Abstract
Casein kinase 1 (CK1) plays an important role in eukaryotic signaling pathways, and their substrates include key regulatory proteins involved in cell differentiation, proliferation and chromosome segregation. The Leishmania genome encodes six potential CK1 isoforms, of which five have orthologs in other trypanosomatidae. Leishmania donovani CK1 isoform 4 (Ldck1.4, orthologous to LmjF27.1780) is unique to Leishmania and contains a putative secretion signal peptide. The full-length gene and three shorter constructs were cloned and expressed in E. coli as His-tag proteins. Only the full-length 62.3 kDa protein showed protein kinase activity indicating that the N-terminal and C-terminal domains are essential for protein activity. LdCK1.4-FLAG was stably over expressed in L. donovani, and shown by immunofluorescence to be localized primarily in the cytosol. Western blotting using anti-FLAG and anti-CK1.4 antibodies showed that this CK1 isoform is expressed and secreted by promastigotes. Over expression of LdCK1.4 had a significant effect on promastigote growth in culture with these parasites growing to higher cell densities than the control parasites (wild-type or Ld:luciferase, P<0.001). Analysis by flow cytometry showed a higher percentage, ∼4–5-fold, of virulent metacyclic promastigotes on day 3 among the LdCK1.4 parasites. Finally, parasites over expressing LdCK1.4 gave significantly higher infections of mouse peritoneal macrophages compared to wild-type parasites, 28.6% versus 6.3%, respectively (p = 0.0005). These results suggest that LdCK1.4 plays an important role in parasite survival and virulence. Further studies are needed to validate CK1.4 as a therapeutic target in Leishmania.
Highlights
Leishmania are protozoan parasites responsible for a variety of human diseases ranging from simple self-healing cutaneous leishmaniasis to the fatal visceral form of the disease
DNA sequences for the L. major and L. infantum ck1.4 orthologs (LmjF27.1780 and LinJ.27.1680, respectively) were aligned, and oligonucleotide primers (MFCK1 and MRCK1) to the conserved 59- and 39- regions of the gene used to amplify the gene from L. donovani
The DNA sequence obtained for Ldck1.4 shows high homology to orthologs in other Leishmania species (99, 95, 95 and 82% homology over 568 amino acids for L. infantum, L. major, L. mexicana and L. braziliensis, respectively)
Summary
Leishmania are protozoan parasites responsible for a variety of human diseases ranging from simple self-healing cutaneous leishmaniasis to the fatal visceral form of the disease. Leishmania have a digenetic life-cycle existing as extracellular flagellated promastigotes in sand fly vectors; and as intracellular aflagellated amastigotes in the macrophages and dendritic cells of their mammalian hosts [3,4]. It was demonstrated that protein kinases, including casein kinase 1 (CK1) and casein kinase 2 (CK2), are released/secreted by promastigotes of several Leishmania species [8,9,10,11,12,13]. Constitutive or induced release of CK1 and CK2 from promastigotes could be modulated by temperature and pH [11], two important environmental cues for leishmanial differentiation from promastigotes to amastigotes and visa versa. Modification of temperature (34–37uC) and acidic pH, have been utilized for axenic amastigote propagation in vitro and studies on parasite differentiation [14,15]
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