Critical Care Medicine Department, Clinical Center, National Institutes of Health, Building 10, Room 7D43, MSC 1662, Bethesda, MD 20892-1662, USA
Correspondence
Joseph A. Kovacs
jkovacs{at}nih.gov
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ABSTRACT |
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Present address: Gentofte University Hospital, Internal Medicine Dept F, Niels Andersens Vej 65, 2900 Hellerup, Denmark.
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INTRODUCTION |
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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-2'-deoxyuridine (BrdU) was administered for 14 days to Pneumocystis-infected rats and mice. BrdU is a thymidine analogue which can be incorporated into DNA during the S-phase of cell cycle if the cells have a salvage pathway. Incorporation can be readily detected histologically, since a monoclonal antibody (mAb) is available against BrdU. Incorporated BrdU in mammalian cells appears to be stable and can be detected for many weeks without affecting cell function (Dolbeare, 1995a
, b
; Dolbeare, 1996
; Kovacs et al., 2001b
). Studies were conducted in animals because of the difficulty in culturing Pneumocystis in vitro and concerns that lack of incorporation ex vivo may be due to lack of proliferation of Pneumocystis.
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METHODS |
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At day 14, the animals were killed and the lungs were removed. One lung was used for making impression smears that were stained with Diff-Quik (Dade Behring) to confirm the presence of infection. The other lung was inflated with Histochoice (Amresco) with 20 % ethanol and subsequently stored in the fixative until embedding in paraffin. A portion of the small intestine, processed in a similar manner, served as a positive control for BrdU incorporation (Kellett et al., 1992).
For immunohistopathology, which was performed by contract (SAIC, Frederick, MD), 5 µm thick tissue samples were deparaffinized and stained for Pneumocystis or for BrdU. Staining for Pneumocystis was performed by using mAb 4D7, which is specific for a surface protein of Pneumocystis (Angus et al., 1996; Mei et al., 1995
), together with the Mouse Elite Kit (Vector Laboratories) according to the manufacturer's recommendations. Staining for BrdU incorporation in rat tissue used the same kit together with an anti-BrdU mAb (clone Bu20a; DAKO). For mouse tissue, BrdU staining was performed using the Animal Research Kit (DAKO). In addition, BrdU staining was performed using the tyramide signal amplification technique with the TSA-Indirect kit (NEN Life Science Products) according to the manufacturer's recommendations. Slides prepared with no primary mAb served as negative controls. For BrdU staining, after deparaffinization the slides were placed in 4 M HCl at 37 °C for 20 min to partially denature the DNA and provide access for the anti-BrdU mAb, and then rinsed in boric acid/borate buffer, pH 7·6. Next, slides were placed in 0·01 % trypsin at 37 °C for 3 min, then rinsed in PBS, following which the manufacturer's instructions for the individual kits were followed. The anti-BrdU mAb was used at a dilution of 1 : 500 to 1 : 1000.
The use of animals for the experimental protocol was approved by the Animal Care and Use Committee, Clinical Center, National Institutes of Health.
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RESULTS |
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DISCUSSION |
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The source of thymidine for DNA synthesis varies among different micro-organisms. Two pathogens in which the thymidine pathways are well characterized are Giardia lamblia and Plasmodium falciparum. Whereas G. lamblia depends solely on salvaging thymidine for DNA synthesis (Jarroll et al., 1989), P. falciparum only utilizes de novo synthesis of this pyrimidine (Sherman, 1979
). In contrast, mammalian cells possess both pathways. It is noteworthy that the two fungi most closely related to Pneumocystis, as assessed by comparing the amino acid sequences of homologous proteins, Schizosaccharomyces pombe and Saccharomyces cerevisiae, both lack thymidine kinase and the ability to efficiently take up exogenous deoxyribonucleosides, and thus cannot utilize exogenous thymidine (Hodson et al., 2003
; Vernis et al., 2003
). Both fungi have been genetically engineered to salvage thymidine and incorporate BrdU, which can be detected by immunofluorescent assays (Hodson et al., 2003
; Vernis et al., 2003
).
If Pneumocystis organisms possess only a de novo synthetic pathway for thymidine, thymidylate synthase is an especially crucial enzyme for DNA synthesis in this micro-organism. Thymidylate synthase functions in the reductive methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP). Inhibition of Pneumocystis thymidylate synthase activity, in the absence of salvaged thymidine, would lead to cell cycle arrest and thereby cell death. Thymidylate synthase inhibitors such as 5-fluororuracil have been used for more than 40 years as anti-neoplastic agents (Danenberg et al., 1999). Thus, targeting this enzyme is a feasible approach for developing chemotherapeutic agents.
It is possible that the sensitivity of the techniques that we utilized was too low to detect BrdU incorporated by Pneumocystis, as a result of inaccessibility of the DNA to the anti-BrdU mAb, or to the smaller size of the Pneumocystis genome. However, Pneumocystis DNA is accessible to in situ hybridization by oligonucleotide probes in fixed tissues similar to those we utilized here (Edman et al., 1988), and BrdU incorporation has been detected by immunofluorescent staining in the ciliate Colpoda inflata (Gutierrez et al., 2000
), and in Schizosaccharomyces pombe and Saccharomyces cerevisiae that have been genetically engineered to salvage thymidine (Hodson et al., 2003
; Vernis et al., 2003
).
It is also possible that the dose of BrdU that we utilized was too low, or inaccessible to the organisms in the lung, although BrdU is a small molecule that should readily diffuse throughout the body, and DNA in cells immediately adjacent to clusters of Pneumocystis organisms incorporated BrdU (Fig. 1B).
The crystal structure of thymidylate synthase from rat-derived P. carinii, which was cloned and expressed in Escherichia coli, has been published (Anderson et al., 2000). Comparing the structure to the structure of human thymidylate synthase suggested the potential for designing Pneumocystis-specific inhibitors which would not inhibit human thymidylate synthase. If Pneumocystis cells lack salvage pathways, as suggested by the current study, such an approach, perhaps combined with co-administration of thymidine to rescue host cells (Jiang et al., 2000
), may be an effective therapy for PCP.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Angus, C. W., Tu, A., Vogel, P., Qin, M. & Kovacs, J. A. (1996). Expression of variants of the major surface glycoprotein of Pneumocystis carinii. J Exp Med 183, 12291234.[Abstract]
Danenberg, P. V., Malli, H. & Swenson, S. (1999). Thymidylate synthase inhibitors. Semin Oncol 26, 621631.[Medline]
Dolbeare, F. (1995a). Bromodeoxyuridine: a diagnostic tool in biology and medicine, part II: oncology, chemotherapy and carcinogenesis. Histochem J 27, 923964.[Medline]
Dolbeare, F. (1995b). Bromodeoxyuridine: a diagnostic tool in biology and medicine, part I: historical perspectives, histochemical methods and cell kinetics. Histochem J 27, 339369.[Medline]
Dolbeare, F. (1996). Bromodeoxyuridine: a diagnostic tool in biology and medicine, part III. Proliferation in normal, injured and diseased tissue, growth factors, differentiation, DNA replication sites and in situ hybridization. Histochem J 28, 531575.[Medline]
Edman, J. C., Kovacs, J. A., Masur, H., Santi, D. V., Elwood, H. J. & Sogin, M. L. (1988). Ribosomal RNA sequence shows Pneumocystis carinii to be a member of the fungi. Nature 334, 519522.[CrossRef][Medline]
Edman, U., Edman, J. C., Lundgren, B. & Santi, D. V. (1989). Isolation and expression of the Pneumocystis carinii thymidylate synthase gene. Proc Natl Acad Sci U S A 86, 65036507.[Abstract]
Flynn, K. J., Riberdy, J. M., Christensen, J. P., Altman, J. D. & Doherty, P. C. (1999). In vivo proliferation of naive and memory influenza-specific CD8+ T cells. Proc Natl Acad Sci U S A 96, 85978602.
Gutierrez, J. C., Callejas, S., Borniquel, S. & Martin-Gonzalez, A. (2000). DNA methylation in ciliates: implications in differentiation processes. Int Microbiol 3, 139146.[Medline]
Helweg-Larsen, J., Benfield, T. L., Eugen-Olsen, J., Lundgren, J. D. & Lundgren, B. (1999). Effects of mutations in Pneumocystis carinii dihydropteroate synthase gene on outcome of AIDS-associated P. carinii pneumonia. Lancet 354, 13471351.[CrossRef][Medline]
Hodson, J. A., Bailis, J. M. & Forsburg, S. L. (2003). Efficient labeling of fission yeast Schizosaccharomyces pombe with thymidine and BUdR. Nucleic Acids Res 31, e134.
Hughes, W. T. (1998). Use of dapsone in the prevention and treatment of Pneumocystis carinii pneumonia: a review. Clin Infect Dis 27, 191204.[Medline]
Hughes, W., Leoung, G., Kramer, F. & 14 other authors (1993). Comparison of atovaquone (566C80) with trimethoprim-sulfamethoxazole to treat Pneumocystis carinii pneumonia in patients with AIDS. N Engl J Med 328, 15211527.
Jarroll, E. L., Manning, P., Berrada, A., Hare, D. & Lindmark, D. G. (1989). Biochemistry and metabolism of Giardia. J Protozool 36, 190197.[Medline]
Jiang, L., Lee, P. C., White, J. & Rathod, P. K. (2000). Potent and selective activity of a combination of thymidine and 1843U89, a folate-based thymidylate synthase inhibitor, against Plasmodium falciparum. Antimicrob Agents Chemother 44, 10471050.
Kazanjian, P., Armstrong, W., Hossler, P. A., Burman, W., Richardson, J., Lee, C. H., Crane, L., Katz, J. & Meshnick, S. R. (2000). Pneumocystis carinii mutations are associated with duration of sulfa or sulfone prophylaxis exposure in AIDS patients. J Infect Dis 182, 551557.[CrossRef][Medline]
Kellett, M., Potten, C. S. & Rew, D. A. (1992). A comparison of in vivo cell proliferation measurements in the intestine of mouse and man. Epithelial Cell Biol 1, 147155.[Medline]
Kovacs, J. A. & Masur, H. (2000). Prophylaxis against opportunistic infections in patients with human immunodeficiency virus infection. N Engl J Med 342, 14161429.
Kovacs, J. A., Halpern, J. L., Swan, J. C., Moss, J., Parrillo, J. E. & Masur, H. (1988). Identification of antigens and antibodies specific for Pneumocystis carinii. J Immunol 140, 20232031.
Kovacs, J. A., Allegra, C. J. & Masur, H. (1990). Characterization of dihydrofolate reductase of Pneumocystis carinii and Toxoplasma gondii. Exp Parasitol 71, 6068.[CrossRef][Medline]
Kovacs, J. A., Gill, V. J., Meshnick, S. & Masur, H. (2001a). New insights into transmission, diagnosis, and drug treatment of Pneumocystis carinii pneumonia. JAMA (J Am Med Assoc) 286, 24502460.
Kovacs, J. A., Lempicki, R. A., Sidorov, I. A. & 17 other authors (2001b). Identification of dynamically distinct subpopulations of T lymphocytes that are differentially affected by HIV. J Exp Med 194, 17311741.
Ma, L., Borio, L., Masur, H. & Kovacs, J. A. (1999). Pneumocystis carinii dihydropteroate synthase but not dihydrofolate reductase gene mutations correlate with prior trimethoprim-sulfamethoxazole or dapsone use. J Infect Dis 180, 19691978.[CrossRef][Medline]
Martinez, A. & Kovacs, J. A. (1993). Development and characterization of a rapid screening assay for identifying antipneumocystis agents. Antimicrob Agents Chemother 37, 16741678.[Abstract]
Mei, Q., Wang, Q., Chen, Y., Liu, Y., Fan, W. & Li, P. (1995). Experimental and clinical study on pneumocystosis. IV. Use of a 4D7 monoclonal antibody to detect Pneumocystis carinii by immunoperoxidase staining. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi 13, 130133 (in Chinese).[Medline]
Navin, T. R., Beard, C. B., Huang, L. & 7 other authors (2001). Effect of mutations in Pneumocystis carinii dihydropteroate synthase gene on outcome of P. carinii pneumonia in patients with HIV-1: a prospective study. Lancet 358, 545549.[CrossRef][Medline]
Phair, J., Munoz, A., Detels, R., Kaslow, R., Rinaldo, C. & Saah, A. (1990). The risk of Pneumocystis carinii pneumonia among men infected with human immunodeficiency virus type 1. Multicenter AIDS Cohort Study Group. N Engl J Med 322, 161165.[Abstract]
Roblot, F., Godet, C., Le Moal, G. & 9 other authors (2002). Analysis of underlying diseases and prognosis factors associated with Pneumocystis carinii pneumonia in immunocompromised HIV-negative patients. Eur J Clin Microbiol Infect Dis 21, 523531.[CrossRef][Medline]
Santi, D. V., Edman, U., Minkin, S. & Greene, P. J. (1991). Purification and characterization of recombinant Pneumocystis carinii thymidylate synthase. Protein Expr Purif 2, 350354.[Medline]
Sattler, F. R., Cowan, R., Nielsen, D. M. & Ruskin, J. (1988). Trimethoprim-sulfamethoxazole compared with pentamidine for treatment of Pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome. A prospective, noncrossover study. Ann Intern Med 109, 280287.[Medline]
Sherman, I. W. (1979). Biochemistry of Plasmodium (malarial parasites). Microbiol Rev 43, 453495.[Medline]
Vasconcelles, M. J., Bernardo, M. V., King, C., Weller, E. A. & Antin, J. H. (2000). Aerosolized pentamidine as pneumocystis prophylaxis after bone marrow transplantation is inferior to other regimens and is associated with decreased survival and an increased risk of other infections. Biol Blood Marrow Transplant 6, 3543.[Medline]
Vernis, L., Piskur, J. & Diffley, J. F. (2003). Reconstitution of an efficient thymidine salvage pathway in Saccharomyces cerevisiae. Nucleic Acids Res 31, e120.
Walzer, P. D., Runck, J., Steele, P., White, M., Linke, M. J. & Sidman, C. L. (1997). Immunodeficient and immunosuppressed mice as models to test anti-Pneumocystis carinii drugs. Antimicrob Agents Chemother 41, 251258.[Abstract]
Received 5 November 2003;
revised 6 February 2004;
accepted 9 February 2004.
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