The Leishmania donovani chaperone cyclophilin 40 is essential for intracellular infection independent of its stage‐specific phosphorylation status

Abstract

SummaryDuring its life cycle, the protozoan pathogen Leishmania donovani is exposed to contrasting environments inside insect vector and vertebrate host, to which the parasite must adapt for extra-and intracellular survival. Combining null mutant analysis with phosphorylation site-specific mutagenesis and functional complementation we genetically tested the requirement of the L. donovani chaperone cyclophilin 40 (LdCyP40) for infection. Targeted replacement of LdCyP40 had no effect on parasite viability, axenic amastigote differentiation, and resistance to various forms of environmental stress in culture, suggesting important functional redundancy to other parasite chaperones. However, ultrastructural analyses and video microscopy of cyp40−/− promastigotes uncovered important defects in cell shape, organization of the subpellicular tubulin network and motility at stationary growth phase. More importantly, cyp40−/− parasites were unable to establish intracellular infection in murine macrophages and were eliminated during the first 24 h post infection. Surprisingly, cyp40−/− infectivity was restored in complemented parasites expressing a CyP40 mutant of the unique S274 phosphorylation site. Together our data reveal non-redundant CyP40 functions in parasite cytoskeletal remodelling relevant for the development of infectious parasites in vitro independent of its phosphorylation status, and provide a framework for the genetic analysis of Leishmania-specific phosphorylation sites and their role in regulating parasite protein function.

Phenotypic Characterization of a Leishmania donovani Cyclophilin 40 Null Mutant

“…cyp40 −/− null mutant parasites were generated as previously described by sequential replacement of the endogenous CYP40 alleles using two targeting constructs comprising puromycin and bleomycin resistance genes flanked by approximately 1,000 bp of the 5′ and 3′UTRs of the L. infantum CYP40 gene (GeneDB accession number LinJ.35.4830; Yau et al. ). For genetic complement of cyp40 −/− null mutants, a cyp40 −/− /+ “add‐back” line was established using a knock in strategy integrating the CyP40 ORF and 3′UTR into the ribosomal locus (Yau et al.…”

“…For genetic complement of cyp40 −/− null mutants, a cyp40 −/− /+ “add‐back” line was established using a knock in strategy integrating the CyP40 ORF and 3′UTR into the ribosomal locus (Yau et al. ).…”

“…Ablation of this protein in L. donovani did not affect parasite viability in logarithmically growing culture but caused dramatic morphological defects in parasites entering stationary growth phase that could be linked to a disorganization of the parasite subpellicular tubulin network (Yau et al. ). Cyp40 −/− parasites failed to establish intracellular infection despite expression of metacyclic markers, revealing important nonredundant functions for this cyclophilin in parasite virulence.…”

The genetics of Leishmania virulence

“…A putative co-chaperone protein, cyclophilin 40 (CyP40), is a true virulence factor since it is essential both for the formation of the infective, metacyclic promastigote and for the parasite's establishment within macrophages [117]. The mechanisms by which CyP40 exerts its effects on metacyclogenesis and on intracellular survival are still in the dark.…”

The Emerging Role of TPR-Domain Immunophilins in the Mechanism of Action of Steroid Receptors

“…In contrast to the FKBP subfamily that bind the immunosuppressive macrolide FK506, CyP40 belongs to the cyclophilin subfamily and binds the cyclic undecapeptide cyclosporine A. CyP40 is not recovered with native GR and MR, but with PR and ER [37,164,165]. Because CyP40 is also known to form complexes with dynein/dynactin [60] and associates to the cytoskeleton [166], it is entirely possible that this TPRdomain IMM plays a redundant role with FKBP52 and FKBPL/WisP39 in receptor trafficking. It has been shown that CyP40 is overexpressed in breast cancer tissues when it is compared to normal breast tissue [139] and also that the breast cancer cell line MCF-7 shows 75-fold higher level of Cyp40 mRNA expression in response to high temperature stress and a marked redistribution of Cyp40 protein from a predominantly nucleolar location to nuclear accumulation [167].…”