Leishmania donovani 90 kD heat shock protein - impact of phosphosites on parasite fitness, infectivity and casein kinase affinity

Antje Hombach-Barrigah, Katharina Bartsch, Despina Smirlis, Heidi Rosenqvist, Andrea MacDonald, Florent Dingli, Damarys Loew, Gerald F. Späth, Najma Rachidi, Martin Wiese, Joachim Clos

Research output: Contribution to journalArticle

1 Citation (Scopus)

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.

LanguageEnglish
Article number5074
Number of pages16
JournalScientific Reports
Volume9
Issue number1
DOIs
Publication statusPublished - 25 Mar 2019

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Casein Kinases
HSP90 Heat-Shock Proteins
Leishmania donovani
Parasites
Phosphorylation
Leishmania
Mutation
Threonine
Heat-Shock Proteins
Aspartic Acid
Serine
Signal Transduction
Mass Spectrometry
Proteins
Macrophages
In Vitro Techniques
Growth
Infection
Genes

Keywords

  • Leishmania donovani
  • Leishmania parasites
  • phosphosites
  • kinase substrate protiens

Cite this

Hombach-Barrigah, A., Bartsch, K., Smirlis, D., Rosenqvist, H., MacDonald, A., Dingli, F., ... Clos, J. (2019). Leishmania donovani 90 kD heat shock protein - impact of phosphosites on parasite fitness, infectivity and casein kinase affinity. Scientific Reports, 9(1), [5074]. https://doi.org/10.1038/s41598-019-41640-0
Hombach-Barrigah, Antje ; Bartsch, Katharina ; Smirlis, Despina ; Rosenqvist, Heidi ; MacDonald, Andrea ; Dingli, Florent ; Loew, Damarys ; Späth, Gerald F. ; Rachidi, Najma ; Wiese, Martin ; Clos, Joachim. / Leishmania donovani 90 kD heat shock protein - impact of phosphosites on parasite fitness, infectivity and casein kinase affinity. In: Scientific Reports. 2019 ; Vol. 9, No. 1.
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Hombach-Barrigah, A, Bartsch, K, Smirlis, D, Rosenqvist, H, MacDonald, A, Dingli, F, Loew, D, Späth, GF, Rachidi, N, Wiese, M & Clos, J 2019, 'Leishmania donovani 90 kD heat shock protein - impact of phosphosites on parasite fitness, infectivity and casein kinase affinity' Scientific Reports, vol. 9, no. 1, 5074. https://doi.org/10.1038/s41598-019-41640-0

Leishmania donovani 90 kD heat shock protein - impact of phosphosites on parasite fitness, infectivity and casein kinase affinity. / Hombach-Barrigah, Antje; Bartsch, Katharina; Smirlis, Despina; Rosenqvist, Heidi; MacDonald, Andrea; Dingli, Florent; Loew, Damarys; Späth, Gerald F.; Rachidi, Najma; Wiese, Martin; Clos, Joachim.

In: Scientific Reports, Vol. 9, No. 1, 5074, 25.03.2019.

Research output: Contribution to journalArticle

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T1 - Leishmania donovani 90 kD heat shock protein - impact of phosphosites on parasite fitness, infectivity and casein kinase affinity

AU - Hombach-Barrigah, Antje

AU - Bartsch, Katharina

AU - Smirlis, Despina

AU - Rosenqvist, Heidi

AU - MacDonald, Andrea

AU - Dingli, Florent

AU - Loew, Damarys

AU - Späth, Gerald F.

AU - Rachidi, Najma

AU - Wiese, Martin

AU - Clos, Joachim

PY - 2019/3/25

Y1 - 2019/3/25

N2 - 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.

AB - 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.

KW - Leishmania donovani

KW - Leishmania parasites

KW - phosphosites

KW - kinase substrate protiens

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DO - 10.1038/s41598-019-41640-0

M3 - Article

VL - 9

JO - Scientific Reports

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JF - Scientific Reports

SN - 2045-2322

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