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Leishmania phosphatase PP5 is a regulator of HSP83 phosphorylation and essential for parasite pathogenicity

  • Brianna Norris-Mullins
  • Joseph S. Krivda
  • Kathryn L. Smith
  • Micah J. Ferrell
  • Miguel A. Morales
Original Paper

Abstract

Leishmania parasites are responsible for important neglected diseases in humans and animals, ranging from self-healing cutaneous lesions to fatal visceral manifestations. During the infectious cycle, Leishmania differentiates from the extracellular flagellated promastigote to the intracellular pathogenic amastigote. Parasite differentiation is triggered by changes in environmental cues, mainly pH and temperature. In general, extracellular signals are translated into stage-specific gene expression by a cascade of reversible protein phosphorylation regulated by protein kinases and phosphatases. Though protein kinases have been actively studied as potential anti-parasitic drug targets, our understanding of the biology of protein phosphatases in Leishmania is poor. We have previously reported the principal analysis of a novel protein phosphatase 5 (PP5) in Leishmania species. Here, we assessed the role of PP5 in parasite pathogenicity, where we uncovered, using transgenic PP5 over-expressing and PP5 null-mutant parasites, its importance in metacyclogeneisis, maintaining HSP83 phosphorylation homeostasis and virulence. All together, our results indicate the importance of PP5 in regulating parasite stress and adaptation during differentiation, making this protein an attractive potential target for therapeutic intervention.

Keywords

Leishmania Stress response Phosphatase Virulence Drug target 

Notes

Acknowledgements

This work was supported by the Eck Institute of Global Health and capitalization funds from the University of Notre Dame to MAM. All authors declare no financial/commercial conflict of interest. We thank Prof. Steve Beverley (Washington University of St Louis, USA) for the pXNG system.

Compliance with ethical standards

Ethics statement

The University of Notre Dame is credited through the Animal Welfare Assurance (#A3093-01). All animal studies were conducted according to the Institutional Animal Care and Use Committee (IACUC) guidelines. The protocol for the infection of mice with Leishmania was approved by the University’s IACUC (September 27, 2017, protocol #15-10-2708).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

436_2018_5994_MOESM1_ESM.pptx (51 kb)
Supplementary Figure 1 Tracking negative selection system through western blot. Western blot of protein extracts from L. donovani WT, WT/pXNG_PP5 and two clonal ∆PP5 lines. As a proof of the negative selection principle, PP5 expression increases when electroporating pXNG_PP5 into WT background (lane 2) and subsequently decreases when subjecting this same line to GCV (lane 3). Samples were separated by SDS-PAGE, transferred onto a PVDF membrane and assayed with an anti-PP5 antibody. The membrane was stripped and re-probed with anti-tubulin as a loading control. (PPTX 50 kb)
436_2018_5994_MOESM2_ESM.pptx (44 kb)
Supplementary Figure 2 L. major PP5 null-mutant and Add Back (AB) lines. Western blot of protein extracts from L. major WT, ∆PP5 and AB lines. Samples were separated by SDS-PAGE, transferred onto a PVDF membrane and assayed with an anti-PP5 antibody. The membrane was stripped and re-probed with anti-tubulin as a loading control. (PPTX 43 kb)

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Authors and Affiliations

  1. 1.Eck Institute for Global Health, Department of Biological Sciences, 278 Galvin Life ScienceUniversity of Notre DameNotre DameUSA

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