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

Genes ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 1159
Author(s):  
Vanessa Adaui ◽  
Constanze Kröber-Boncardo ◽  
Christine Brinker ◽  
Henner Zirpel ◽  
Julie Sellau ◽  
...  
Keyword(s):  
New World ◽  
Reverse Genetics ◽  
Leishmania Major ◽  
Genetic Manipulation ◽  
Protozoan Parasite ◽  
Single Copy ◽  
Leishmania Braziliensis ◽  
Old World ◽  
Tegumentary Leishmaniasis ◽  
Null Mutants

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. majorHSP23 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.

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.

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.

Histone deacetylases inhibitors as new potential drugs against Leishmania braziliensis, the main causative agent of new world tegumentary leishmaniasis

2020 ◽  
Vol 180 ◽  
pp. 114191
Author(s):  
Luciana Ângelo de Souza ◽  
Matheus Silva e Bastos ◽  
Joice de Melo Agripino ◽  
Thiago Souza Onofre ◽  
Lourdes Fanny Apaza Calla ◽  
...  
Keyword(s):  
Causative Agent ◽  
Histone Deacetylases ◽  
New World ◽  
Leishmania Braziliensis ◽  
Histone Deacetylases Inhibitors ◽  
Tegumentary Leishmaniasis

scholarly journals First molecular-based detection of mucocutaneous leishmaniasis caused by Leishmania major in Iran

2013 ◽  
Vol 7 (05) ◽  
pp. 413-416 ◽  
Author(s):  
Abdolvahab Alborzi ◽  
Gholam R Pouladfar ◽  
Abdolkarim Ghadimi Moghadam ◽  
Armin Attar ◽  
Nima Drakhshan ◽  
...  
Keyword(s):  
Buccal Mucosa ◽  
New World ◽  
Leishmania Major ◽  
Leishmania Braziliensis ◽  
Kinetoplast Dna ◽  
Lower Lip ◽  
Mucocutaneous Leishmaniasis ◽  
High Prevalence ◽  
Amphotericin B Deoxycholate ◽  
Mucosal Specimen

Mucocutaneous leishmaniasis, which mostly occurs in the New World, is mainly associated with Leishmania braziliensis and to a lesser degree L. panamensis and L. amazonensis infections. Primary mucosal leishmaniasis is very rare in Iran in spite of high prevalence of cutaneous and visceral leishmanisis. A nine-year-old boy had cutaneous leishmaniaisis for five years involving the left side of his face; he then developed swelling and ulceration of the lip and left side buccal mucosa five months before hospital admission. He had severe swelling of the lower lip and there was ulceration and bleeding of the buccal mucosa. Direct smear revealed leishman bodies and nested PCR confirmed the presence of kinetoplast DNA of L. major in the oral mucosal specimen. The patient received amphotericin B deoxycholate 1 mg/kg/day for one month. The lip and face inflammatory reaction disappeared to nearly normal after one month of therapy. The patient was discharged with ketoconazole (5mg/kg/day) for six weeks. To our knowledge, this is the first report of mucocutaneous leishmaniasis caused by L. major in Iran.

scholarly journals The long and winding road of reverse genetics in Trypanosoma cruzi

2021 ◽  
Vol 8 (9) ◽  
pp. 203-207
Author(s):  
Miguel A. Chiurillo ◽  
Noelia Lander
Keyword(s):  
Trypanosoma Cruzi ◽  
Trypanosoma Brucei ◽  
Reverse Genetics ◽  
Genetic Manipulation ◽  
Genetically Modify ◽  
Protozoan Parasite ◽  
Future Directions ◽  
Distinctive Features ◽  
Biologically Relevant ◽  
Leishmania Spp

Trypanosomes are early divergent protists with distinctive features among eukaryotic cells. Together with Trypanosoma brucei and Leishmania spp., Trypanosoma cruzi has been one of the most studied members of the group. This protozoan parasite is the causative agent of Chagas disease, a leading cause of heart disease in the Americas, for which there is no vaccine or satisfactory treatment available. Understanding T. cruzi biology is crucial to identify alternative targets for antiparasitic interventions. Genetic manipulation of T. cruzi has been historically challenging. However, the emergence of CRISPR/Cas9 technology has significantly improved the ability to generate genetically modified T. cruzi cell lines. Still, the system alone is not sufficient to answer all biologically relevant questions. In general, current genetic methods have limitations that should be overcome to advance in the study of this peculiar parasite. In this brief historic overview, we highlight the strengths and weaknesses of the molecular strategies that have been developed to genetically modify T. cruzi, emphasizing the future directions of the field.

scholarly journals Amerigo Vespucci's Contribution to the Modernization of Cartographic Representation

2020 ◽  
Author(s):  
Gyula Pápay
Keyword(s):  
New World ◽  
De Novo ◽  
Old World ◽  
University Library ◽  
The City

AbstractIn 2019, the Rostock University Library acquired the report by Amerigo Vespucci (1454–1512) on transatlantic discoveries, which was published in 1505 by the city secretary Hermann Barckhusen (c 1460–1528/29) in Rostock under the title “Epistola Albericij. De novo mundo” [1505] and, unlike other editions, was published with a map. The special feature of the map is that it is the oldest map with a globular projection. Vespucci reported in a letter dated July 18, 1500 to Lorenzo di Pierfrancesco de' Medici about his voyage 1499–1500, which is an important source for the fact that his longitude determinations contributed to the realization that the transatlantic discoveries were about a continent. The letter also contains evidence that Vespucci was the originator of the globular projection. This marked the beginning of a departure from ancient traditions regarding the projections for world maps. To enable the combined representation of the “old world” together with the “new world” in one map, Vespucci's projection was later modified into an oval map, which was used, for example, by Franzesco Rosselli, Sebastian Münster and Abraham Ortelius.

scholarly journals SINE Insertions in Cladistic Analyses and the Phylogenetic Affiliations of Tarsius bancanus to Other Primates

Genetics ◽  
2001 ◽  
Vol 157 (2) ◽  
pp. 777-784
Author(s):  
Jürgen Schmitz ◽  
Martina Ohme ◽  
Hans Zischler
Keyword(s):  
New World ◽  
World Monkey ◽  
Old World ◽  
Alu Elements ◽  
Alu Sequences ◽  
Short Interspersed Elements ◽  
Divergence Point ◽  
Tarsius Bancanus ◽  
Cladistic Analyses ◽  
The Common

Abstract Transpositions of Alu sequences, representing the most abundant primate short interspersed elements (SINE), were evaluated as molecular cladistic markers to analyze the phylogenetic affiliations among the primate infraorders. Altogether 118 human loci, containing intronic Alu elements, were PCR analyzed for the presence of Alu sequences at orthologous sites in each of two strepsirhine, New World and Old World monkey species, Tarsius bancanus, and a nonprimate outgroup. Fourteen size-polymorphic amplification patterns exhibited longer fragments for the anthropoids (New World and Old World monkeys) and T. bancanus whereas shorter fragments were detected for the strepsirhines and the outgroup. From these, subsequent sequence analyses revealed three Alu transpositions, which can be regarded as shared derived molecular characters linking tarsiers and anthropoid primates. Concerning the other loci, scenarios are represented in which different SINE transpositions occurred independently in the same intron on the lineages leading both to the common ancestor of anthropoids and to T. bancanus, albeit at different nucleotide positions. Our results demonstrate the efficiency and possible pitfalls of SINE transpositions used as molecular cladistic markers in tracing back a divergence point in primate evolution over 40 million years old. The three Alu insertions characterized underpin the monophyly of haplorhine primates (Anthropoidea and Tarsioidea) from a novel perspective.

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