pneumocystis carinii polyamine catabolism
1Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, New York 10010, USA. merals01@popmail.med.nyu.edu
Abstract:
DL-alpha-Difluoromethylornithine (DFMO) causes polyamines of the AIDS-associated opportunistic pathogen Pneumocystis carinii to diminish 15 times more rapidly than mammalian host cells. The proposed mechanism was that, unlike mammalian cells, P. carinii is unable to regulate polyamine catabolism when synthesis is blocked. To test this, the responses of the polyamine catabolic enzymes spermidine/spermine acetyltransferase (SSAT) and polyamine oxidase (PAO) were determined using a new high-performance liquid chromatography assay to measure the products of these enzymes. The specific activities in untreated Pneumocystis carinii were 1.78 +/- 0.5 pmol min(-1) mg protein(-1) for SSAT, similar to mammalian cells, and 6.42 +/- 0.8 pmol min(-1) mg protein(-1) for PAO, 19% of that of mammalian cells. DFMO treatment for 12 h caused reductions of only 11 and 4% in SSAT and PAO, respectively, despite polyamine reductions of 94, 96, and 90% for putrescine, spermidine, and spermine, respectively. The P. carinii SSAT K(m) value of 25 microM spermidine is 20% of that of mammalian cells, and the PAO K(m) value of 14 nM N(1)-acetylspermidine is 0.01% of that of mammalian cells. Acetylated polyamines continue to be lost from P. carinii even when exposed to DFMO. Collectively, these results support the hypothesis that P. carinii is unable to regulate polyamine catabolism.
Insights
DL-alpha-Difluoromethylornithine (DFMO) rapidly depletes Pneumocystis carinii polyamines. This study shows P. carinii cannot regulate polyamine breakdown, unlike mammalian cells, explaining DFMO
Area of Science:
- Microbiology and Infectious Diseases
- Biochemistry and Molecular Biology
- Drug Discovery and Development
Background:
- Pneumocystis carinii, an AIDS-associated opportunistic pathogen, is sensitive to DL-alpha-Difluoromethylornithine (DFMO).
- DFMO inhibits polyamine synthesis, leading to rapid polyamine depletion in P. carinii compared to mammalian cells.
- A proposed mechanism suggests P. carinii's inability to regulate polyamine catabolism contributes to this sensitivity.
Purpose of the Study:
- To investigate the hypothesis that P. carinii cannot regulate polyamine catabolism when synthesis is blocked by DFMO.
- To compare the activity and kinetic properties of key polyamine catabolic enzymes (SSAT and PAO) in P. carinii and mammalian cells.
Main Methods:
- Developed a high-performance liquid chromatography (HPLC) assay to measure products of spermidine/spermine acetyltransferase (SSAT) and polyamine oxidase (PAO).
- Determined specific activities and kinetic parameters (Km) of SSAT and PAO in untreated and DFMO-treated P. carinii.
- Compared enzyme activities and kinetics with those reported for mammalian cells.
Main Results:
- P. carinii exhibited significantly lower specific activity for polyamine oxidase (PAO) compared to mammalian cells.
- DFMO treatment caused minimal reduction in SSAT and PAO activity despite drastic polyamine depletion in P. carinii.
- P. carinii showed lower Km values for SSAT and PAO substrates than mammalian cells, indicating altered enzyme kinetics.
Conclusions:
- The results support the hypothesis that P. carinii is deficient in regulating polyamine catabolism.
- Impaired polyamine catabolism regulation, coupled with inhibited synthesis, contributes to the rapid polyamine loss observed in DFMO-treated P. carinii.
- This understanding of P. carinii's unique polyamine metabolism could inform therapeutic strategies against Pneumocystis pneumonia.