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Casein kinase 1.2 over expression restores stress resistance to Leishmania donovani HSP23 null mutants.

ABSTRACT: Leishmania donovani is a trypanosomatidic parasite and causes the lethal kala-azar fever, a neglected tropical disease. The Trypanosomatida are devoid of transcriptional gene regulation and rely on gene copy number variations and translational control for their adaption to changing conditions. To survive at mammalian tissue temperatures, L. donovani relies on the small heat shock protein HSP23, the loss of which renders the parasites stress sensitive and impairs their proliferation. Here, we analysed a spontaneous escape mutant with wild type-like in vitro growth. Further selection of this escape strains resulted in a complete reversion of the phenotype. Whole genome sequencing revealed a correlation between stress tolerance and the massive amplification of a six-gene cluster on chromosome 35, with further analysis showing over expression of the casein kinase 1.2 gene as responsible. In vitro phosphorylation experiments established both HSP23 and the related P23 co-chaperone as substrates and modulators of casein kinase 1.2, providing evidence for another crucial link between chaperones and signal transduction protein kinases in this early branching eukaryote.

SUBMITTER: Krober-Boncardo C

PROVIDER: S-EPMC7525241 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Casein kinase 1.2 over expression restores stress resistance to Leishmania donovani HSP23 null mutants.

Kröber-Boncardo Constanze C Lorenzen Stephan S Brinker Christine C Clos Joachim J

Scientific reports 20200929 1

Leishmania donovani is a trypanosomatidic parasite and causes the lethal kala-azar fever, a neglected tropical disease. The Trypanosomatida are devoid of transcriptional gene regulation and rely on gene copy number variations and translational control for their adaption to changing conditions. To survive at mammalian tissue temperatures, L. donovani relies on the small heat shock protein HSP23, the loss of which renders the parasites stress sensitive and impairs their proliferation. Here, we ana ...[more]

PMID: 32994468

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Project description:BackgroundVisceral leishmaniasis is the most clinically relevant and dangerous form of human leishmaniasis. Most traditional drugs for treatment of leishmaniasis are toxic, possess many adverse reactions and drug resistance is emerging. Therefore, there is urgent need for identification of new therapeutic targets. Recently, we found that mice with an inactivating knock-in mutation in the p110δ isoform of pi3k, (p110δ(d910a)) are hyper resistant to L. major, develop minimal cutaneous lesion and rapidly clear their parasite. Here, we investigated whether pi3k signaling also regulates resistance to L. donovani, one of the causative agents of visceral leishmaniasis.Methodology/principal findingsWT and p110δ(D910A) mice (on a BALB/c background) were infected with L. donovani. At different time points, parasite burden and granuloma formation were assessed. T and B cell responses in the liver and spleen were determined. In addition, Tregs were expanded in vivo and its impact on resistance was assessed. We found that p110δ(D910A) mice had significantly reduced splenomegaly and hepatomegaly and these organs harbored significantly fewer parasites than those of WT mice. Interestingly, infected p110δ(D910A) mice liver contains fewer and less organized granulomas than their infected WT counterparts. Cells from p110δ(D910A) mice were significantly impaired in their ability to produce cytokines compared to WT mice. The percentage and absolute numbers of Tregs in infected p110δ(D910A) mice were lower than those in WT mice throughout the course of infection. In vivo expansion of Tregs in infected p110δ(D910A) mice abolished their enhanced resistance to L. donovani infection.Conclusions/significanceOur results indicate that the enhanced resistance of p110δ(D910A) mice to L. donovani infection is due to impaired activities of Tregs. They further show that resistance to Leishmania in the absence of p110δ signaling is independent of parasite species, suggesting that targeting the PI3K signaling pathway may be useful for treatment of both visceral and cutaneous leishmaniasis.

Project description:BackgroundResistance emergence against antileishmanial drugs, particularly Sodium Antimony Gluconate (SAG) has severely hampered the therapeutic strategy against visceral leishmaniasis, the mechanism of resistance being indistinguishable. Cysteine leucine rich protein (CLrP), was recognized as one of the overexpressed proteins in resistant isolates, as observed in differential proteomics between sensitive and resistant isolates of L. donovani. The present study deals with the characterization of CLrP and for its possible connection with SAG resistance.Methodology and principal findingsIn pursuance of deciphering the role of CLrP in SAG resistance, gene was cloned, over-expressed in E. coli system and thereafter antibody was raised. The expression profile of CLrP and was found to be over-expressed in SAG resistant clinical isolates of L. donovani as compared to SAG sensitive ones when investigated by real-time PCR and western blotting. CLrP has been characterized through bioinformatics, immunoblotting and immunolocalization analysis, which reveals its post-translational modification along with its dual existence in the nucleus as well as in the membrane of the parasite. Further investigation using a ChIP assay confirmed its DNA binding potential. Over-expression of CLrP in sensitive isolate of L. donovani significantly decreased its responsiveness to SAG (SbV and SbIII) and a shift towards the resistant mode was observed. Further, a significant increase in its infectivity in murine macrophages has been observed.Conclusion/significanceThe study reports the differential expression of CLrP in SAG sensitive and resistant isolates of L. donovani. Functional intricacy of CLrP increases with dual localization, glycosylation and DNA binding potential of the protein. Further over-expressing CLrP in sensitive isolate of L. donovani shows significantly decreased sensitivity towards SAG and increased infectivity as well, thus assisting the parasite in securing a safe niche. Results indicates the possible contribution of CLrP to antimonial resistance in L. donovani by assisting the parasite growth in the macrophages.

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Casein kinase 1.2 over expression restores stress resistance to Leishmania donovani HSP23 null mutants.

Publications

Casein kinase 1.2 over expression restores stress resistance to Leishmania donovani HSP23 null mutants.

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