Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene.

Abstract

Pneumocystis carinii dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) cDNA sequences have been isolated by their ability to confer trimethoprim resistance to Escherichia coli. Consistent with the recent conclusion that P. carinji is a member of the Fungi, sequence analysis and chromosomal localization show that DHFR is neither physically nor genetically linked to thymidylate synthase. Expression of recombinant P. carinji DHFR in heterologous hosts provides an abundant source of the enzyme that may form a basis for the development of new therapies for this enigmatic pathogen. Studies with the recombinant enzyme show that trimethoprim is a very poor inhibitor of P. carinji DHFR and, in fact, is a more potent inhibitor of human DHFR.Pneumocystis carinii pneumonia is a leading cause of morbidity and mortality in AIDS (1, 2). Since the onset of the AIDS epidemic, the incidence of P. carinii pneumonia has risen from ==200 to >25,000 cases per year in the United States. Due to the lack of a continuous in vitro culture system and the cumbersome nature of the rat model of P. carinii pneumonia, the development of anti-P. carinji therapy has rested largely on the assumption that antiprotozoan agents were likely to be effective (3-5). Despite the fact that P. carinii has recently been shown to be a member of the Fungi (6), the two principal therapeutic modalities, trimethoprim/ sulfamethoxazole and pentamidine, were developed on this basis. Before the AIDS epidemic, these agents were sufficient for treatment of the rare cases of P. carinii pneumonia. However, in the human immunodeficiency virus-positive patient, therapy and prophylaxis with the standard anti-P. carinii agents are complicated by frequent toxic and allergic side effects (7). New compounds active against P. carinii are clearly needed.The inability to propagate P. carinii reliably in vitro and the limited quantities of P. carinii enzymes that can be purified from infected rat lungs have hindered the search for anti-P. carinii agents. Purification and characterization of the intracellular targets for such agents would enable the development of new therapies for P. carinii pneumonia. Of the known anti-P. carinii agents, the dihydrofolate reductase (DHFR;5,6,7,3) inhibitors are the most thoroughly characterized. DHFR plays a central role in the de novo synthesis of nucleic acid precursors. DHFR inhibitors (methotrexate, trimethoprim, pyrimethamine) are effective antineoplastic, antibacterial, and antiprotozoal agents. P. carinii pneumonia clearly responds to the combination of a DHFR inhibitor (trimethoprim or pyrimethamine) and a sulfonamide. Despite their obvious efficacy when used in conjunction with a sulfonamide, available information suggests that trimethoprim and pyrimethamine, are in themselves poor inhibitors of P. carinii DHFR (IC50 values of 39,600 and 2400 nM, respectively, compared with 8 and 2500 nM for Escherichia coli DHFR at similar substrate concentrations; refs. 8 and 9). Other antifolates have b...

Isolation and expression of the Pneumocystis carinii thymidylate synthase gene.

“…carinii TS did not reveal proximal open reading frames that could encode dihydrofolate reductase. Further, TAFE demonstrated that TS and dihydrofolate reductase reside on different P. carinji chromosomes (35). Thus, our results support the contention that P. carinii is not a protozoan.…”

Dihydrofolate reductase gene polymorphisms in Pneumocystis carinii f. sp. hominis in Japan

“…Edman et al [13] determined the nucleotide sequences of P. carinii DHFR gene derived from five rat and two human specimens to evaluate the natural variability of DHFR sequences from different sources of this organism. The single base change in the sequence was shown to be an A to G transition at position 654 in the 39-untranslated region of rat-derived P. carinii.…”

Characterization of variants of the gene encoding the p55 antigen in Pneumocystis from rats and mice

“…Based on regions conserved between the previously reported p55 sequence (Smulian et al, 1992(Smulian et al, , 1993 and a DNA fragment that we identified by sequencing randomly picked clones from a P. carinii cDNA library (Edman et al, 1989) (GenBank accession no. AF494450), we designed the primer pair PU947 and PD1637 (Table 1).…”

“…Two major groups of antigens have been identified in this organism by immunoblotting. One group, which is present as a broad band of 90-120 kDa, has been well-characterized as the major surface glycoprotein (MSG) in Pneumocystis organisms derived from rats, humans, mice, ferrets and rabbits (Graves et al, 1986;Walzer & Linke, 1987;Gigliotti et al, 1988;Kovacs et al, 1989Kovacs et al, , 1993Bauer et al, 1993;Kutty et al, 2001). The second group, which appears as a broad band of 45-55 kDa in P. carinii and 35-45 kDa in human-derived Pneumocystis jiroveci (Stringer et al, 2002), is less well-studied (Walzer & Linke, 1987;Smulian et al, 1992Smulian et al, , 1993Smulian et al, , 2000.…”