This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Amit, Z.
Right arrow Articles by Kaneshiro, E. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Amit, Z.
Right arrow Articles by Kaneshiro, E. S.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, March 2001, p. 1137-1139, Vol. 39, No. 3
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.3.1137-1139.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Heterogeneity of Pneumocystis Sterol Profiles of Samples from Different Sites in the Same Pair of Lungs Suggests Coinfection by Distinct Organism Populations

Zunika Amit and Edna S. Kaneshiro*

Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio 45221

Received 17 July 2000/Returned for modification 12 October 2000/Accepted 12 December 2000


    ABSTRACT
Top
Abstract
Text
References

Sterol profiles of samples taken from different sites of a Pneumocystis-infected human lung showed large variations in pneumocysterol similar to those that occur among samples from different patients. Thus, the influence of diet or drugs on pneumocysterol accumulation was ruled out, suggesting distinct phenotypic populations as the basis for the heterogeneity.


    TEXT
Top
Abstract
Text
References

Pneumocystis carinii synthesizes a number of distinct Delta 7 and Delta 8 24-alkylsterols but not ergosterol (the target of several antimycotics), and the organism scavenges cholesterol from its mammalian host (5, 6, 14-16, 18, 24). Most fungal sterols have an alkyl group consisting of one carbon at C-24 of the side chain. In contrast, the sterols of P. carinii and many plants have either one or two carbons at that site (C28 and C29 sterols). The P. carinii sterols are excellent chemotherapeutic targets because mammals cannot synthesize 24-alkylsterols.

The C32 24-alkylated lanosterol compound pneumocysterol (17) was detected in only trace amounts in organisms isolated from the corticosteroid-immunosuppressesd rat P. carinii pneumonia (PCP) model. The sterol profiles of organisms isolated from this animal model are consistent and reproducible from preparation to preparation (5, 14, 15, 18). In contrast, pneumocysterol was found in various percentages, from trace levels to 50% of the noncholesterol sterols, in organisms isolated from cryopreserved human lungs (16). Replicate analyses of these human-derived samples were consistent and reproducible, suggesting biochemical differences in organism populations. No correlation between human immunodeficiency virus infection (HIV) and high levels of pneumocysterol was found. However, in that earlier study, the possible effects of diet, nonprescribed drugs, or other factors could not be ruled out as the basis for broad variations in the accumulation of this sterol. In the present study, variations in pneumocysterol in samples from the same pair of human lungs were noted, and thus, more than six different sites in the lungs from the same individual were analyzed.

A formalin-fixed pair of lungs from an AIDS patient who died from PCP was provided by M. Pereira (Tufts Medical School, Boston, Mass.). Approximately 100-g pieces were excised from different sites and homogenized with distilled water in a Waring blender for 2 min, and total lipids were extracted (2) at room temperature for at least 2 h. The sterols were prepared and analyzed by gas-liquid chromatographic (GLC) methods as previously described (16-18).

Formalin does not have functions that would interact with sterols, and it was experimentally shown in a previous study that formalin fixation did not alter the sterols of rat lungs (19). Also, formalin-fixed lung tissue (17, 19) and Pneumocystis organisms isolated from cryopreserved human lungs (16) contained the same steroidal compounds. In the present study, the GLC components designated peaks 13, 16, 19, 20 and 24 were considered the organism's signature sterol profile [24-methylcholest-7-en-3beta -ol (fungisterol), 24-ethylcholestadiene-3beta -ol, 24-ethylcholest-7-en-3beta -ol, 24-ethylidenecholesta-7,24(28)- diene-3beta -ol, and 24-ethylidenelanost-8,24(28)-diene-3beta -ol (pneumocysterol), respectively]. In addition to cholesterol, GLC peaks 5, 8, 10, and 15 (desmosterol, campesterol, cholest-5-en-3-one, and beta -sitosterol, respectively) are believed to originate from the host.

In the samples taken from different sites of the Pneumocystis-infected human lung, the same major Pneumocystis sterols were detected, demonstrating qualitative similarities among the sampled areas (three examples are shown in Table 1). However, the relative proportions of these sterols at each site were different. The variations were most dramatically illustrated in the proportions of the distinct P. carinii sterols. In this individual with PCP, pneumocysterol composed only 29% of the noncholesterol sterols at one site, whereas it composed 49 and 54% of the noncholesterol sterols at the two other sites. Fungisterol composed 20% of the noncholesterol sterols at one site, whereas it accounted for only 2 and 6% of the noncholesterol sterols at the other sites. These findings were similar to those obtained for organisms isolated from human lungs from different subjects with PCP (16). Pneumocysterol was found in concentrations up to 50% of the noncholesterol sterols in some samples, whereas it was present in only trace amounts in other organism preparations (16); the sterol profiles of three independent organism preparations are shown in Table 1. In all analyses performed thus far on organisms from the experimental rat PCP model, pneumocysterol has been found in only trace amounts (14, 15, 18).

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1.   Sterol profiles of samples from different sites of the same heavily infected pair of lungs of a PCP patient and of P. carinii f. sp. hominis organisms isolated from cryopreserved lung samples from different patients

Pneumocystis is now recognized as a group of genetically diverse organisms (species and strains) which are specific for a given host species (7-9, 13, 22, 25). There is strong evidence for more than one distinct species or strain infecting the same mammalian host species and the same animal (3, 4, 13). Recent data obtained by nucleotide sequence analyses have demonstrated distinct genotypes infecting humans, and these organism populations can coexist in the same individual (10-12, 20, 21, 23).

It is unlikely that the results of the present study are due to varying proportions of different life cycle stages (e.g., trophic and cystic stages). The sterol compositions of trophic forms (24) and mixed life cycle stages (6, 15, 18) of P. carinii f. sp. carinii do not differ. A more likely explanation of the wide range of pneumocysterol and fungisterol in the samples from different regions of the same human lung is that it represents biochemical differences between distinct Pneumocystis genotypes coinfecting the same individual. These regional differences may result from infections that were initiated at distinct loci (clones) by individual invading organisms. This suggestion is supported by the identification within the same experimental rat lung of foci consisting of distinct populations (1). These foci represent either genetically distinct organism populations or populations expressing different major surface glycoprotein antigens.

In the previous report of Pneumocystis sterols in organisms isolated from cryopreserved human lungs, the samples were from HIV-positive and HIV-negative individuals (16). No correlation was observed between the accumulation of pneumocysterol and the HIV infection status of the patients. It was unlikely that diet or drug therapy (or possible unprescribed drugs taken) could cause these vast differences in sterol profiles. Nonetheless, these factors could not be ruled out in that study. In the present study, these factors can be ruled out because the samples were from the lungs of a single individual. Thus, the most parsimonious explanation for differences in sterol profiles at different PCP lung foci and in different organism preparations from different patients is the presence of genetically distinct organism populations that differ in their biochemical makeups. Thus, one population may accumulate large amounts of pneumocysterol and another phenotypic or genotypic population may normally produce or accumulate this sterol in only trace amounts. Intermediate pneumocysterol values can then be explained by the presence of mixed infections consisting of the two phenotypes or genotypes in various proportions.


    ACKNOWLEDGMENTS

We thank R. P. Baughman, M. Basselin, M. A. Wyder, and H. Rudney for valuable discussions of the study and the manuscript.

This work was supported in part by grants RO1 AI38758 and RO1 AI29316 from the National Institute of Allergy and Infectious Diseases (E.S.K.) and a fellowship from the Universiti Malaysia Sarawak (Z.A.).


    FOOTNOTES

* Corresponding author. Mailing address: Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221-0006. Phone: (513) 556-9712. Fax: (513) 556-5280. E-mail: Edna.Kaneshiro{at}uc.edu.


    REFERENCES
Top
Abstract
Text
References

1. Angus, C. W., A. Tu, P. Vogel, M. Qin, and J. A. Kovacs. 1996. Expression of variants of the major surface glycoprotein of Pneumocystis carinii. J. Exp. Med. 183:1229-1234[Abstract/Free Full Text].
2. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917.
3. Cushion, M. T., M. Kaselis, S. L. Stringer, and J. R. Stringer. 1993. Genetic stability and diversity of Pneumocystis carinii infecting rat colonies. Infect. Immun. 61:4801-4813[Abstract/Free Full Text].
4. Cushion, M. T., J. Zhang, M. Kaselis, D. Giuntoli, S. L. Stringer, and J. R. Stringer. 1993. Evidence for two genetic variants of Pneumocystis carinii coinfecting laboratory rats. J. Clin. Microbiol. 31:1217-1223[Abstract/Free Full Text].
5. Florin-Christensen, M., J. Florin-Christensen, Y.-P. Wu, L. Zhou, A. Gupta, H. Rudney, and E. S. Kaneshiro. 1994. Occurrence of specific sterols in Pneumocystis carinii. Biochem. Biophys. Res. Commun. 198:236-242[CrossRef][Medline].
6. Furlong, S. T., J. A. Samia, R. M. Rose, and J. A. Fishman. 1994. Phytosterols are present in Pneumocystis carinii. Antimicrob. Agents Chemother. 38:2534-2540[Abstract/Free Full Text].
7. Furuta, T., M. Fujita, R. Mukai, I. Sakakibara, T. Sata, K. Miki, M. Hayami, S. Kojima, and Y. Yoshikawa. 1993. Severe pulmonary pneumocystosis in simian acquired immunodeficiency syndrome induced by simian immunodeficiency virus: its characterization by the polymerase chain-reaction method and failure of experimental transmission to immunodeficient animals. Parasitol. Res. 79:624-628[CrossRef][Medline].
8. Georgopapadakou, N. H., and T. J. Walsh. 1994. Human mycoses: drugs and targets for emerging pathogens. Science 264:371-373[Free Full Text].
9. Gigliotti, F., A. G. Harmsen, C. G. Haidaris, and P. J. Haidaris. 1993. Pneumocystis carinii is not universally transmissible between mammalian species. Infect. Immun. 61:2886-2890[Abstract/Free Full Text].
10. Hauser, P. M., D. S. Blane, J. Bille, and P. Francioli. 1998. Typing methods to approach typing of Pneumocystis carinii genetic heterogeneity. FEMS Immunol. Med. Microbiol. 22:27-35[CrossRef][Medline].
11. Hauser, P. M., D. S. Blanc, J. Bille, A. Nahimana, and P. Francioli. 2000. Carriage of Pneumocystis carinii by immunosuppressed patients and molecular typing of the organisms. AIDS 14:461-463[CrossRef][Medline].
12. Helweg-Larsen, J., A. G. Tsolaki, R. F. Miller, B. Lundgren, and A. E. Wakefield. 1998. Clusters of Pneumocystis carinii pneumonia: analysis of person-to-person transmission by genotyping. Quart. J. Med. 91:813-820.
13. Hong, S. T., P. E. Steele, M. T. Cushion, P. D. Walzer, S. L. Stringer, and J. R. Stringer. 1990. Pneumocystis carinii karyotypes. J. Clin. Microbiol. 28:1785-1795[Abstract/Free Full Text].
14. Kaneshiro, E. S. 1998. Lipid metabolism of Pneumocystis carinii: toward the definition of new molecular targets. FEMS Immunol. Med. Microbiol. 22:135-144[CrossRef][Medline].
15. Kaneshiro, E. S. 1998. The lipids of Pneumocystis carinii. Clin. Microbiol. Rev. 11:27-41[Abstract/Free Full Text].
16. Kaneshiro, E. S., Z. Amit, J. Chandra, R. P. Baughman, C. Contini, and B. Lundgren. 1999. The sterol composition of Pneumocystis carinii hominis organisms isolated from human lungs. Clin. Diagn. Lab. Immunol. 6:970-976[Abstract/Free Full Text].
17. Kaneshiro, E. S., Z. Amit, M. M. Swonger, K. Jayasimhulu, G. P. Kreishman, E. E. Brooks, M. Kreishman, D. H. Beach, and E. J. Parish. 1999. Pneumocysterol (24Z-ethylidenelanost-8-en-3beta -ol), a rare sterol detected in the opportunistic pathogen Pneumocystis carinii f. sp. hominis. Proc. Natl. Acad. Sci. USA 96:97-102[Abstract/Free Full Text].
18. Kaneshiro, E. S., J. E. Ellis, K. Jayasimhulu, and D. H. Beach. 1994. Evidence for the presence of `metabolic sterols' in Pneumocystis:identification and initial characterization of Pneumocystis carinii sterols. J. Eukaryot. Microbiol. 41:78-85[Medline].
19. Kaneshiro, E. S., M. M. Swonger, G. Kreishman, E. Brooks, K. Jayasimhulu, E. J. Parish, and D. H. Beach. 1996. Identification of C31 and C32 sterols in Pneumocystis carinii hominis-infected human lungs. J. Eukaryot. Microbiol. 43:36S[Medline].
20. Keely, S., J. R. Stringer, R. P. Baughman, M. J. Linke, P. D. Walzer, and A. G. Smulian. 1995. Genetic variation among Pneumocystis carinii hominis isolates in recurrent pneumocystosis. J. Infect. Dis. 172:595-598[Medline].
21. Latouche, S., P. Roux, J. L. Poirot, I. Lavrard, B. Hermelin, and V. Bertand. 1994. Preliminary results of Pneumocystis carinii strain differentiation by using molecular biology. J. Clin. Microbiol. 32:3052-3053[Abstract/Free Full Text].
22. Mazars, E., K. Guyot, I. Durand, E. Dei-Cas, S. Boucher, S. B. Abderrazak, A. L. Banuls, M. Tibayrenc, and D. Camus. 1997. Isoenzyme diversity in Pneumocystis carinii from rats, mice, and rabbits. J. Infect. Dis. 175:655-660[Medline].
23. Tsolaki, A. G., R. F. Miller, and A. E. Wakefield. 1999. Oropharyngeal samples for genotyping and monitoring response to treatment in AIDS patients with Pneumocystis carinii pneumonia. J. Med. Microbiol. 48:897-905[Abstract/Free Full Text].
24. Urbina, J. A., G. Visbal, L. M. Contreras, G. McLaughlin, and R. Docampo. 1997. Inhibitors of Delta 24(25) sterol methyltransferase block sterol synthesis and cell proliferation in Pneumocystis carinii. Antimicrob. Agents Chemother. 41:1428-1432[Abstract].
25. Weinberg, G. A., and P. J. Durant. 1994. Genetic diversity of Pneumocystis carinii derived from infected rats, mice, ferrets, and cell cultures. J. Eukaryot. Microbiol. 41:223-228[Medline].

Journal of Clinical Microbiology, March 2001, p. 1137-1139, Vol. 39, No. 3
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.3.1137-1139.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • TAKAHASHI, T., ENDO, T., NAKAMURA, T., SAKASHITA, H., KIMURA, K., OHNISHI, K., KITAMURA, Y., IWAMOTO, A. (2002). Dihydrofolate reductase gene polymorphisms in Pneumocystis carinii f. sp. hominis in Japan. J Med Microbiol 51: 510-515 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Amit, Z.
Right arrow Articles by Kaneshiro, E. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Amit, Z.
Right arrow Articles by Kaneshiro, E. S.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»