Gene Disruption of Honey Bee Trypanosomatid Parasite, Lotmaria passim, by CRISPR/Cas9 System

In this study, we first generated L. passim clone expressing fluorescent marker and then attempted to use CRISPR/Cas9 for the genome editing. We will discuss how these approaches can be used to better understand honey bee-trypanosomatid parasite interactions. Materials and Methods Culture of L. passim Lotmaria passim strain SF (PRA-403) was obtained from the American Type Culture Collection (ATCC) and cultured in the modified FP-FB medium (Salathe et al., 2012) at 25°C without CO2. To monitor the growth rate of L. passim, the parasites were first inoculated at 5 × 105/mL and their number during the culture was measured by CASY® Cell Counter together with Analyzer System Model TT (OMNI Life Science). Electroporation of L. passim Followed by the Single Clone Isolation Actively growing L. passim (107/mL) was collected, washed twice, and resuspended in 0.4 mL of Cytomix buffer without EDTA (20 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4, 25 mM HEPES, and 5 mM MgCl2, pH 7.6) (Van Den Hoff et al., 1992; Ngo et al., 1998). 2 × 107 parasites were electroporated with 10 μg of pTrex-Neo-tdTomato (Canavaci et al., 2010) or pTrex-b-NLS-hSpCas9 (Peng et al., 2015) using a Gene Pulser X cell electroporator (Bio-Rad). For co-transfection of sgRNA expression vector and donor DNA, 10 μg of the plasmid DNA and 25 μg of the linearized donor DNA were electroporated. The parasites and DNA were mixed well and maintained on ice for 15 min in a cuvette (2 mm gap) before ele...
Source: Frontiers in cellular and infection microbiology - Category: Microbiology Source Type: research

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TLR4 activates two distinct signaling pathways involving adaptors MyD88 and TRIF to produce proinflammatory cytokines and type-I interferon respectively. How Leishmania donovani suppresses these pathways is not well studied. We earlier reported, TLR4 is hypersialylated due to reduced membrane-bound neuraminidase (Neu1) on infected-macrophages. We hypothesized that such enhanced sialoglycoconjugates on host cells may modulate the interactions with siglecs- which are the inhibitory receptors. Here, we examined the impact of such sialylation on overall TLR4 activation both in murine cell line J774A.1 and primary bone marrow d...
Source: Frontiers in Immunology - Category: Allergy & Immunology Source Type: research
Enucleated cells or cytoplasts (cells whose nucleus is removed in vitro) represent an unexplored biological model for intracellular infection studies due to the abrupt interruption of nuclear processing and new RNA synthesis by the host cell in response to pathogen entry. Using enucleated fibroblasts hosting the protozoan parasite Leishmania amazonensis, we demonstrate that parasite multiplication and biogenesis of large parasitophorous vacuoles in which parasites multiply are independent of the host cell nucleus. Dual RNA sequencing of both host cytoplast and intracellular parasite transcripts identified host transcripts ...
Source: Infection and Immunity - Category: Infectious Diseases Authors: Tags: Cellular Microbiology: Pathogen-Host Cell Molecular Interactions Source Type: research
inker Clos 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...
Source: Genes - Category: Genetics & Stem Cells Authors: Tags: Article Source Type: research
Leishmania infection causes considerable human morbidity and may develop into a deadly visceral form in endemic regions. The parasite infects macrophages where they can replicate intracellularly. Furthermore, they modulate host immune responses by using virulence factors (lipophosphoglycan, glycoprotein-63, and others) that promote survival inside the cells. Extracellular vesicles (EVs) released by parasites are important for cell-cell communication in the proinflammatory milieu modulating the establishment of infection. However, information on the ability of EVs from different Leishmania species to modulate inflammatory r...
Source: Frontiers in cellular and infection microbiology - Category: Microbiology Source Type: research
AbstractIdentifying genetic risk factors for parasitic infections such as the leishmaniases could provide important leads for improved therapies and vaccines. Until recently most genetic studies of human leishmaniasis were underpowered and/or not replicated. Here, we focus on recent genome-wide association studies of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). For VL, analysis across 2287 cases and 2692 controls from three cohorts identified a single major peak of genome-wide significance (Pcombined = 2.76 × 10–17) atHLA-DRB1–HLA-DQA1. HLA-DRB1*1501 and DRB1*1...
Source: Human Genetics - Category: Genetics & Stem Cells Source Type: research
Abstract Immune evasion strategies adopted by Leishmania donovani involve the exploitation of suppressor of cytokine signaling (SOCS) proteins that are well-known negative regulators of the JAK/STAT pathway. However, the cellular mechanism underpinning the induction of SOCS isoforms and their role in breaching the multilevel regulatory circuit connecting the innate and adaptive arms of immunity are still ambiguous during experimental visceral leishmaniasis. Using bone marrow-derived macrophages (BMMфs) and CD4+ T cells, we observed that L. donovani preferentially upregulates SOCS1 and SOCS3 expression in macropha...
Source: Journal of Immunology - Category: Allergy & Immunology Authors: Tags: J Immunol Source Type: research
Altered sialylation is generally maintained by a fine balance between sialidases and sialyltransferases, which plays an essential role during disease pathogenesis. TLR4 is a membrane-bound highly sialylated glycoprotein predominantly having α2,3-linked sialic acids. It is one of the most important client molecules in the anti-leishmanial innate immune arm. Here, we initiated a comprehensive study on the modulation of TLR4 sialylation in Leishmania donovani (L. d)-infected macrophages by a mammalian sialidase/neuraminidase-1 (Neu1) having substrate specificity toward α2,3-linked sialic acids. We observed reduced...
Source: Frontiers in Immunology - Category: Allergy & Immunology Source Type: research
In conclusion, we hope that this review brings about a new and more in-depth understanding of the part that Leishmania exosomes and various infectious agents play in the context of host-parasite interactions, with a particular focus on the establishment of infection. Future research in this field of investigations is critical for the development of new vaccine and diagnostic tools. Author Contributions The first draft was done by MO. AF and GD added new information. Funding Research in MO laboratory is supported by the Canadian Institute of Health Research (grant number: PJT-159765) and the Natural Sciences and Enginee...
Source: Frontiers in cellular and infection microbiology - Category: Microbiology Source Type: research
In this study, we report the novel biphasic role of Toll-interacting protein (Tollip), a negative regulator of the IL-1R/TLR pathway, in the disease progression of experimental visceral leishmaniasis. We observed that during early hours of infection, L. donovani induced phosphorylation of IRAK-1, resulting in the release of Tollip from the IL-1R-associated kinase (IRAK)-1 complex in J774 macrophages, which then acted as an endocytic adaptor on cell surface IL-1R1 and promoted its lysosomal degradation. In the later stage, Tollip shuttled back to IRAK-1, thereby inhibiting IRAK-1 phosphorylation in association with IRAK-M t...
Source: Journal of Immunology - Category: Allergy & Immunology Authors: Tags: J Immunol Source Type: research
The P4 family of P-type ATPases (P4-ATPases) plays an important role in maintaining phospholipid asymmetry in eukaryotic cell membranes. Leishmania miltefosine transporter (LMT) is a plasma membrane (PM) P4-ATPase that catalyses translocation into the parasite of the leishmanicidal drug miltefosine as well as phosphatidylcholine and phosphatidylethanolamine analogues. In the present study, we analysed the role, in LMT, of a series of highly conserved amino acids previously undescribed in the N-terminal region of P4-ATPases. Seven residues were identified and, according to an LMT structural model, five were located in the c...
Source: Biochemical Journal - Category: Biochemistry Authors: Tags: Research Articles Source Type: research
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