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Evidence for multiple sterol methyl transferase pathways in Pneumocystis carinii

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Lipids

Abstract

The sterol composition of Pneumocystis carinii, an opportunistic pathogen responsible for life-threatening pneumonia in immunocompromised patients, was determined. Our purpose was to identify pathway-specific enzymes to impair using sterol biosynthesis inhibitors. Prior to this study, cholesterol 15 (ca. 80% of total sterols), lanosterol 1, and several phytosterols common to plants (sitosterol 31, 24α-ethyl and campesterol, 24α-methyl 30) were demonstrated in the fungus. In this investigation, we isolated all the previous sterols and many new compounds from P. carinii by culturing the microorganism in steroid-immunosuppressed rats. Thirty-one sterols were identified from the fungus (total sterol=100 fg/cell), and seven sterols were identified from rat chow. Unusual sterols in the fungus not present in the diet included, 24(28)-methylenelanosterol 2; 24(28)E-ethylidene lanosterol 3; 24(28)Z-ethylidene lanosterol 4; 24β-ethyllanosta-25(27)-dienol 5; 24β-ethylcholest-7-enol 6; 24β-ethylcholesterol 7; 24β-ethylcholesta-5,25(27)-dienol 8; 24-methyllanosta-7-enol 9; 24-methyldesmosterol 10; 24(28)-methylenecholest-7-enol 11; 24β-methylcholest-7-enol 12; and 24β-methylcholesterol 13. The structural relationships of the 24-alkyl groups in the sterol side chain were demonstrated chromatographically relative to authentic specimens, by MS and high-resolution 1H NMR. The hypothetical order of these compounds poses multiple phytosterol pathways that diverge from a common intermediate to generate 24β-methyl sterols: route 1, 1→2→11→12→13; route 2, 1→2→9→10→13; or 24β-ethyl sterols: route 3, 1→2→4→6→7; route 4, 1→2→5→8→7. Formation of 3 is considered to form an interrupted sterol pathway. Taken together, operation of distinct sterol methyl transferase (SMT) pathways that generate 24β-alkyl sterols in P. carinii with no counterpart in human biochemistry suggests a close taxonomic affinity with fungi and provides a basis for mechanism-based inactivation of SMI enzyme to treat Pneumocystis pneumonia.

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Abbreviations

NSF:

nonsaponifiable lipid fraction

RRT:

relative retention time of cholesterol

SBI:

sterol biosynthesis inhibitor

SMT:

sterol methyl transferase

References

  1. Furlong, S.T., Samia, J.A., Rose, R.M., and Fishman, J.A. (1994) Phytosterols Are Present in Pneumocystis carinii, Antimicrob. Agents Chemother. 38, 2534–2540.

    PubMed  CAS  Google Scholar 

  2. Kaneshiro, E.S., Ellis, J.E., Jayasimhulu, K., and Beach, D.H. (1994) Evidence for the Presence of “Metabolic Sterols” in Pneumocystis: Identification and Initial Characterization of Pneumocystis carinii Sterols, J. Eukaryot. Microbiol. 4, 78–85.

    Google Scholar 

  3. Kaneshiro, E.S., Amit, Z., Swonger, M.M., Kreishman, G.P., Brooks, E.E., Kreishman, M., Jayasimhulu, K., Parish, E.J., Sun, H., Kizito, S.A. et al. (1999) Pneumocysterol [(24Z)-ethylidenelanost-8-en-3β-ol], a Rare Sterol Detected in the Opportunistic Pathogen Pneumocystis carinii hominis: Structural Identity and Chemical Synthesis, Proc. Natl. Acad. Sci. USA 96, 97–102.

    Article  PubMed  CAS  Google Scholar 

  4. Urbina, J.A., Visbal, G., Contreras, L.M., McLaughlin, G., and Docampo, R. (1997) Inhibitors of Δ24(25)-Sterol Methyl Transferase Block Sterol Synthesis and Cell Proliferation in Pneumocystis carinii, Antimicrob. Agents Chemother. 41, 1428–1432.

    PubMed  CAS  Google Scholar 

  5. Nes, W.R., and Nes, W.D. (1980) Lipids in Evolution, Plenum Press, New York, 244 pp.

    Google Scholar 

  6. Gargas, A., DePriest, P.T., Grube, M., and Tehler, A. (1995) Multiple Origins of Lichen Symbioses in Fungi Suggested by SSU rDNA Phylogeny, Science 268, 1492–1495.

    Article  PubMed  CAS  Google Scholar 

  7. Cushion, M.T. (1998) Taxonomy, Genetic Organization, and Life Cycle of Pneumocystis carinii, Semin. Respir. Infect. 13, 304–312.

    PubMed  CAS  Google Scholar 

  8. Bartlett, M.S., Queener, S.F., Shaw, M.M., Richardson, J.D., and Smith, J.W. (1994) Pneumocystis carinii Is Resistant to Imidazole Antifungal Agents, Antimicrob. Agents Chemother. 38, 1859–1861.

    PubMed  CAS  Google Scholar 

  9. Hartman, M. (1998) Plant Sterols and the Membrane Environment, Trends Plant Sci. 3, 170–174.

    Article  Google Scholar 

  10. Nes, W.D. (1987) Biosynthesis and Requirement for Sterols in the Growth and Reproduction of Oomycetes, Am. Chem. Soc. Symp. Ser. 325, 304–328.

    CAS  Google Scholar 

  11. Nes, W.D., Janssen, G.G., Crumley, F.G., Kalinowska, M., and Akihisa, T. (1993) The Structural Requirements of Sterol for Membrane Function in Saccharomyces cerevisiae, Arch. Biochem. Biophys. 300, 724–733.

    Article  PubMed  CAS  Google Scholar 

  12. White, R.H., and McMorris, T.C. (1978) Biosynthetic Intermediates in the Conversion of Fucosterol and Oogoniol, Phytochemistry 17, 1800–1802.

    Article  CAS  Google Scholar 

  13. Weete, J.D., Fuller, M.S., Huang, H.Q., and Gandhi, S. (1989) Fatty and Sterols of Selected Hypochytriomycetes and Chytridromycetes, Exp. Mycol. 13, 183–195.

    Article  CAS  Google Scholar 

  14. Weete, J.D., and Gandhi, S.R. (1997) Sterols of the Phylum Zygomycota: Phylogenetic Implications, Lipids 32, 1309–1316.

    Article  PubMed  CAS  Google Scholar 

  15. Patterson, G.W. (1994) Phylogenetic Distribution of Sterols, Am. Chem. Soc. Symp. Ser. 562, 90–108.

    CAS  Google Scholar 

  16. Herber, R., Villoutreix, J., Granger, P., and Chapelle, S. (1983) Influence of l'Anaerobiose sur la Composition en Sterols de Mucor hiemalis, Can. J. Microbiol. 29, 606–611.

    Article  PubMed  CAS  Google Scholar 

  17. Abreu, P.M., Lobo, M., and Prabhakar, S. (1991) Revision of C-22, C-23 Configurations of Two Triterpenoids of the Fungus Pisolithus tinctorius, Phytochemistry 30, 3818–3819.

    Article  Google Scholar 

  18. Li, S. (1996) Stereochemical Studies on the Metabolism of Sterols by Saccharomyces cerevisiae Strain GL7. Master's Thesis. Texas Tech University, Lubbock, pp. 1–123.

    Google Scholar 

  19. Nes, W.D., Norton, R.A., Crumley, F.G., Madigan, S.J., and Katz, E.R. (1990) Sterol Phylogenesis and Algal Evolution, Proc. Natl. Acad. Sci. USA 87, 7565–7569.

    Article  PubMed  CAS  Google Scholar 

  20. Chen, F., and Cushion, M.T. (1994) Use of an ATP Bioluminescent Assay to Evaluate Viability of Pneumocystis carinii from Rats, J. Clin. Microbiol. 32, 2791–2800.

    PubMed  CAS  Google Scholar 

  21. Cushion, M.T., Chen, F., and Kloepfer, N. (1997) A Cytotoxicity Assay for Evaluation of Candidate Anti-Pneumocystis Agents, Antimicrob. Agents. Chemother. 41, 379–384.

    PubMed  CAS  Google Scholar 

  22. Norton, R.A., and Nes, W.D. (1991) Identification of Ergosta-6(7),8(14),25(27)-trien-3β-ol and Ergosta-5(6),7(8),25(27)-trien-3β-ol, Two New Steroidal Trienes Synthesized by Prototheca wickerhamii, Lipids 26, 247–249.

    CAS  Google Scholar 

  23. Guo, D., Venkatramesh, M., and Nes, W.D. (1995) Developmental Regulation of Sterol Biosynthesis in Zea mays, Lipids 30, 203–219.

    PubMed  CAS  Google Scholar 

  24. Venkatramesh, M., Guo, D., Jia, Z., and Nes, W.D. (1996) Mechanism and Structural Requirements for Transformation of Substrates by the (S)-Adenosyl-l-methionine:Δ24(25)-Sterol Methyl Transferase from Saccharomyces cerevisiae, Biochim. Biophys. Acta 1299, 313–324.

    PubMed  Google Scholar 

  25. Nes, W.D., and Le, P.H. (1990) Evidence for Separate Intermediates in the Biosynthesis of 24β-Methyl Sterols End Products by Gibberella fujikuori, Biochim. Biophys. Acta 1042, 119–125.

    CAS  Google Scholar 

  26. Nes, W.D., Xu, S., and Haddon, W.F. (1988) Evidence for Similarities and Differences in the Biosynthesis of Fungal Sterols, Steroids 53, 533–558.

    Article  Google Scholar 

  27. Dennis, A.L., and Nes, W.D. (2002) Sterol Methyl Transferase. Evidence for Successive C-Methyl Transfer Reactions Generating Δ24(28)-Sterol- and Δ25(27)-Sterol by a Single Plant Enzyme, Tetrahedron Lett. 43, 7017–7021.

    Article  CAS  Google Scholar 

  28. Nes, W.R., Krevitz, K., and Behzadan, S. (1976) Configuration at C-24 of 24-Methyl and 24-Ethylcholesterol, Lipids 11, 118–126.

    CAS  Google Scholar 

  29. Nes, W.R., Dhanuka, I.C., and Pinto, W.J. (1986) Evidence for Facilitated Transport in the Absorption of Sterols by Saccharomyces cerevisiae, Lipids 21, 102–106.

    PubMed  CAS  Google Scholar 

  30. Pinto, W.J., and Nes, W.R. (1983) Stereochemical Specificity for Sterols in Saccharomyces cerevisiae, J. Biol. Chem. 258, 4472–4476.

    PubMed  CAS  Google Scholar 

  31. Florin-Christensen, M., Florin-Christensen, J., Wu, Y.P., Zhou, L., Gupta, A., Rudney, H., and Kaneshiro, E.S. (1994) Occurrence of Specific Sterols in Pneumocystis carinii, Biochem. Biophys. Res. Commun. 14, 236–242.

    Article  Google Scholar 

  32. Kaneshiro, E.S., Rosenfeld, J.A., Basselin-Eiweida, M., Stringer, J.R., Keely, S.P., Smulian, A.G., and Giner, J.-L. (2002) The Pneumocystis carinii Drug Target S-Adenosyl-l-methionine: Sterol C-24 Methyl Transferase Has a Unique Substrate Preference, Mol. Microbiol. 44, 989–999.

    Article  PubMed  CAS  Google Scholar 

  33. Nes, W.D. (2000) Sterol Methyl Transferase: Enzymology and Inhibition, Biochim. Biophys. Acta 1529, 63–88.

    PubMed  CAS  Google Scholar 

  34. Nes, W.D., McCourt, B.S., Zhou, W., Ma, J., Marshall, J.A., Peek, L.-A., and Brennan, M. (1998) Overexpression, Purification, and Stereochemical Studies of the Recombinant (S)-Adenosyl-l-methionine: Δ24(25)- to Δ24(28)-Sterol Methyl Transferase from Saccharomyces cerevisiae, Arch. Biochem. Biophys. 353, 297–311.

    Article  PubMed  CAS  Google Scholar 

  35. Nes, W.D., McCourt, B.S., Marshall, J.A., Ma, J., Dennis, A.L., Lopez, M., and Le, H. (1999) Site-Directed Mutagenesis of the Sterol Methyl Transferase Active Site from Saccharomyces cerevisiae Results in Formation of Novel 24-Ethyl Sterols, J. Org. Chem. 64, 1535–1542.

    Article  PubMed  CAS  Google Scholar 

  36. Nes, W.D., Marshall, J.A., Jia, Z., Jaradat, T.T., Song, Z., and Jayasimha, P. (2002) Active Site Mapping and Substrate Channeling in the Sterol Methyl Transferase Pathway, J. Biol. Chem. 277, 42549–42556.

    Article  PubMed  CAS  Google Scholar 

  37. Kagan, R.M., and Clarke, S. (1994) Widespread Occurrence of Three Sequence Motifs in Diverse S-Adenosylmethionine-Dependent Methyl Transferases Suggests a Common Structure for These Enzymes, Arch. Biochem. Biophys. 310, 41–427.

    Article  Google Scholar 

  38. Niewmierzycka, A., and Clarke, S. (1999) S-Adenosylmethionine-Dependent Methylation in Saccharomyces cerevisiae, J. Biol. Chem. 274, 814–824.

    Article  PubMed  CAS  Google Scholar 

  39. Hrmova, M., and Fincher, G.B. (2001) Plant Enzyme Structure. Explaining Substrate Specificity and the Evolution of Function, Plant Physiol. 125, 54–57.

    Article  PubMed  CAS  Google Scholar 

  40. Bouvier-Nave, P., Husselstein, T., and Benveniste, P. (1998) Two Families of Sterol Methyl Transferase Are Involved in the First and the Second Methylation Steps of Plant Sterol Biosynthesis, Eur. J. Biochem. 256, 88–96.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Texas Tech University, 79409-1061, Lubbock, Texas

    Wenxu Zhou, Thi Thuy Minh Nguyen, Margaret S. Collins & W. David Nes

  2. Department of Internal Medicine, University of Cincinnati College of Medicine, 45267, Cincinnati, Ohio

    Melanie T. Cushion

  3. Veterans Administration Medical Center, Cincinnati, Ohio

    Melanie T. Cushion

Authors
  1. Wenxu Zhou
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  2. Thi Thuy Minh Nguyen
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  3. Margaret S. Collins
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  4. Melanie T. Cushion
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  5. W. David Nes
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Correspondence to W. David Nes.

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Zhou, W., Nguyen, T.T.M., Collins, M.S. et al. Evidence for multiple sterol methyl transferase pathways in Pneumocystis carinii . Lipids 37, 1177–1186 (2002). https://doi.org/10.1007/s11745-002-1018-8

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  • DOI: https://doi.org/10.1007/s11745-002-1018-8

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