Phosphorylation of Sli15 by Ipl1 is important for proper CPC localization and chromosome stability in <em>Saccharomyces cerevisiae</em>

The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here we have identified multiple sites of CPC autophosphorylation on yeast Sli15 that are located within its central microtubule-binding domain and examined the functional significance of their phosphorylation by Ipl1 through mutation of these sites, either to non-phosphorylatable alanine (sli15-20A) or to acidic residues to mimic constitutive phosphorylation (sli15-20D). Both mutant sli15 alleles confer chromosome instability, but this is mediated neither by changes in the capacity of Sli15 to activate Ipl1 kinase nor by decreased efficiency of chromosome biorientation, a key process in cell division that requires CPC function. Instead, we find that mimicking constitutive phosphorylation of Sli15 on the Ipl1 phosphorylation sites causes delocalization of the CPC in metaphase, whereas blocking phosphorylation of Sli15 on the Ipl1 sites drives excessive localization of Sli15 to the mitotic spindle in pre-anaphase cells. Consistent with these results, direct interaction of Sli15 with microtubules in vitro is greatly reduced either following phosphorylation by Ipl1 or when constitutive phosphorylation at the Ipl1-dependent phosphorylation sites is mimicked by aspartate or glutamate substitutions. Furthermore, we find that mimicking Ipl1 phosphorylation of Sli15 interferes with the 'tension checkpoint'--the CPC-dependent mechanism through which cells activate the spindle assembly checkpoint to delay anaphase in the absence of tension on kinetochore-microtubule attachments. Ipl1-dependent phosphorylation of Sli15 therefore inhibits its association with microtubules both in vivo and in vitro and may negatively regulate the tension checkpoint mechanism

Transgenic analysis of the Leishmania MAP kinase MPK10 reveals an auto-inhibitory mechanism crucial for stage-regulated activity and parasite viability

Protozoan pathogens of the genus Leishmania have evolved unique signaling mechanisms that can sense changes in the host environment and trigger adaptive stage differentiation essential for host cell infection. The signaling mechanisms underlying parasite development remain largely elusive even though Leishmania mitogen-activated protein kinases (MAPKs) have been linked previously to environmentally induced differentiation and virulence. Here, we unravel highly unusual regulatory mechanisms for Leishmania MAP kinase 10 (MPK10). Using a transgenic approach, we demonstrate that MPK10 is stage-specifically regulated, as its kinase activity increases during the promastigote to amastigote conversion. However, unlike canonical MAPKs that are activated by dual phosphorylation of the regulatory TxY motif in the activation loop, MPK10 activation is independent from the phosphorylation of the tyrosine residue, which is largely constitutive. Removal of the last 46 amino acids resulted in significantly enhanced MPK10 activity both for the recombinant and transgenic protein, revealing that MPK10 is regulated by an auto-inhibitory mechanism. Over-expression of this hyperactive mutant in transgenic parasites led to a dominant negative effect causing massive cell death during amastigote differentiation, demonstrating the essential nature of MPK10 auto-inhibition for parasite viability. Moreover, phosphoproteomics analyses identified a novel regulatory phospho-serine residue in the C-terminal auto-inhibitory domain at position 395 that could be implicated in kinase regulation. Finally, we uncovered a feedback loop that limits MPK10 activity through dephosphorylation of the tyrosine residue of the TxY motif. Together our data reveal novel aspects of protein kinase regulation in Leishmania, and propose MPK10 as a potential signal sensor of the mammalian host environment, whose intrinsic pre-activated conformation is regulated by auto-inhibition

Dangerous Duplicity: The Dual Functions of Casein Kinase 1 in Parasite Biology and Host Subversion

International audienceCasein Kinase 1 (CK1) family members are serine/threonine protein kinases that are involved in many biological processes and highly conserved in eukaryotes from protozoan to humans. Even though pathogens exploit host CK1 signaling pathways to survive, the role of CK1 in infectious diseases and host/pathogen interaction is less well characterized compared to other diseases, such as cancer or neurodegenerative diseases. Here we present the current knowledge on CK1 in protozoan parasites highlighting their essential role for parasite survival and their importance for host-pathogen interactions. We also discuss how the dual requirement of CK1 family members for parasite biological processes and host subversion could be exploited to identify novel antimicrobial interventions

A touch of Zen: post-translational regulation of the Leishmania stress response

International audienceAcross bacterial, archaeal and eukaryotic kingdoms, heat shock proteins (HSPs) are defined as a class of highly conserved chaperone proteins that are rapidly induced in response to temperature increase through dedicated heat shock transcription factors. While this transcriptional response governs cellular adaptation of fungal, plant and animal cells to thermic shock and other forms of stress, early-branching eukaryotes of the kinetoplastid order, including trypanosomatid parasites, lack classical mechanisms of transcriptional regulation and show largely constitutive expression of HSPs, thus raising important questions on the function of HSPs in the absence of stress and the regulation of their chaperone activity in response to environmental adversity. Understanding parasite-specific mechanisms of stress-response regulation is especially relevant for protozoan parasites of the genus Leishmania that are adapted for survival inside highly toxic phagolysosomes of host macrophages causing the various immuno-pathologies of leishmaniasis. Here we review recent advances on the function and regulation of chaperone activities in these kinetoplastid pathogens and propose a new model for stress-response regulation through a reciprocal regulatory relationship between stress kinases and chaperones that may be relevant for parasite-adaptive differentiation and infectivity

Released Parasite-Derived Kinases as Novel Targets for Antiparasitic Therapies

International audienceThe efficient manipulation of their host cell is an essential feature of intracellular parasites. Most molecular mechanisms governing the subversion of host cell by protozoan parasites involve the release of parasite-derived molecules into the host cell cytoplasm and direct interaction with host proteins. Among these released proteins, kinases are particularly important as they govern the subversion of important host pathways, such as signalling or metabolic pathways. These enzymes, which catalyse the transfer of a phosphate group from ATP onto serine, threonine, tyrosine or histidine residues to covalently modify proteins, are involved in numerous essential biological processes such as cell cycle or transport. Although little is known about the role of most of the released parasite-derived kinases in the host cell, they are examples of kinases hijacking host cellular pathways such as signal transduction or apoptosis, which are essential for immune response evasion as well as parasite survival and development. Here we present the current knowledge on released protozoan kinases and their involvement in host-pathogen interactions. We also highlight the knowledge gaps remaining before considering those kinases - involved in host signalling subversion - as antiparasitic drug targets

CRISPR in Parasitology: Not Exactly Cut and Dried!

International audienceCRISPR/Cas9 technology has been developing rapidly in the field of parasitology, allowing for the dissection of molecular processes with unprecedented efficiency. Optimization and implementation of a new technology like CRISPR, especially in nonmodel organisms, requires communication and collaboration throughout the field. Recently, a 'CRISPR in Parasitology' symposium was held at the Institut Pasteur Paris, bringing together scientists studying Leishmania, Plasmodium, Trypanosoma, and Anopheles. Here we share technological advances and challenges in using CRISPR/Cas9 in the parasite and vector systems that were discussed. As CRISPR/Cas9 continues to be applied to diverse parasite systems, the community should now focus on improvement and standardization of the technique as well as expanding the CRISPR toolkit to include Cas9 alternatives/derivatives for more advanced applications like genome-wide functional screens

Studying two complementary infection models to identify common mechanisms of intracellular parasite survival: The roles of Leishmania and Eimeria exo-kinases in subversion of host cell signalling

International audienceUpon infection, parasites secrete effector molecules particularly kinases, that modify their host cells to create a permissive environment. For instance, Leishmania secretes casein kinase 1.2 (L-CK1.2) into the host cell via exosomes. Previous studies in the lab demonstrated that L-CK1.2 interacts with and phosphorylates more than 200 host proteins in vitro. Moreover, L-CK1.2 when ectopically expressed alters the phosphorylation of 771 proteins in J774 macrophages. Pathway analysis of these host targets indicated that L-CK1.2 might have an impact on a multitude of host pathways, including a few known to be modified during Leishmania infection. During Eimeriatenella infection, rhoptry kinase 1 (EtROP1) inhibits host apoptosis by interacting with host p53. On comparison, it appears that these two exo-kinases from divergent parasites, impact similar host pathways such as apoptosis and cell cycle. Our hypothesis states that there might exist convergence in the host pathways modified by Leishmania and Eimeria. To investigate this hypothesis, we will perform phosphoproteomic and transcriptomic studies. For Leishmania, we have just established the first ever phosphoproteome for L donovani infected mouse BMDMs. We quantified about 12,000 phosphosites comparing infected versus uninfected, including 663 sites with a p-value below 0.05. In order to uncover L-CK1.2 specific effects, we also had infected cells treated or untreated with the L-CK1.2 inhibitor, D4476. Further analysis is ongoing. For E. tenella, chicken lung epithelial cells (CLEC213) were infected with either wild type or EtROP1 overexpressing parasites; the samples were collected and will soon be sent for transcriptomic analysis. Once we have the complete set, including the phosphoproteome for Eimeria-infected CLEC213 cells and the transcriptome for L. donovani-infected macrophages, we will then assess which pathways are similarly or differently modified during both infections and decipher their role by interfering with important genes of these pathways using CRISPR-Cas knockout, RNAi or overexpression

Studying two complementary infection models to identify common mechanisms of intracellular parasite survival: The roles of Leishmania and Eimeria exo-kinases in subversion of host cell signalling

International audienceUpon infection, parasites secrete effector molecules particularly kinases, that modify their host cells to create a permissive environment. For instance, Leishmania secretes casein kinase 1.2 (L-CK1.2) into the host cell via exosomes. Previous studies in the lab demonstrated that L-CK1.2 interacts with and phosphorylates more than 200 host proteins in vitro. Moreover, L-CK1.2 when ectopically expressed alters the phosphorylation of 771 proteins in J774 macrophages. Pathway analysis of these host targets indicated that L-CK1.2 might have an impact on a multitude of host pathways, including a few known to be modified during Leishmania infection. During Eimeriatenella infection, rhoptry kinase 1 (EtROP1) inhibits host apoptosis by interacting with host p53. On comparison, it appears that these two exo-kinases from divergent parasites, impact similar host pathways such as apoptosis and cell cycle. Our hypothesis states that there might exist convergence in the host pathways modified by Leishmania and Eimeria. To investigate this hypothesis, we will perform phosphoproteomic and transcriptomic studies. For Leishmania, we have just established the first ever phosphoproteome for L donovani infected mouse BMDMs. We quantified about 12,000 phosphosites comparing infected versus uninfected, including 663 sites with a p-value below 0.05. In order to uncover L-CK1.2 specific effects, we also had infected cells treated or untreated with the L-CK1.2 inhibitor, D4476. Further analysis is ongoing. For E. tenella, chicken lung epithelial cells (CLEC213) were infected with either wild type or EtROP1 overexpressing parasites; the samples were collected and will soon be sent for transcriptomic analysis. Once we have the complete set, including the phosphoproteome for Eimeria-infected CLEC213 cells and the transcriptome for L. donovani-infected macrophages, we will then assess which pathways are similarly or differently modified during both infections and decipher their role by interfering with important genes of these pathways using CRISPR-Cas knockout, RNAi or overexpression