Characterization of De Novo Folate Synthesis in Pneumocystis cannu and Toxoplasma gondii: Potential for Screening Therapeutic Agents

Abstract

Drug therapy studies imply that Pneumocystis carinii and Toxoplasma gondii possess the enzymes necessary for de novo folate synthesis. To verify this, incorporation of [3H]paraaminobenzoic acid [( 3H]PABA) into reduced folates by P. carinii and T. gondii was investigated. Both organisms synthesized tritiated reduced folates. In P. carinii, 10-formyltetrahydrofolate and tetrahydrofolate, and in T. gondii, 5-formyltetrahydrofolate were the major synthesized folates. P. carinii remained metabolically active in vitro for only a few days. Because current systems for screening antipneumocystis agents are cumbersome, the utility of this assay system for screening therapeutic agents was investigated. Sulfonamides and pentamidine efficiently inhibited de novo folate synthesis in P. carinii. Inhibitors of dihydrofolate reductase such as trimethoprim and trimetrexate were poor inhibitors for P. carinii but efficient inhibitors for T. gondii. This study demonstrates the first unambiguous evidence of metabolic activity in P. carinii, and provides a potential assay for efficiently screening antipneumocystis drugs in vitro.

Epidemiology and clinical relevanceofPneumocystis jiroveciiFrenkel, 1976 dihydropteroate synthase gene mutations

“…In Europe, fourteen studies covering a period between 1989 and 2007, reported P. jirovecii DHPS gene mutation rates of 3.7-57 % (mean 23 %) (Table II). In Denmark, it was reported a prevalence of P. jirovecii DHPS mutations of 20 %, in HIV-infected patients with PcP followed between 1989(Helweg-Larsen et al, 1999. In a study conducted in the UK, a decline in the proportion of P. jirovecii DHPS mutations was observed for the two time periods 1992-1993 (36 %) and 2000-2001 (17 %) of the study.…”

“…U nlike the vertebrate host that exogenously acquired folic acid, P. jirovecii is unable to scavenge folates from the environment, being absolutely dependent on its own mechanism for de novo synthesis of this molecule (Kovacs et al, 1989). Folic acid is required in several metabolic pathways, including the synthesis of purines, thymidine, glycine and methionine.…”

Interaction of sulfonamide and sulfone compounds with Toxoplasma gondii dihydropteroate synthase.

“…The potency of the various inhibitors was assessed in intact organisms according to published methods (18). These methods quantitated the ability of drugs to inhibit the metabolism of [3HjpABA to intracellular folates.…”

“…A variety of factors may alter the effectiveness of a given compound in intact cells including compound stability under physiologic conditions, membrane transport, and intracellular metabolism. We used the ability ofthe intact organisms to incorporate radiolabeled pABA into their folate pools as a measure ofthe antifolate's effect on cellular metabolism according to methods previously described in detail (18).…”

Microculture screening assay for primary in vitro evaluation of drugs against Pneumocystis carinii

“…Rat lung cell contamination was visualized and indirect evidence of preparation viability was obtained by the differential uptake and metabolism of fluorogenic substrates (fluorescein diacetate and ethidium bromide [2,14]). Bacterial contamination was assessed by culturing on sheep blood agar (17). The maximum amount of P. carinii suspension which could be readily filtered and washed on the assay plate membranes [MultiScreen 0.45-,m-pore-size hydrophiic polyvinylidene difluoride membranes in 96-well microtiter plates catalog no.…”