Gene Replacement by Homologous Recombination

Lorenzen

Brinker

et al. 2020

Sci Rep

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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.

Section: Methodsmentioning

confidence: 99%

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

Lorenzen

Brinker

et al. 2020

Sci Rep

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“…Electrotransfection and selection was performed as described [ 31 ]. Clonal parasite populations were obtained by limiting dilution in 96-well plates with an initial inoculum of 0.5 parasites/well in a final volume of 200 µL M199+ medium supplemented with the respective antibiotics and 1× penicillin/streptomycin (Sigma Aldrich, Munich, Germany).…”

Section: Methodsmentioning

confidence: 99%

The Leishmania donovani SENP Protease Is Required for SUMO Processing but Not for Viability

Bea

Sandhu

et al. 2020

Genes

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The protozoan parasite Leishmania donovani is part of an early eukaryotic branch and depends on post-transcriptional mechanisms for gene expression regulation. This includes post-transcriptional protein modifications, such as protein phosphorylation. The presence of genes for protein SUMOylation, i.e., the covalent attachment of small ubiquitin-like modifier (SUMO) polypeptides, in the Leishmania genomes prompted us to investigate the importance of the sentrin-specific protease (SENP) and its putative client, SUMO, for the vitality and infectivity of Leishmania donovani. While SENP null mutants are viable with reduced vitality, viable SUMO null mutant lines could not be obtained. SUMO C-terminal processing is disrupted in SENP null mutants, preventing SUMO from covalent attachment to proteins and nuclear translocation. Infectivity in vitro is not affected by the loss of SENP-dependent SUMO processing. We conclude that SENP is required for SUMO processing, but that functions of unprocessed SUMO are critical for Leishmania viability.

“…While Gene replacement using homologous recombination has proven a useful tool for testing gene function in Old World Leishmania spp. [ 5 , 6 , 7 ], yet—to our knowledge—no gene replacement analyses have been reported for L. braziliensis . However, a functional RNA interference (RNAi) machinery, predicted from the L. braziliensis genome sequence [ 8 ], was corroborated experimentally [ 9 ], allowing gene function analysis in this species [ 9 , 10 ].…”

Section: Introductionmentioning

confidence: 99%

Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23

Adaui

Brinker

et al. 2020

Genes

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The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR–Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene (eGFP) and two L. braziliensis single-copy genes (HSP23 and HSP100). We obtained homozygous Cas9-free HSP23- and HSP100-null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null mutants. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. major HSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of L. braziliensis by CRISPR–Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite’s biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis.