Leishmania major parasites express cyclophilin isoforms with an unusual interaction with calcineurin

Abstract

The immunosuppressive effects of the fungal metabolite cyclosporin A (CsA) are mediated primarily by binding to cyclophilins (Cyps). The resulting CsA-Cyp complex inhibits the Ca2+-regulated protein phosphatase calcineurin and down-regulates signal transduction events. Previously we reported that CsA is a potent inhibitor of infections transmitted by the human pathogenic protozoan parasite Leishmania major in vitro and in vivo, but does not effect the extracellular growth of L. major itself. It is unknown how L. major exerts this resistance to CsA. Here we report that a major Cyp, besides additional isoforms with the same N-terminal amino acid sequence, was expressed in L. major. The cloned and sequenced gene encodes a putative 174-residue protein called L. major Cyp 19 (LmCyp19). The recombinant LmCyp19 exhibits peptidyl-prolyl cis/trans isomerase activity with a substrate specificity and an inhibition by CsA that are characteristic of other eukaryotic Cyps. To determine whether calcineurin is involved in the discrimination of the effects of CsA we also examined the presence of a parasitic calcineurin and tested the interaction with Cyps. Despite the expression of functionally active calcineurin by L. major, neither LmCyp19 nor other L. major Cyps bound to its own or mammalian calcineurin. The amino acid sequence of most Cyps includes an essential arginine residue around the calcineurin-docking side. In LmCyp19 this is replaced by an asparagine residue. This exchange and additional charged residues are apparently responsible for the lack of LmCyp19 interaction with calcineurin. These observations indicate that resistance of L. major to CsA in vitro is mediated by the lack of complex formation with calcineurin despite CsA binding by parasitic Cyp.

Cyclosporin A Treatment of Leishmania donovani Reveals Stage-Specific Functions of Cyclophilins in Parasite Proliferation and Viability

“…The observation that CsA has no overt anti-microbial activity against L. major promastigotes in culture, but efficiently kills amastigotes in infected macrophages, provided support to the idea that the toxic effect of CsA on intracellular parasites depends on inhibition of host rather than Leishmania CyPs. This hypothesis was further supported by findings showing that the phosphatase calcineurin, the prime target of the inhibitory CsA/CyP complex, is expressed at very low levels and is not recognized by Leishmania LmaCyP19 (corresponding to LmaCyP1 according to our nomenclature), although this protein efficiently bound CsA [60], [61]. In contrast to these previous reports, our data provide several lines of evidence for a direct action of CsA on Leishmania CyPs.…”

Cyclosporin A-mediated killing of Leishmania major by macrophages is independent of reactive nitrogen and endogenous TNF-α and is not inhibited by IL-10 and 13

“…Although L. major parasites express two Cyps (20 and 22 kDa) and CsA inhibited enzyme activity of these Cyps, pretreatment of L. major parasites with CsA did not result in the reduction of a subsequent macrophage infection (Rascher et al 1998). In contrast, by the use of antisense oligonucleotides host-cell CypA was found to be critically involved in the intracellular replication of L. major parasites in murine macrophages (Hoerauf et al 1997).…”

Application of a green fluorescent fusion protein to study protein-protein interactions by electrophoretic methods

“…Cell extracts from E. coli grown in 26YT medium and Jurkat T cells grown in RPMI 1640 medium with 10% fetal serum albumin were prepared as described previously [26]. Drosophila melanogaster was cultured and characterized as reported elsewhere [25].…”