Margaret M. Shaw

Pneumocystis carinii is resistant to imidazole antifungal agents.

Because Pneumocystis carinii is closely related to fungi, drugs useful for treating mycoses have been considered for use in the treatment of P. carinii pneumonia. Six antifungal imidazole drugs were tested for their activities against P. carinii in a short-term culture screen and in animals. None of the imidazoles tested was effective in inoculated infected rats, and only miconazole showed slight effects in culture at the high concentration of 10 micrograms/ml. Analysis of cell membranes from culture-grown P. carinii showed that ergosterol, the target for this class of antifungal agents, was absent, so that the lack of effect of these agents is rational.

Evaluation of potent inhibitors of dihydrofolate reductase in a culture model for growth of Pneumocystis carinii.

Many antifolates are known to inhibit dihydrofolate reductase from murine Pneumocystis carinii, with 50% inhibitory concentrations (IC50s) ranging from 10(-4) to 10(-11) M. The relationship of the potency against isolated enzyme to the potency against intact murine P. carinii cells was explored with 17 compounds that had proven selectivity for or potency against P. carinii dihydrofolate reductase. Pyrimethamine and one analog were inhibitory to P. carinii in culture at concentrations two to seven times the IC50s for the enzyme, suggesting that the compounds may enter P. carinii cells in culture. Methotrexate was a potent inhibitor of P. carinii dihydrofolate reductase, but the concentrations effective in culture were more than 1,000-fold higher than IC50s for the enzyme, since P. carinii lacks an uptake system for methotrexate. Analogs of methotrexate in which chlorine, bromine, or iodine was added to the phenyl ring had improved potency against the isolated enzyme but were markedly less effective in culture; polyglutamation also lowered the activity in culture but improved activity against the enzyme. Substitution of a naphthyl group for the phenyl group of methotrexate produced a compound with improved activity against the enzyme (IC50, 0.00019 microM) and excellent activity in culture (IC50, 0.1 microM). One trimetrexate analog in which an aspartate or a chlorine replaced two of the methoxy groups of trimetrexate was much more potent and was much more selective toward P. carinii dihydrofolate reductase than trimetrexate; this analog was also as active as trimetrexate in culture. These studies suggest that modifications of antifolate structures can be made that facilitate activity against intact organisms while maintaining the high degrees of potency and the selectivities of the agents can be made.

Albendazole inhibits Pneumocystis carinii proliferation in inoculated immunosuppressed mice.

Albendazole, a benzimidazole derivative widely used for treating helminth infections, was successfully used to treat and prevent development of Pneumocystis carinii pneumonia in transtracheally inoculated immunosuppressed mice. For treatment, 3 weeks postinoculation, albendazole at 300 and 600 mg/kg of body weight per day was administered in food for 3 weeks. For prophylaxis, albendazole was begun on the same day as inoculation at 300 mg/kg/day for 7 days, and then the dose was reduced to 150 mg/kg/day for 35 additional days. With these regimens, albendazole was effective both for treatment and prophylaxis. Both dexamethasone-immunosuppressed and L3T4+ monoclonal antibody-immunosuppressed mouse models were used, and albendazole inhibited P. carinii infection in both.

Activities and Conformational Fitting of 1,4-Naphthoquinone Derivatives and Other Cyclic 1,4-Diones Tested In Vitro against Pneumocystis carinii

ABSTRACT Atovaquone is a chemotherapeutic agent used to treat pneumonia caused by Pneumocystis carinii in some immunocompromised patients. A set of cyclic 1,4-diones were tested in vitro for ability to inhibit growth of P. carinii, including 22 variously substituted 1,4-naphthoquinones, one bis-1,4-naphthoquinone, and three other quinones. For comparison, the antipneumocystic primaquine and its 5-hydroxy-6-desmethyl metabolite were also tested. At 1.0 μg/ml, seven compounds inhibited growth by at least 39%, with atovaquone at 92%; of these seven, five are 2-hydroxy-1,4-naphthoquinones, while one is a 2-chloro- and another is a 2-methyl-1,4-naphthoquinone. At 0.1 μg/ml, however, the most active compound tested was the primaquine metabolite, which inhibited growth by more than 42% at this concentration. To ascertain a structure-activity relationship, all 1,4-naphthoquinones were compared conformationally by means of computer-based molecular modeling (Spartan) incorporating the Sybyl force field. Without exception, for all 21 monomers tested, the substituent at position 3 of the 1,4-naphthoquinone favored activity most strongly when it simultaneously occupied (i) space centered at about 3 Å from position 3, without projecting steric bulk from the area encompassed by atovaquones cyclohexyl ring, and (ii) roughly planar space at about 7.3 Å from position 3, without projecting steric bulk perpendicularly. This structure-activity relationship may prove useful in the rational design of better antipneumocystis agents.

Close association of Pneumocystis carinii from infected rat lung with culture cells as shown by light and electron microscopy

Studies of the association of rat-originPneumocystis carinii with culture cells were performed both to learn more about the role of cells inP. carinii culture and to evaluate additional cell lines in an effort to improve culture methods. Proliferation of trophozoites ofP. carinii from rat lung in cultures with six lung cell lines was demonstrated by light microscopic evaluations of both Giemsa-stained and immune-specific-stained culture samples. Scanning electron microscopy and transmission electron microscopy were used to study the organisms interaction with culture cells and demonstrated a close association ofP. carinii with cells in cell lines that supported growth. Proliferation with the MVILU line was suboptimal and there was less organism interaction with these cells than with other cell lines that allowed proliferation. Two cell lines evaluated, Chinese Hamster ovary CHOKI and CHOLEKI, did not allow proliferation and had no association ofP. carinii with cells. Scanning and transmission electron micrographs demonstrated the close association of organisms with rat fetal lung (RFL), human embryonic lung (HEL), human diploid lung (HFL), and feline embryonic lung (AKD) culture cells. It appears that the association of rat-originP. carinii with cells is essential for parasite proliferation in short-term culture.

Alteration in expression of the rat mitochondrial ATPase 6 gene during Pneumocystis carinii infection

BackgroundPneumocystis carinii causes pneumonia in immunocompromised patients with a high morbidity and mortality rate, but the interaction between this organism and the host cell is not well understood. The purpose of this research was to study the response of host cells to P. carinii infection on a molecular level.ResultsThe technique of mRNA differential display was used to detect genes whose expression may be affected by P. carinii infection. The nucleotide sequence of one differentially displayed DNA fragment was found to be identical to that of the rat mitochondrial ATPase 6 gene, which is a subunit of the F0F1-ATP synthase complex. A four-fold increase in expression of this gene was verified by Northern blot analysis of total RNA extracted from P. carinii- infected rat lung versus that from mock-infected rat lung. Localization of the cells containing ATPase 6 mRNA was accomplished by in situ hybridization. In sections of non-infected rat lung, these cells were found lining the distal parts of the respiratory tree and in apical areas of the alveoli. Histological location of these cells suggested that they were Clara cells and type II pneumocytes. This hypothesis was confirmed by co-localizing the mRNAs for ATPase 6 and surfactant protein B (SP-B) to the same cells by two-color fluorescent in situ hybridization.ConclusionsThe ATPase 6 gene is over expressed during P. carinii infection, and type II pneumocytes and Clara cells are the cell types responsible for this over-expression.

Demonstration of Esterase Activity in Pneumocystis carinii by Cleavage of 4-Methylumbelliferyl Substrates

Pneurnocystis carinii is a principal cause of nonbacterial pneumonia in severely immunocompromised patients. Both morbidity and mortality related to Pneurnocystis pneumonia have prompted investigations to better understand mechanisms of host cell injury and metabolism of the organism. Pneurnocystis infection has been noted to cause extensive tissue damage in a substantial proportion of AIDS patients, but the mechanism is unclear [7]. P. carinii is also thought to take significant amounts of lipids from its environment in the lung, but again the mechanism is unclear [3,4]. Because esterases could play a role in breakdown of host cell membranes and scavenging of lipids, we surveyed esterase activity in P. carinii, using a convenient fluorescent assay based on cleavage of 4-methylumbelliferyllinked (MUF) substrates. MATERIALS AND METHODS Substrates tested were 4methylumbelliferyl esters of heptanoate, propionate, stearate, palmitate, elaidate and oleate obtained from Sigma. Stock solutions of 200mM concentration were made in dimethyl sulfoxide then serially diluted in either 0.05 M ammonium acetate pH 5.5 or phosphate buffered saline (PBS) pH 7.4. P. carinii organisms were obtained from spinner flask cultures [2], washed twice and resuspended in PBS. Organisms were quantitated by sampling 1Op1, staining with Giemsa and counting at 1OOOX. The concentration of organisms used in the experiments ranged from 4.4 x lo5 to 7.2 x lo6 trophozoites per ml. Microtiter plates were used for combining lop1 of each substrate with 9Op1 of a suspension of organisms. Uninfected human embryonic lung (HEL) cells, as well as organisms without substrate were included as controls. Samples were incubated at 35C for various time points up to 18 h. The cleavage of MUF substrates by esterases produced a fluorescent product (4-methylumbelliferone) that was excited in a Cambridge fluorometer at 360 nm and quantitated by its emission at 460 nm. RESULTS AND DISCUSSION A method to detect enzymes in Candida using MUF-labeled substrates [1,5] was applied to P. carinii harvested from spinner flask cultures. The cleavage of heptanoate and propionate MUF substrates by P. carinii was concentration dependent, time dependent and dependent upon the number of organisms present. Figure 1 shows how the cleavage of MUF-heptanoate at 80 mM substrate varies with numbers of organism and time. Similar results were obtained for 80 mM MUF-