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. 2019 Mar 25;9(1):5074.
doi: 10.1038/s41598-019-41640-0.

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

Antje Hombach-Barrigah  1 , Katharina Bartsch  1 , Despina Smirlis  2   3 , Heidi Rosenqvist  4   5 , Andrea MacDonald  1 , Florent Dingli  6 , Damarys Loew  6 , Gerald F Späth  2 , Najma Rachidi  2 , Martin Wiese  4 , Joachim Clos  7
Affiliations

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

Antje Hombach-Barrigah et al. Sci Rep. .

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Schematic representation of the targeted phosphorylation sites and domain organisation of HSP90. (A) HSP90 sequence alignment for L. donovani (LdBPK_330360.1) and L. mexicana (LmxM.32.0314). Phosphorylation sites (see Table 1) are highlighted. (B) Putative domain architecture of HSP90 and distribution of phosphorylation sites over N-terminal (ND), charged linker (CLD), middle (MD) and C-terminal domain (CD). Standard one-letter amino acid codes apply.
Figure 2
Figure 2
Impact of phosphorylation site mutations on the in vitro proliferation. L. donovani promastigotes were seeded at 1 × 106 cells ml-1 in Medium199 containing radicicol at IC90 (batch-dependent; 0,5-1,25 μg/ml) and allowed to proliferate for 96 h. Median (±range) cell density was recorded and plotted as [%] relative to the cell density of promastigotes expressing HSP90rr. Standard one-letter amino acid codes apply. (n.s.): not significant, (***): p ≤ 0.001 by U-test; n = 8. (A) ATP-binding pocket mutations. (B) Charged linker domain mutations. (C) Middle domain mutations. (D) C-terminal domain mutations.
Figure 3
Figure 3
Morphological analysis of recombinant L. donovani populations. L. donovani promastigotes were seeded at 1 × 106 cells ml-1 in Medium199 containing radicicol at IC90 (batch-dependent; 0,5-1,25 μg/ml) and allowed to proliferate for 72 h. The cells were fixed and then visualised by SEM or fluorescence microscopy using anti-α-tubulin mAb. By using image analysis, the cell body lengths of 50 (in µm, upper panels), the cell body widths of 25 (in µm, middle panels) and the flagella lengths (in µm, lower panels) of 25 randomly selected cells were analysed and mean values were compiled. Bars represent the mean measurements with SEM, n = 25. (n.d.): not detectable, (n.s.): not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, respectively, by U-test. (A) analysis performed by using SEM images. (B) analysis performed by using fluorescence microscopy images. (C) SEM images of HSP90rr and HSP90rr-T100D expressing cells -RAD and + RAD.
Figure 4
Figure 4
Effects of HSP90 phosphorylation site mutations on the in vitro infectivity (A–F). Recombinant parasites were first pre-treated with RAD for 48 h at IC90 and then used to infect bone marrow-derived macrophages (MOI 10:1). After 4 h, free parasites were removed and the infected macrophage cultures were further incubated at 37 °C and 5% CO2 for 44 h. Genomic DNAs were isolated from free and attached macrophages and subjected to a duplex qPCR against mouse and Leishmania actin DNA (Bifeld et al., 2016). The median parasite:host DNA ratio was then compiled. Abbreviations: (n.s.): not significant, *p ≤ 0.05, **p ≤ 0.01 in a one-tailed, paired Student’s t test (A), or in a two-tailed U-test (B–F); n = 6.
Figure 5
Figure 5
Protein kinase of HSP90. (A) Three protein kinases, rLmCK1.2, rDYRK 1, and rGSK 1, were incubated with γ-32P-ATP with MBP or with HSP90wt as substrate as indicated below and analysed by SDS-PAGE and Coomassie Brilliant Blue staining. The positions of protein size markers are indicated to the left, the positions of the kinases and of HSP90 are indicated on the right. (B) As in (A), but autoradiograph. (C,D) In vitro protein kinase assay of rLmCK1.2 with HSP90 and 5 phosphosite mutants thereof, using γ-32P-ATP. (C) shows a Coomassie Brilliant Blue stain, (D) shows the corresponding autoradiograph. (E,F) As in (C,D), but with 4 other phosphosite mutants of HSP90. (G) Mass spectrometry-based quantification of the phosphoserine/serine ratio at Ser289 without (−D4476) or with (+D4476) a CK1.2-specific inhibitor. The y-axis is in log10 scale. All greyscale images were cropped from contiguous parts of the original image. Digital enhancements, using Adobe Photoshop CS3, were performed over the entire greyscale images and restricted to tonality optimisation and size adjustments.
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