Abstract
Leishmaniasis is a severe public health problem, caused by the protozoan Leishmania. This parasite has two developmental forms, extracellular promastigote in the insect vector and intracellular amastigote in the mammalian host where it resides inside the phagolysosome of macrophages. Little is known about the virulence factors that regulate host-pathogen interactions and particularly host signalling subversion. All the proteomes of Leishmania extracellular vesicles identified the presence of Leishmania casein kinase 1 (L-CK1.2), a signalling kinase. L-CK1.2 is essential for parasite survival and thus might be essential for host subversion. To get insights into the functions of L-CK1.2 in the macrophage, the systematic identification of its host substrates is crucial, we thus developed an easy method to identify substrates, combining phosphatase treatment, in vitro kinase assay and Stable Isotope Labelling with Amino acids in Cell (SILAC) culture-based mass spectrometry. Implementing this approach, we identified 225 host substrates as well as a potential novel phosphorylation motif for CK1. We confirmed experimentally the enrichment of our substratome in bona fide L-CK1.2 substrates and showed they were also phosphorylated by human CK1δ. L-CK1.2 substratome is enriched in biological processes such as “viral and symbiotic interaction,” “actin cytoskeleton organisation” and “apoptosis,” which are consistent with the host pathways modified by Leishmania upon infection, suggesting that L-CK1.2 might be the missing link. Overall, our results generate important mechanistic insights into the signalling of host subversion by these parasites and other microbial pathogens adapted for intracellular survival.
Highlights
Leishmania causes immuno-pathological diseases including cutaneous, muco-cutaneous, and visceral leishmaniasis, leading to severe morbidity and mortality
L-CK1.2 was shown to be part of the core cargo of exosomal proteins (Silverman et al, 2011; Silverman et al, 2010), present in exosomes released by promastigotes in the insect vector (Atayde et al, 2015) and enriched in exosomes released by amphotericin B-resistant parasites but not in that released by miltefosine- or antimonyresistant parasites (Douanne et al, 2020; Rachidi et al, 2021), suggesting that it has an important role for parasite survival in the insect and mammalian hosts
In order to increase the number of sites available for de novo L-CK1.2 phosphorylation prior in vitro kinase assay (IVKA), we depleted the lysates in ATP before treating them with Antarctic phosphatase
Summary
Leishmania causes immuno-pathological diseases including cutaneous, muco-cutaneous, and visceral leishmaniasis, leading to severe morbidity and mortality This parasite has two developmental stages, in the insect vector as an extracellular promastigote form, and in the mammalian host as an intracellular amastigote form where it resides inside the phagolysosome of macrophages. The low stoichiometry of protein phosphorylation, the presence of endogenous kinases as well as the reversibility of the phosphorylation by phosphatases render systematic mapping of the cellular substratome extremely challenging This is true when handling pleiotropic signalling kinases such as CK1 family members, able to phosphorylate hundreds of substrates (Knippschild et al, 2014). We developed a technology, applicable to other protein kinases, that allows efficient identification of substrates Applying this pipeline on L-CK1.2, we identified 225 host substrates that shed important new lights on parasite immune and biological subversion of its host. We validated our approach, which might become a powerful new tool to study mechanisms of host-pathogen interactions and might provide host targets for host-directed therapy against Leishmaniasis
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
