Inability of Pneumocystis organisms to incorporate bromodeoxyuridine suggests the absence of a salvage pathway for thymidine

Abstract

Because thymidine metabolism is a potential target for therapy of Pneumocystis pneumonia, it was investigated whether Pneumocystis organisms have a salvage pathway for thymidine by administering 5-bromo-29-deoxyuridine (BrdU) to mice and rats with Pneumocystis pneumonia. Although BrdU incorporation was detected in host cells, no incorporation was seen in Pneumocystis organisms infecting either rats or mice. This suggests that Pneumocystis organisms do not have a salvage pathway for thymidine, and that inhibitors of de novo synthesis, such as thymidylate synthase inhibitors, may be effective drugs for treating Pneumocystis pneumonia. INTRODUCTIONPneumocystis jirovecii is an atypical fungus that infects patients suffering from a variety of immunosuppressive diseases, such as AIDS and cancer (Kovacs et al., 2001a;Roblot et al., 2002). Although there has been a decrease in morbidity among AIDS patients due to the availability of potent anti-retroviral regimens, Pneumocystis pneumonia (PCP) continues to be the most common life-threatening opportunistic infection among HIV-infected patients. Therapy with the combination of trimethoprim and sulfamethoxazole (TMP/SMX) is the preferred approach for primary prophylaxis in susceptible patients (i.e. for AIDS patients with CD4 + count levels <200 mm 23 ) as well as for treatment of active PCP (Kovacs & Masur, 2000;Phair et al., 1990). These drugs target the enzymes dihydrofolate reductase and dihydropteroate synthase (DHPS), respectively. Other drugs including dapsone, atovaquone and pentamidine are alternatives to TMP/SMX, but they are either more toxic or less active against P. jirovecii (Hughes Hughes, 1998;Kovacs et al., 2001a;Sattler et al., 1988; Vasconcelles et al., 2000). Unfortunately, recent studies have identified mutations in the gene encoding DHPS suggesting the emergence of resistance in P. jirovecii to sulfa drugs (dapsone and SMX) (Helweg-Larsen et al., 1999;Kazanjian et al., 2000;Ma et al., 1999;Navin et al., 2001). Development of new drugs to treat or prevent PCP thus remains an important priority for research.P. jirovecii is closely related to Pneumocystis species that infect rats (Pneumocystis carinii) and mice (P. carinii f. sp. muris). A better understanding of the metabolic pathways of Pneumocystis species may lead to the identification of novel therapeutic targets. Thymidine metabolism has been partially characterized in Pneumocystis: thymidylate synthase has been identified, isolated, cloned and crystallized (Anderson et al., 2000; Edman et al., 1989;Kovacs et al., 1990;Santi et al., 1991). Thus, Pneumocystis organisms possess de novo synthetic pathways for thymidine, a target of chemotherapy in other infections. Therapies that target de novo thymidylate synthesis can potentially be bypassed using thymidine salvage pathways. To see if Pneumocystis organisms possessed a salvage pathway for thymidine, 5-bromo-29-deoxyuridine (BrdU) was administered for 14 days to Pneumocystis-infected rats and mice. BrdU is a thymidine analogue which can be incorpora...

A Molecular Window into the Biology and Epidemiology of Pneumocystis spp

“…Nucleotide metabolism is focused solely on de novo biosynthesis of fundamental cellular components. The Pneumocystis genomes have retained the complete de novo biosynthesis pathways for purine and pyrimidine nucleotides but have lost nucleotide salvage and degradation pathways (69,145), consistent with the reported absence of a thymidine salvage pathway (153). This is unusual for a highly compact genome, since the de novo biosynthetic pathways involve substantially more chemical reactions and thus require more energy than those for salvage pathways.…”

Pneumocystis Species Co-evolution: State-of-the-Art Review

“…Moreover, most biotrophs are obligate parasites, meaning they cannot survive without their hosts and cannot be cultured axenically in the laboratory [24]. In addition, data obtained from the genome analysis of Pneumocystis species revealed very compact genomes, suggesting that these organisms have lost several families of genes and metabolic pathways during the course of evolution, and whose products they scavenge from the lung environment of the host [7,9,22,25,26]. Consistently, Pneumocystis pneumonia seems to be rare in wild mammals and only low rates of Pneumocystis organisms are usually detected in their lungs [1][2][3][4][5][6].…”

DNA replication during endospore development in Metabacterium polyspora

“…BrdU is taken up and incorporated into the DNA of many, but not all, growing wild-type bacteria (Urbach et al, 1999;Pernthaler et al, 2002;Vestereng and Kovacs, 2004). In Escherichia coli and B. subtilis, the most efficient incorporation is seen in strains in which the thymidylate synthetase gene, thyA, has been mutated (Frisch and Visser, 1960;Yoshikawa, 1967).…”