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.
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 µM 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 µM, 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.
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.
Visceral leishmanisasis, also known as Kala Azar, is caused by the protozoan parasite Leishmania donovani. The L. donovani infectious cycle comprises two developmental stages, a motile promastigote stage that proliferates inside the digestive tract of the phlebotomine insect host, and a non-motile amastigote stage that differentiates inside the macrophages of mammalian hosts. Intracellular parasite survival in mouse and macrophage infection assays has been shown to be strongly compromised in the presence of the inhibitor cyclosporin A (CsA), which binds to members of the cyclophilin (CyP) protein family. It has been suggested that the toxic effects of CsA on amastigotes occurs indirectly via host cyclophilins, which may be required for intracellular parasite development and growth. Using a host-free L. donovani culture system we revealed for the first time a direct and stage-specific effect of CsA on promastigote growth and amastigote viability. We provided evidence that parasite killing occurs through a heat sensitivity mechanism likely due to direct inhibition of the co-chaperone cyclophilin 40. Our data allow important new insights into the function of the Leishmania CyP protein family in differentiation, growth, and intracellular survival, and define this class of molecules as important drug targets.