Cyclosporin A treatment of Leishmania donovani reveals stage-specific functions of cyclophilins in parasite proliferation and viability.

Publisher: Public Library of Science Country of Publication: United States NLM ID: 101291488 Publication Model: Electronic Cited Medium: Internet ISSN: 1935-2735 (Electronic) Linking ISSN: 19352727 NLM ISO Abbreviation: PLoS Negl Trop Dis Subsets: MEDLINE

Original Publication: San Francisco, CA : Public Library of Science

Cyclophilins/*physiology ; Cyclosporine/*pharmacology ; Leishmania donovani/*drug effects ; Protozoan Proteins/*physiology; Amino Acid Sequence ; Animals ; Binding Sites ; Cells, Cultured ; Cluster Analysis ; Computational Biology ; Cyclophilins/genetics ; Cyclophilins/metabolism ; Gene Expression ; Genome, Protozoan ; Heat-Shock Response ; Humans ; Leishmania donovani/genetics ; Leishmania donovani/growth & development ; Life Cycle Stages ; Models, Molecular ; Molecular Sequence Data ; Phylogeny ; Protozoan Proteins/genetics ; Protozoan Proteins/metabolism ; Sequence Alignment

Background: Cyclosporin A (CsA) has important anti-microbial activity against parasites of the genus Leishmania, suggesting CsA-binding cyclophilins (CyPs) as potential drug targets. However, no information is available on the genetic diversity of this important protein family, and the mechanisms underlying the cytotoxic effects of CsA on intracellular amastigotes are only poorly understood. Here, we performed a first genome-wide analysis of Leishmania CyPs and investigated the effects of CsA on host-free L. donovani amastigotes in order to elucidate the relevance of these parasite proteins for drug development.
Methodology/principal Findings: Multiple sequence alignment and cluster analysis identified 17 Leishmania CyPs with significant sequence differences to human CyPs, but with highly conserved functional residues implicated in PPIase function and CsA binding. CsA treatment of promastigotes resulted in a dose-dependent inhibition of cell growth with an IC50 between 15 and 20 microM as demonstrated by proliferation assay and cell cycle analysis. Scanning electron microscopy revealed striking morphological changes in CsA treated promastigotes reminiscent to developing amastigotes, suggesting a role for parasite CyPs in Leishmania differentiation. In contrast to promastigotes, CsA was highly toxic to amastigotes with an IC50 between 5 and 10 microM, revealing for the first time a direct lethal effect of CsA on the pathogenic mammalian stage linked to parasite thermotolerance, independent from host CyPs. Structural modeling, enrichment of CsA-binding proteins from parasite extracts by FPLC, and PPIase activity assays revealed direct interaction of the inhibitor with LmaCyP40, a bifunctional cyclophilin with potential co-chaperone function.
Conclusions/significance: The evolutionary expansion of the Leishmania CyP protein family and the toxicity of CsA on host-free amastigotes suggest important roles of PPIases in parasite biology and implicate Leishmania CyPs in key processes relevant for parasite proliferation and viability. The requirement of Leishmania CyP functions for intracellular parasite survival and their substantial divergence form host CyPs defines these proteins as prime drug targets.

Science. 2005 Jul 15;309(5733):436-42. (PMID: 16020728)
EMBO J. 1997 May 15;16(10):2576-89. (PMID: 9184205)
Trends Cell Biol. 2002 Apr;12(4):164-72. (PMID: 11978535)
Clin Haematol. 1982 Oct;11(3):743-61. (PMID: 6217010)
J Immunol. 1986 Apr 15;136(8):3067-75. (PMID: 3958489)
EMBO Rep. 2001 Jun;2(6):511-8. (PMID: 11415984)
Nature. 1993 Jan 7;361(6407):91-4. (PMID: 8421501)
Parasitology. 1992;105 Suppl:S25-40. (PMID: 1308927)
Am J Trop Med Hyg. 1985 Sep;34(5):861-5. (PMID: 3929634)
Biochemistry. 2005 Dec 13;44(49):16026-34. (PMID: 16331962)
Front Biosci. 2004 Sep 01;9:2420-46. (PMID: 15353296)
Biochemistry. 2004 Sep 21;43(37):11862-72. (PMID: 15362872)
Mol Biochem Parasitol. 1998 Mar 1;91(1):145-52. (PMID: 9574932)
Acta Crystallogr D Biol Crystallogr. 2005 May;61(Pt 5):513-9. (PMID: 15858260)
Eur J Biochem. 1995 Apr 1;229(1):188-93. (PMID: 7744028)
Drugs. 2001;61(13):1957-2016. (PMID: 11708766)
Exp Parasitol. 1991 Oct;73(3):326-34. (PMID: 1915747)
Biochemistry. 2006 Jan 10;45(1):263-71. (PMID: 16388602)
Science. 2005 Jul 15;309(5733):409-15. (PMID: 16020725)
Parasitol Res. 1994;80(6):537-42. (PMID: 7528916)
Lancet Infect Dis. 2007 Sep;7(9):581-96. (PMID: 17714672)
Adv Parasitol. 1993;32:161-259. (PMID: 8237615)
Mol Biochem Parasitol. 1996 Jun;78(1-2):79-90. (PMID: 8813679)
Clin Exp Immunol. 1989 Jan;75(1):141-6. (PMID: 2702771)
J Mol Biol. 2000 Sep 8;302(1):205-17. (PMID: 10964570)
Org Lett. 2006 Nov 9;8(23):5247-50. (PMID: 17078689)
Proteins. 2004 Sep 1;56(4):808-20. (PMID: 15281132)
Parasitol Res. 2003 Feb;89(3):221-7. (PMID: 12541065)
Proc Natl Acad Sci U S A. 2010 May 4;107(18):8381-6. (PMID: 20404152)
EMBO J. 2001 Mar 1;20(5):1020-32. (PMID: 11230126)
J Parasitol. 1984 Aug;70(4):561-2. (PMID: 6502359)
Parasitol Today. 1995 Oct;11(10):385-8. (PMID: 15275401)
J Biol Chem. 2001 Jun 1;276(22):19294-300. (PMID: 11278494)
Biochemistry. 2009 Nov 3;48(43):10423-36. (PMID: 19785464)
Mol Biochem Parasitol. 1998 Sep 1;95(1):9-20. (PMID: 9763285)
Protein Sci. 1992 Sep;1(9):1092-9. (PMID: 1338979)
Mol Microbiol. 1997 Apr;24(2):421-9. (PMID: 9159527)
J Immunol. 1986 Jul 15;137(2):702-7. (PMID: 3487578)
J Immunol Methods. 1981;43(3):269-75. (PMID: 7019344)
Science. 1996 Dec 6;274(5293):1713-5. (PMID: 8939862)
Comp Immunol Microbiol Infect Dis. 2004 Sep;27(5):305-18. (PMID: 15225981)
EMBO Rep. 2007 Jan;8(1):40-5. (PMID: 17203101)
Infect Immun. 1990 Sep;58(9):3151-3. (PMID: 2387638)
Proteomics. 2008 Jan;8(2):350-63. (PMID: 18203260)
Biochem J. 1998 Sep 15;334 ( Pt 3):659-67. (PMID: 9729475)
Mol Biol Cell. 2001 Nov;12(11):3307-16. (PMID: 11694568)
Cell Mol Life Sci. 1999 Mar;55(3):423-36. (PMID: 10228556)
Mol Biochem Parasitol. 1983 Sep;9(1):15-28. (PMID: 6199667)
Biochem J. 1995 Apr 1;307 ( Pt 1):5-8. (PMID: 7717993)
J Biol Chem. 1990 Dec 5;265(34):21011-5. (PMID: 1701173)
Mol Biochem Parasitol. 2003 Aug 11;130(1):31-42. (PMID: 14550894)
Science. 2005 Jul 15;309(5733):416-22. (PMID: 16020726)
Int J Parasitol. 2006 Mar;36(3):261-76. (PMID: 16443228)
Transplantation. 1986 May;41(5):611-5. (PMID: 3705159)
Nature. 1989 Feb 2;337(6206):476-8. (PMID: 2492638)
Curr Top Med Chem. 2003;3(12):1315-47. (PMID: 12871165)
Adv Parasitol. 1999;44:1-39. (PMID: 10563394)
J Biol Chem. 2002 Dec 6;277(49):47451-60. (PMID: 12244046)
Biol Pharm Bull. 1996 Apr;19(4):506-11. (PMID: 8860948)
Structure. 2001 May 9;9(5):431-8. (PMID: 11377203)
Mol Cell Biol. 1994 May;14(5):2975-84. (PMID: 7545921)
Nat Protoc. 2009;4(1):1-13. (PMID: 19131951)
Acta Crystallogr D Biol Crystallogr. 2009 Nov;65(Pt 11):1187-95. (PMID: 19923714)
Clin Immunol Immunopathol. 1996 Sep;80(3 Pt 2):S40-5. (PMID: 8811062)
Cell. 1991 Aug 23;66(4):807-15. (PMID: 1715244)
J Biol Chem. 1992 Jul 5;267(19):13115-8. (PMID: 1618811)
Cell Mol Life Sci. 2006 Dec;63(24):2889-900. (PMID: 17075696)
J Biol Chem. 1996 Feb 9;271(6):2961-5. (PMID: 8621687)
PLoS One. 2007 May 09;2(5):e437. (PMID: 17487282)
FEBS Lett. 1994 Sep 26;352(2):180-4. (PMID: 7925971)
Cell Stress Chaperones. 2004 Summer;9(2):167-81. (PMID: 15497503)
Mol Biochem Parasitol. 2005 May;141(1):99-108. (PMID: 15811531)
EMBO Rep. 2000 Nov;1(5):428-34. (PMID: 11258483)
Trends Cell Biol. 1992 Sep;2(9):272-6. (PMID: 14731520)

0 (Protozoan Proteins)
83HN0GTJ6D (Cyclosporine)
EC 5.2.1.- (Cyclophilins)

Date Created: 20100709 Date Completed: 20100924 Latest Revision: 20211020

20231215

PMC2894131

10.1371/journal.pntd.0000729

20614016