scholarly journals Construction of PX-LmGP63 Using CRISPR-Cas9 as Primary Goal for GP63 gene Knockout in Leishmania major and Leishmanization

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Saeedeh Ebrahimi ◽  
Hamzeh Alipour ◽  
Kourosh Azizi ◽  
Mohsen Kalantari
Keyword(s):  
Leishmania Major ◽  
Gene Knockout ◽  
Public Health Problem ◽  
Protozoan Parasite ◽  
Potential Method ◽  
Effective Vaccine ◽  
Mammalian Host ◽  
Signaling Mechanism ◽  
Final Confirmation ◽  
Intracellular Protozoan Parasite

Background: Leishmania is an intracellular protozoan parasite that uses complex methods for destroying the innate immune response in mammalian host macrophage cells. Many factors have been identified that play a role in the severity of the parasite’s pathogenicity. One of the factors is the GP63, which is a group of metalloproteinases that disrupts the signaling mechanism of the host cell. Objectives: The aim of this study was to construct PX-LMGP63 vector through CRISPR-Cas9 for GP63 gene knockout in Leishmania major as a potential method for leishmanization. Methods: A pair of gRNAs were designed based on the mRNA sequence of the GP63. Then annealing primers were cloned into the linearized vector PX-459 and transformed into the DH5ɑ competent cells. Then, PCR assay was performed with gene-specific and vector primers to confirm the colonies. In addition, the constructed plasmid was sequenced for final confirmation. Results: The expected size band of 270 was confirmed by PCR. The plasmid sequence showed that the gRNA789 was ligated in the vector. The created structure was named PX-LMGP63 and will be transfected into the promastigote cell in the next step. Conclusions: Owing to the prevalence of cutaneous Leishman as a public health problem in most countries and the lack of an effective vaccine for leishmaniasis, the use of the CRISPR method may make it possible to achieve an effective vaccine in the future.

scholarly journals Characterization of glycoinositol phospholipids in the amastigote stage of the protozoan parasite Leishmania major

1993 ◽  
Vol 295 (2) ◽  
pp. 555-564 ◽  
Author(s):  
P Schneider ◽  
J P Rosat ◽  
A Ransijn ◽  
M A J Ferguson ◽  
M J McConville
Keyword(s):  
Leishmania Major ◽  
Developmental Stages ◽  
Protozoan Parasite ◽  
Base Hydrolysis ◽  
Mammalian Host ◽  
Amastigote Stage ◽  
Head Groups ◽  
Host Cell Membranes ◽  
Intracellular Amastigote

The major macromolecules on the surface of the parasitic protozoan Leishmania major appear to be down-regulated during transformation of the parasite from an insect-dwelling promastigote stage to an intracellular amastigote stage that invades mammalian macrophages. In contrast, the major parasite glycolipids, the glycoinositol phospholipids (GIPLs), are shown here to be expressed at near-constant levels in both developmental stages. The structures of the GIPLs from tissue-derived amastigotes have been determined by h.p.l.c. analysis of the deaminated and reduced glycan head groups, and by chemical and enzymic sequencing. The deduced structures appear to form a complete biosynthetic series, ranging from Man alpha 1-4GlcN-phosphatidylinositol (PI) to Gal alpha 1-3Galf beta 1-3Man alpha 1-3Man alpha 1-4GlcN-PI (GIPL-2). A small proportion of GIPL-2 was further extended by addition of a Gal residue in either alpha 1-6 or beta 1-3 linkage. From g.c.-m.s. analysis and mild base treatment, all the GIPLs were shown to contain either alkylacylglycerol or lyso-alkylglycerol lipid moieties, where the alkyl chains were predominantly C18:0, with lower levels of C20:0, C22:0 and C24:0. L. major amastigotes also contained at least two PI-specific phospholipase C-resistant glycolipids which are absent from promastigotes. These neutral glycolipids were resistant to both mild acid and mild base hydrolysis, contained terminal beta-Gal residues and were not lost during extensive purification of amastigotes from host cell membranes. It is likely that these glycolipids are glycosphingolipids acquired from the mammalian host. The GIPL profile of L. major amastigotes is compared with the profiles found in L. major promastigotes and L. donovani amastigotes.

Leishmanicidal Activity of Plant Extracts from Sefrou, a Moroccan Focus of Leishmaniasis, against Various Leishmania Parasites in the Promastigote Stage

2018 ◽  
Vol 17 (2) ◽  
pp. 83-89 ◽  
Author(s):  
I. Zeouk ◽  
A. Et-Touys ◽  
M. Balouiri ◽  
H. Fellah ◽  
A. El Ouali Lalami ◽  
...  
Keyword(s):  
Cutaneous Leishmaniasis ◽  
Plant Extracts ◽  
Leishmania Infantum ◽  
Leishmania Major ◽  
Local Population ◽  
Public Health Problem ◽  
World Health ◽  
Total Phenolic ◽  
Leishmania Tropica ◽  
Cistus Salviifolius

According to the World Health Organization, leishmaniasis remains a major worldwide public health problem. The province of Sefrou located in the center of Morocco is a focus of cutaneous leishmaniasis. The present study aims at evaluating the antileishmanial potential of Berberis sp.,Crataegus oxyacantha, Cistus salviifolius, Ephedra altissima and Lavandula dentatafrequently used by the local population. Methanolic extracts were tested against the promastigote form ofLeishmania tropica, Leishmania majorandLeishmania infantumusing tetrazolium-based colorimetric (MTT) assay. The total phenol and flavonoids content of all extracts were determined using the Folin–Ciocalteu reagent, aluminum chloride, and potassium acetate solutions respectively. The plant extracts exhibited antileishmanial activity with variability depending on the tested strain and the plant species compared to Glucantime® used as control (IC50 (the half maximal inhibitory concentration) > 1,000 μg/mL). The best inhibition was observed with Berberis sp., againstLeishmania major(IC50 = 394.40 ± 3.02 μg/ml), andEphedra altissima(reported for the first time) againstLeishmania infantum(IC50 = 490.84 ± 3.15 μg/mL).Leishmania tropicahas shown the same sensitivity behavior toward the five extracts (in average IC50 = 540 ± 11.20 μg/mL). The total phenolic content was higher forCrataegus oxyacanthaandCistus salviifolius(140.67 ± 3.17 μg eq Gallic Acid (GA)/ mg of Extract (E) and 133.83 ± 9.03 μg eq GA/mg of E respectively), while flavonoid was higher forCistus salviifoliusandLavandula dentata(57.92 ± 2.46 μg eq Quercetin (Que)/ mg of Extract (E) and 41.53 ± 1.74 μg eq Que/mg of E). All the tested extracts present some promising aspects that may cure cutaneous leishmaniasis in the center of Morocco; further bioguided assays are needed to isolate the fractions and the bioactive molecule.

scholarly journals Metabolic reprogramming during the Trypanosoma brucei life cycle

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 683 ◽  
Author(s):  
Terry K. Smith ◽  
Frédéric Bringaud ◽  
Derek P. Nolan ◽  
Luisa M. Figueiredo
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Model Organism ◽  
Protozoan Parasite ◽  
Tsetse Fly ◽  
Metabolic Reprogramming ◽  
Mammalian Host ◽  
Insect Vector ◽  
Environmental Signals ◽  
Cellular Metabolic Activity

Cellular metabolic activity is a highly complex, dynamic, regulated process that is influenced by numerous factors, including extracellular environmental signals, nutrient availability and the physiological and developmental status of the cell. The causative agent of sleeping sickness, Trypanosoma brucei, is an exclusively extracellular protozoan parasite that encounters very different extracellular environments during its life cycle within the mammalian host and tsetse fly insect vector. In order to meet these challenges, there are significant alterations in the major energetic and metabolic pathways of these highly adaptable parasites. This review highlights some of these metabolic changes in this early divergent eukaryotic model organism.

scholarly journals Comparisons of Mutants Lacking the Golgi UDP-Galactose or GDP-Mannose Transporters Establish that Phosphoglycans Are Important for Promastigote but Not Amastigote Virulence in Leishmania major

2007 ◽  
Vol 75 (9) ◽  
pp. 4629-4637 ◽  
Author(s):  
Althea A. Capul ◽  
Suzanne Hickerson ◽  
Tamara Barron ◽  
Salvatore J. Turco ◽  
Stephen M. Beverley
Keyword(s):  
Golgi Apparatus ◽  
Protective Immunity ◽  
Leishmania Major ◽  
Protozoan Parasite ◽  
Macrophage Infection ◽  
Nucleotide Sugar ◽  
Infectious Cycle ◽  
Null Mutants

ABSTRACT Abundant surface Leishmania phosphoglycans (PGs) containing [Gal(β1,4)Man(α1-PO4)]-derived repeating units are important at several points in the infectious cycle of this protozoan parasite. PG synthesis requires transport of activated nucleotide-sugar precursors from the cytoplasm to the Golgi apparatus. Correspondingly, null mutants of the L. major GDP-mannose transporter LPG2 lack PGs and are severely compromised in macrophage survival and induction of acute pathology in susceptible mice, yet they are able to persist indefinitely and induce protective immunity. However, lpg2 − L. mexicana amastigotes similarly lacking PGs but otherwise normal in known glycoconjugates remain able to induce acute pathology. To explore this further, we tested the infectivity of a new PG-null L. major mutant, which is inactivated in the two UDP-galactose transporter genes LPG5A and LPG5B. Surprisingly this mutant did not recapitulate the phenotype of L. major lpg2 −, instead resembling the L. major lipophosphoglycan-deficient lpg1 − mutant. Metacyclic lpg5A −/lpg5B − promastigotes showed strong defects in the initial steps of macrophage infection and survival. However, after a modest delay, the lpg5A − /lpg5B − mutant induced lesion pathology in infected mice, which thereafter progressed normally. Amastigotes recovered from these lesions were fully infective in mice and in macrophages despite the continued absence of PGs. This suggests that another LPG2-dependent metabolite is responsible for the L. major amastigote virulence defect, although further studies ruled out cytoplasmic mannans. These data thus resolve the distinct phenotypes seen among lpg2 − Leishmania species by emphasizing the role of glycoconjugates other than PGs in amastigote virulence, while providing further support for the role of PGs in metacyclic promastigote virulence.

Temperature, Mitochondria, and Reye's Syndrome

PEDIATRICS ◽  
1983 ◽  
Vol 71 (6) ◽  
pp. 985-985
Author(s):  
RIF S. EL-MALLAKH
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Protozoan Parasite ◽  
Mitochondrial Enzyme ◽  
Reye's Syndrome ◽  
Electron Microscopic ◽  
Microscopic Studies ◽  
Mitochondrial Defect ◽  
Intracellular Protozoan Parasite

To the Editor.— Mitochondrial failure, manifest by changes in mitochondrial enzyme activity1-3 and morphology,4-5 is central to Reye's syndrome (RS).6 Although it has been variously hypothesized that the mitochondrial changes are secondary to an exogenous toxin,7-12 or an intrinsic mitochondrial defect,6 the actual cause remains obscure. Electron microscopic studies have shown sweelling and loss of cristate in mitochondria of patients with RS. It is interesting that very similar changes occur in Trypanosoma cruzi.13-16 T cruzi is an extracellular/intracellular protozoan parasite which causes Chagas' disease.17

The structural mechanics of cell division in Trypanosoma brucei

2008 ◽  
Vol 36 (3) ◽  
pp. 421-424 ◽  
Author(s):  
Sue Vaughan ◽  
Keith Gull
Keyword(s):  
Cell Division ◽  
Trypanosoma Brucei ◽  
Mammalian Cells ◽  
Reverse Genetics ◽  
Protozoan Parasite ◽  
Cell Types ◽  
Single Copy ◽  
Structural Mechanics ◽  
Sub Saharan Africa ◽  
Mammalian Host

Undoubtedly, there are fundamental processes driving the structural mechanics of cell division in eukaryotic organisms that have been conserved throughout evolution and are being revealed by studies on organisms such as yeast and mammalian cells. Precision of structural mechanics of cytokinesis is however probably no better illustrated than in the protozoa. A dramatic example of this is the protozoan parasite Trypanosoma brucei, a unicellular flagellated parasite that causes a devastating disease (African sleeping sickness) across Sub-Saharan Africa in both man and animals. As trypanosomes migrate between and within a mammalian host and the tsetse vector, there are periods of cell proliferation and cell differentiation involving at least five morphologically distinct cell types. Much of the existing cytoskeleton remains intact during these processes, necessitating a very precise temporal and spatial duplication and segregation of the many single-copy organelles. This structural precision is aiding progress in understanding these processes as we apply the excellent reverse genetics and post-genomic technologies available in this system. Here we outline our current understanding of some of the structural aspects of cell division in this fascinating organism.

scholarly journals Generation of a CRISPR/Cas9-Based Vector Specific for Gene Manipulation in Leishmania major

2019 ◽  
Author(s):  
Ghodratollah SALEHI SANGANI ◽  
Vahid JAJARMI ◽  
Ali KHAMESIPOUR ◽  
Mahmoud MAHMOUDI ◽  
Abdolmajid FATA ◽  
...  
Keyword(s):  
Resistance Gene ◽  
Leishmania Major ◽  
Gene Knockout ◽  
Ease Of Use ◽  
Selection Marker ◽  
Gene Manipulation ◽  
Resistance Proteins ◽  
U6 Promoter ◽  
Overlap Extension ◽  
Leishmania Parasites

Background: Gene manipulation strategies including gene knockout and editing are becoming more sophisticated in terms of mechanism of action, efficacy and ease of use. In classical molecular methods of gene knockout, homologous arms are designed for induction of crossing over event in double strand DNA. Recently, CRISPR/Cas9 system has been emerged as a precise and powerful tool for gene targeting. In this effort, we aimed to generate a CRISPR/Cas9-based vector specific for targeting genes in Leishmania parasites. Methods: U6 and DHFR promoters and neomycin-resistance gene were amplified from genome of L. major (MHRO/IR/75/ER) and pEGFP-N1, respectively. U6 promoter was cloned in pX330 vector which is named as pX330-U6. DHFR promoter and neo resistance gene sequence fragments were fused using a combination of SOE (Splicing by overlap extension)-PCR and T/A cloning techniques. To generate pX-leish, fused fragments su-bcloned into the pX330-U6. Two sgRNAs were designed to target the gp63 gene and cloned in pX-leish. Results: The pX-leish vector was designed for simultaneous expression of cas9 and G418 resistance proteins along with a self-cleaving 2A peptide for efficient separation of the two proteins. In this study pX-leish was designed with 3 features: 1) Compatible promoters with Leishmania parasites. 2) Insertion of antibiotic selection marker 3) Designing an all-in-one vector containing all components required for CRISPR/Cas9 system. Conclusion: This modified system would be valuable in genome manipulation studies in Leishmania for vaccine research in future.

Leishmania type II dehydrogenase is essential for parasite viability irrespective of the presence of an active complex I

2021 ◽  
Vol 118 (42) ◽  
pp. e2103803118
Author(s):  
Margarida Duarte ◽  
Cleide Ferreira ◽  
Gurleen Kaur Khandpur ◽  
Tamara Flohr ◽  
Jannik Zimmermann ◽  
...  
Keyword(s):  
Complex I ◽  
Nadh Dehydrogenase ◽  
Leishmania Major ◽  
Proton Translocation ◽  
Protozoan Parasite ◽  
Type I ◽  
Type Ii ◽  
Active Complex ◽  
Mitochondrial Inner Membrane ◽  
Candidate Target

Type II NADH dehydrogenases (NDH2) are monotopic enzymes present in the external or internal face of the mitochondrial inner membrane that contribute to NADH/NAD+ balance by conveying electrons from NADH to ubiquinone without coupled proton translocation. Herein, we characterize the product of a gene present in all species of the human protozoan parasite Leishmania as a bona fide, matrix-oriented, type II NADH dehydrogenase. Within mitochondria, this respiratory activity concurs with that of type I NADH dehydrogenase (complex I) in some Leishmania species but not others. To query the significance of NDH2 in parasite physiology, we attempted its genetic disruption in two parasite species, exhibiting a silent (Leishmania infantum, Li) and a fully operational (Leishmania major, Lm) complex I. Strikingly, this analysis revealed that NDH2 abrogation is not tolerated by Leishmania, not even by complex I–expressing Lm species. Conversely, complex I is dispensable in both species, provided that NDH2 is sufficiently expressed. That a type II dehydrogenase is essential even in the presence of an active complex I places Leishmania NADH metabolism into an entirely unique perspective and suggests unexplored functions for NDH2 that span beyond its complex I–overlapping activities. Notably, by showing that the essential character of NDH2 extends to the disease-causing stage of Leishmania, we genetically validate NDH2—an enzyme without a counterpart in mammals—as a candidate target for leishmanicidal drugs.

Infection with the intracellular protozoan parasite Theileria parva induces constitutively high levels of NF-kappa B in bovine T lymphocytes

1989 ◽  
Vol 9 (11) ◽  
pp. 4677-4686
Author(s):  
V Ivanov ◽  
B Stein ◽  
I Baumann ◽  
D A Dobbelaere ◽  
P Herrlich ◽  
...  
Keyword(s):  
T Cells ◽  
Phorbol Esters ◽  
Protozoan Parasite ◽  
Lymphoproliferative Disease ◽  
Host Cells ◽  
Specific Gene ◽  
Theileria Parva ◽  
Nf Kappa B ◽  
Intracellular Protozoan Parasite

The intracellular protozoan parasite Theileria parva causes a lymphoproliferative disease of T cells in cattle and uncontrolled lymphocyte proliferation in culture. We have identified and characterized in infected cells the transcriptional activator, NF-kappa B, whose recognition motifs have been identified in several gene enhancers important for lymphocyte-specific gene expression. NF-kappa B is normally constitutively activated in nuclear extracts derived from B cells and can be induced in T cells and nonlymphoid cells by phorbol esters. Theileria-infected lymphocytes contained constitutively high levels of activated NF-kappa B in nuclear fractions and inactive NF-kappa B in cytoplasmic fractions. The inactive cytoplasmic precursor could be activated by treatment of extracts with deoxycholate, which was shown previously to dissociate NF-kappa B from an inhibitor, I kappa B. Treatment of lymphocyte extracts with 3 mM GTP stimulated NF-kappa B binding to its recognition motif in vitro, thereby distinguishing it from a related nuclear factor, H2-TF1. Selective killing of the parasite, which left the host cells intact, resulted in a rapid loss of NF-kappa B from the nuclear fractions and a slower loss from the cytoplasmic fractions. In parasitized cells, NF-kappa B could not be further stimulated by treatment with 12-O-tetradecanoylphorbol-13-acetate whereas in cells treated to remove the parasite, this compound stimulated elevated levels of NF-kappa B. We propose that high levels of activated NF-kappa B are maintained by the presence of the parasite in infected T cells. Similarly, we propose that the high levels of inactive cytoplasmic precursor are a result of increased synthesis due to the presence of the parasite.

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