Leishmania donovani 90 kD Heat Shock Protein – Impact of Phosphosites on Parasite Fitness, Infectivity and Casein Kinase Affinity

Abstract

Leishmania parasites are thought to control protein activity at the post-translational level, e.g. by protein phosphorylation. In the pathogenic amastigote, the mammalian stage of Leishmania parasites, heat shock proteins show increased phosphorylation, indicating a role in stage-specific signal transduction. Here we investigate the impact of phosphosites in the L. donovani heat shock protein 90. Using a chemical knock-down/genetic complementation approach, we mutated 11 confirmed or presumed phosphorylation sites and assessed the impact on overall fitness, morphology and in vitro infectivity. Most phosphosite mutations affected the growth and morphology of promastigotes in vitro, but with one exception, none of the phosphorylation site mutants had a selective impact on the in vitro infection of macrophages. Surprisingly, aspartate replacements mimicking the negative charge of phosphorylated serines or threonines had mostly negative impacts on viability and infectivity. HSP90 is a substrate for casein kinase 1.2-catalysed phosphorylation in vitro. While several putative phosphosite mutations abrogated casein kinase 1.2 activity on HSP90, only Ser289 could be identified as casein kinase target by mass spectrometry. In summary, our data show HSP90 as a downstream client of phosphorylation-mediated signalling in an organism that depends on post-transcriptional gene regulation.

The low complexity regions in the C-terminus are essential for the subcellular localisation of Leishmania casein kinase 1 but not for its activity

“…In human, these two chaperones are phosphorylated by human CK1 to control the balance between folding and degradation [34]. Likewise, Leishmania CK1.2 was recently shown to phosphorylate Leishmania Hsp90 [35] [34]. To investigate whether CK1.2 co-localises with Hsp90 and Hsp70 to the flagellar pocket, we performed immunofluorescence microscopy using detergent treatment to remove the cytoplasmic fraction of these chaperones.…”

“…We showed for the first time that Hsp90 is located at the flagellar pocket and more specifically to the neck, where it co-localises with CK1.2. Because CK1.2 phosphorylates Hsp90, both proteins may be involved in functions associated with the FPN such as facilitating the entry of macromolecules or regulating endocytosis [35, 36, 60]. However, given the localisation of these two proteins, an involvement in the regulation of FAZ proteins cannot be excluded and will be further investigated.…”

Identification of differential protein recognition pattern between Naegleria fowleri and Naegleria lovaniensis

“…In the case of heat shock protein, many members of this superfamily are regulated by post‐translational modifications, such as phosphorylation 67 . In this regard for Leishmania parasites, an increased phosphorylation of heat shock proteins has been reported at the stage of pathogenic amastigote; thus, it is believed that at the post‐translational level, there is a control of the fundamental protein activity 68 . Therefore, in N fowleri , the protein phosphorylation could be a key mechanism in the regulation of some proteins implicated in the pathogenicity of these amoeba.…”

Leishmania dual‐specificity tyrosine‐regulated kinase 1 (DYRK1) is required for sustaining Leishmania stationary phase phenotype

“…Differences in highly conserved sequences (glycine rich loop, cation binding site) of the kinase catalytic domain for specific Leishmania DYRK family members indicate that these are either pseudokinases or kinases with atypical features mirroring the unique biology of the parasite. Lin DYRK1 is one of the genes that shares all the features of an active kinase, and recombinant Lin DYRK1 has been shown to be indeed an active kinase (Hombach‐Barrigah et al, ).…”

“…For cell cycle analysis, logarithmic parasites were synchronised in the G 1 /S boundary with 2.5 mM of HU (Sigma Aldrich) for 12 hr, as previously reported. The DNA content of promastigotes was analysed by PI staining of RNAse A‐treated and ethanol‐fixed parasites, as previously described (Hombach‐Barrigah et al, ; Smirlis et al, ). More specifically, cells were fixed by addition of ice‐cold ethanol and parasitic DNA content was measured by flow cytometry following the incubation with 50 μg/ml PI and 100 μg/ml RNase A (Invitrogen) in PBS in the FL2 or 610/20BP channels.…”