Continuous axenic cultivation of
            <i>Pneumocystis carinii</i>

Merali, Salim; Frevert, Ute; Williams, Jonathan; Chin, K; Bryan, Richard T.; Clarkson, Allen B.

doi:10.1073/pnas.96.5.2402

Cited by 87 publications

(94 citation statements)

References 27 publications

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“…AdoMet is synthesized in a one-step condensation of methionine and ATP catalyzed by AdoMet synthetase (methionine ATP transferase, MAT, EC 2.5.1.6). Pneumocystis is highly unusual in lacking this enzymatic activity (22). With the exception of Pneumocystis, a Rickettsia (23,24) and an aberrant protozoan (25,26), every other cell studied is able to synthesize AdoMet.…”

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confidence: 99%

Effect of Nicotine on Lung S-Adenosylmethionine and Development of Pneumocystis Pneumonia

Shivji

Burger

Moncada

et al. 2005

Journal of Biological Chemistry

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Because S-adenosylmethionine (AdoMet) is required by Pneumocystis carinii in vitro, Pneumocystis infection depletes plasma AdoMet of rats and humans, nicotine reduces AdoMet of guinea pig lungs, and smoking correlates with reduced episodes of Pneumocystis pneumonia (PCP) in AIDS patients, we tested the effect of nicotine treatment on PCP using a rat model. Intraperitoneal infusion of 400 g of R-(؉) nicotine kg ؊1 h ؊1 intraperitoneal for 21 days caused a 15-fold reduction in lung AdoMet although neither plasma nor liver were changed. Infusion of 4 and 400 g kg ؊1 h ؊1 into immunosuppressed rats, beginning when rats were inoculated with P. carinii, caused 85 and 99.88% reductions, respectively, in P. carinii cysts at sacrifice 21 days later; P. carinii nuclei were reduced by 91.2 and >99.99%, respectively. This effect was reversed by concomitant administration of AdoMet with nicotine. Treatment with AdoMet alone increased infection intensity. We conclude that AdoMet is a critical and limiting nutrient for Pneumocystis thus can serve as a therapeutic target for PCP. Regarding the mechanism, nicotine treatment caused no change in rat lung activity of AdoMet synthesizing methionine ATP transferase activity nor was there any evidence of increased AdoMet utilization for methylation reactions. Except of a doubling of putrescine, nicotine treatment also did not change lung polyamine content. However, key polyamine anabolic and catabolic enzymes were upregulated, and there were corresponding changes in polyamine metabolic intermediates. We conclude that chronic nicotine treatment increases lung polyamine catabolic/anabolic cycling and/or excretion leading to increased AdoMet-consuming polyamine biosynthesis and depletion of lung AdoMet.

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confidence: 99%

Effect of Nicotine on Lung S-Adenosylmethionine and Development of Pneumocystis Pneumonia

Shivji

Burger

Moncada

et al. 2005

Journal of Biological Chemistry

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“…Most of the work reported here was done with cells grown in a new culture system for P. carinii (17). One critical experiment that had been done previously with cells isolated from infected animals was repeated with cultured cells for comparison and for validation of other results obtained with cultured cells; i.e.…”

Section: Resultsmentioning

confidence: 99%

“…(12) were used for development of the N 1 -acetylspermidine HPLC method. All other experiments were performed with cells grown in a newly developed continuous axenic culture system for P. carinii (17). Extracts were prepared by suspending P. carinii in NKPD buffer (2.68 mM KCl, 1.47 mM KH 2 PO 4 , 51.1 mM Na 2 HPO4, 7.43 mM NaH 2 PO 4 , 62 mM NaCl, 1 mM EDTA, and 1.0 mM dithiothreitol), sonicating for 20 min at 40 watts and 70% duty cycle (Heat System, Ultrasonics Inc., Plainview, NY), clarifying by centrifugation at 30,000 ϫ g for 30 min, and dialyzing the supernatant against two changes of 1000 ml of NKPD buffer.…”

Section: Methodsmentioning

confidence: 99%

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Pneumocystis carinii Polyamine Catabolism

Merali

1999

Journal of Biological Chemistry

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DL-␣-Difluoromethylornithine (DFMO) causes polyamines of the AIDS-associated opportunistic pathogenPneumocystis 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 M 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.Polyamines are small molecular weight, positively charged compounds that are ubiquitous in all living cells. The most important polyamines are putrescine, spermidine, and spermine. Although much of the evidence is indirect, it is clear that polyamines play many important roles in cell growth and differentiation (1-3). Consequently, inhibition of polyamine synthesis, as well as other means of manipulating polyamine concentrations, has been used as therapeutic approaches for the treatment of cancer and infectious diseases.The most commonly used compound for manipulating polyamine metabolism is difluoromethylornithine (DFMO, 1 eflornithine, Ornidyl ® ). DFMO is an enzyme-activated irreversible inhibitor of ornithine decarboxylase, an enzyme that catalyzes the first step in polyamine synthesis and is rate-limiting. Thus DFMO can completely block de novo polyamine synthesis. This drug is currently approved for treatment of human African sleeping sickness (4) and has been used as an investigational drug for cancer chemotherapy and for treatment of Pneumocystis carinii pneumonia (PCP), the most common opportunistic infection associated with AIDS. DFMO is effective against PCP, in an animal model (5) and in patients (6). Although clinical trials showed this drug to be less effective than drugs already approved, it was also less toxic (7). Animal studies, however, demonstrated that continuous infusion of DFMO greatly enhances the anti-PCP activity as compared with the intermittent dosage obtained by inclusion of the drug in the drinking water (8). The activity of DFMO against PCP demonstrates that polyamine metabolism is a valid therapeutic target for this disease. A better understanding of P....

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“…Attempts to cultivate the organism in cell free media, as usual for fungi, succeeded in ten-fold amplifications of the number of cells. However, continuous axenic cultivation of P. carinii is difficult, and has been obtained only by few groups (Merali et al 1999).…”

Section: The Life Cyclementioning

confidence: 99%