Isolation and expression of the Pneumocystis carinii thymidylate synthase gene.

Edman, Ursula; Edman, Jeffrey C.; Lundgren, Bettina; Santi, Daniel V.

doi:10.1073/pnas.86.17.6503

Cited by 122 publications

(56 citation statements)

References 33 publications

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“…In general, the arguments in favor of classifying P. carinii as a protozoan are the amoeboid appearance of the trophic form, susceptibility to antiprotozoal drugs such as pentamidine isethionate or trimethoprim-sulfamethoxazole, insusceptibility to antifungal drugs such as amphotericin B, and the lack of ergosterol in Pneumocystis cell membranes. Arguments in favor of including P. carinii among the fungi include similarities of the sporogenous state to ascospore formation in yeasts, poorly developed mitochondria which show ultrastructural similarities to fungal mitochondria, lack of the internal organelles typically found in protozoa, the encoding of the enzymes thymidylate synthase and dihydrofolate synthase by physically separate genes, and the nucleic acid sequence homologies described above (10,30).…”

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confidence: 99%

Phytosterols are present in Pneumocystis carinii

Furlong

Samia

Rose

et al. 1994

Antimicrob Agents Chemother

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Although originally classified as a protozoan, Pneumocystis carinii is now considered to have fungal characteristics. Drugs typically used for the treatment of fungal infections target ergosterol. Because P. carinii is an important pathogen in AIDS and other immunocompromised patients, knowledge of the sterol content of this organism may be useful as a basis for developing new treatment strategies or for improving diagnosis. P. carinii organisms were harvested from infected rat lungs and were purified by filtration. Control preparations from uninfected animals were identically prepared. Lipids were extracted from the organisms and control preparations and were separated into neutral lipid, glycolipid, and phospholipid fractions by silicic acid chromatography. The neutral lipid fraction was further treated by alkaline hydrolysis and was analyzed by reversed-phase high-pressure liquid chromatography (HPLC), gas chromatography (GC), and GC-mass spectrometry (GC-MS). As shown by HPLC, the neutral lipid fraction from infected rats contained a minimum of six peaks, while in control preparations a single peak with a retention time identical to that of cholesterol was observed. The predominant sterol in these preparations was positively identified by GC-MS as cholesterol and constituted 80 to 90% of the total. The remaining peaks had relative retention times similar to those of phytosterols by both HPLC and GC, and the similarity of these sterols to those derived from plants and fungi was confirmed by MS. Ergosterol, however, was not present. These results provide further evidence for a close phylogenetic relationship between P. carinii and fungi and suggest that these sterols could be used as targets for drug development and for improving diagnosis.Pneumocystis carinii is one of the leading causes of opportunistic infection in AIDS patients (30). Despite the importance of this microorganism as an opportunistic pathogen, much basic information about the parasite is lacking. While originally classified phylogenetically as a protozoan, analyses of P. carinii DNA and mitochondrial and rRNA sequences have generally shown a higher degree of homology to the mitochondrial and rRNA sequences of fungi (yeasts) than to those of protozoa (9,13,27,31,34,35). The exact relationship remains to be determined. Analyses of 5S RNA have suggested a somewhat close phylogenetic relationship to members of the Rhizopoda-Myxomycota-Zygomycota group of "protista fungi." Other studies have suggested similarities to Saccharomyces, Candida, and Neurospora spp. and "red yeast" fungi. In general, the arguments in favor of classifying P. carinii as a protozoan are the amoeboid appearance of the trophic form, susceptibility to antiprotozoal drugs such as pentamidine isethionate or trimethoprim-sulfamethoxazole, insusceptibility to antifungal drugs such as amphotericin B, and the lack of ergosterol in Pneumocystis cell membranes. Arguments in favor of including P. carinii among the fungi include similarities of the sporogenous state to ascospore format...

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mentioning

confidence: 99%

Phytosterols are present in Pneumocystis carinii

Furlong

Samia

Rose

et al. 1994

Antimicrob Agents Chemother

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“…6 Until 1988, N. caninum was misdiagnosed as Toxoplasma gondii because of close structural similarities. Neosporosis has not been recognized to cause clinical disease in adult livestock species.…”

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“…A recent published report of the Pneumocystis TS gene 14 shows only an approximately 80% identity between rat and human P. carinii, but the non-identities were located primarily in the introns. The outer primers of this PCR assay are, however, located in conserved regions of the TS amino acid sequence, 6 and the inner (nested) primers are designed from coding parts of the TS gene. The modified (decreased) annealing temperature of the nested PCR, thus reducing the conditions for stringency, indicates that P. carinii of the dog is genetically distinct from human and rat P. carinii, and it was assumed that the heterogeneity may consist of single base exchanges of the inner primer binding sites of the gene.…”

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confidence: 99%

“…k The PCR amplifications were performed by a nested PCR assay (Olsson et al, manuscript in preparation) based on the thymidylate synthase (TS) gene sequence of rat P. carinii. 6 A single-step PCR was originally developed, 20 by which specific rat and human P. carinii DNA can be detected. The nested PCR assay amplified by 35 + 35 cycles a DNA fragment of 131 bp, as predicted from the TS gene sequence, and was detected on ethidium bromide-stained agarose gel (3% Nusieve-1% agarose 1 ).…”

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confidence: 99%

“…Generally due to the high sensitivity of the PCR, the clinical significance of a positive result must be considered with other findings. A modified nested PCR amplification protocol was used, based on the TS gene sequence of rat P. carinii, 6 for detecting a P. carinii-specific DNA fragment in the lungs of a dog. By use of a nested PCR instead of a labeled P. carinii probe to confirm the specificity of the first PCR, laborious hybridization procedures are avoided, and built-in specificity control is obtained with the inner primers.…”

mentioning

confidence: 99%

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Pneumocystis Carinii Pneumonia in Dogs -a Diagnostic Challenge

Sukura

Saari²,

Järvinen³

et al. 1996

J VET Diagn Invest

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Sources and manufacturers 8:124-130 (1996) Pneumocystis carinii pneumonia in dogs -a diagnostic challenge Antti Sukura, Seppo Saari, Anna-Kaisa Järvinen, Mats Olsson, Marjatta Kärkkäinen, Timo IlvesniemiPneumocystis carinii is an extracellular opportunistic pathogen of the lung with an uncertain taxonomic status. It was classified initially as a protozoan, but more recent DNAFrom the Departments of Basic Veterinary Sciences (Sukura, Saari) and Clinical Sciences (Järvinen, Kärkäinen,

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Topley &Amp; Wilson's Microbiology and Microbial Infections

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Isolation and expression of the Pneumocystis carinii thymidylate synthase gene.

Abstract: The

Cited by 122 publications

References 33 publications

Phytosterols are present in Pneumocystis carinii

Phytosterols are present in Pneumocystis carinii

Pneumocystis Carinii Pneumonia in Dogs -a Diagnostic Challenge

Pneumocystispneumonia

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