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International Journal of Molecular... Mar 2024Twenty 2-(4-alkyloxyphenyl)-imidazolines and 2-(4-alkyloxyphenyl)-imidazoles were synthesized, with the former being synthesized in two steps by using MW and...
Twenty 2-(4-alkyloxyphenyl)-imidazolines and 2-(4-alkyloxyphenyl)-imidazoles were synthesized, with the former being synthesized in two steps by using MW and ultrasonication energy, resulting in good to excellent yields. Imidazoles were obtained in moderate yields by oxidizing imidazolines with MnO and MW energy. In response to the urgent need to treat neglected tropical diseases, a set of 2-(4-alkyloxyphenyl)- imidazolines and imidazoles was tested in vitro on and . The leishmanicidal activity of ten compounds was evaluated, showing an IC < 10 µg/mL. Among these compounds, - were the most active, with IC values < 1 µg/mL (similar to the reference drugs). In the evaluation on epimastigotes of , only and reached an IC < 1 µg/mL, showing better inhibition than both reference drugs. However, compounds , , and also demonstrated attractive trypanocidal activities, with IC values < 10 µg/mL, similar to the values for benznidazole and nifurtimox.
Topics: Humans; Imidazoles; Imidazolines; Trypanosoma cruzi; Leishmania mexicana; Manganese Compounds; Oxides; Antiprotozoal Agents; Chagas Disease
PubMed: 38612484
DOI: 10.3390/ijms25073673 -
Molecular and Biochemical Parasitology Jun 2024In eukaryotic cells, molecular fate and cellular responses are shaped by multicomponent enzyme systems which reversibly attach ubiquitin and ubiquitin-like modifiers to...
In eukaryotic cells, molecular fate and cellular responses are shaped by multicomponent enzyme systems which reversibly attach ubiquitin and ubiquitin-like modifiers to target proteins. The extent of the ubiquitin proteasome system in Leishmania mexicana and its importance for parasite survival has recently been established through deletion mutagenesis and life-cycle phenotyping studies. The ubiquitin conjugating E2 enzyme UBC2, and the E2 enzyme variant UEV1, with which it forms a stable complex in vitro, were shown to be essential for the differentiation of promastigote parasites to the infectious amastigote form. To investigate further, we used immunoprecipitation of Myc-UBC2 or Myc-UEV1 to identify interacting proteins in L. mexicana promastigotes. The interactome of UBC2 comprises multiple ubiquitin-proteasome components including UEV1 and four RING E3 ligases, as well as potential substrates predicted to have roles in carbohydrate metabolism and intracellular trafficking. The smaller UEV1 interactome comprises six proteins, including UBC2 and shared components of the UBC2 interactome consistent with the presence of intracellular UBC2-UEV1 complexes. Recombinant RING1, RING2 and RING4 E3 ligases were shown to support ubiquitin transfer reactions involving the E1, UBA1a, and UBC2 to available substrate proteins or to unanchored ubiquitin chains. These studies define additional components of a UBC2-dependent ubiquitination pathway shown previously to be essential for promastigote to amastigote differentiation.
Topics: Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligases; Protozoan Proteins; Leishmania mexicana; Protein Binding; Protein Interaction Mapping; Immunoprecipitation
PubMed: 38556171
DOI: 10.1016/j.molbiopara.2024.111619 -
Vaccines Mar 2024Immunization with various species lacking induces robust immunity against a homologous and heterologous virulent challenge, making mutants a putative candidate for a...
Immunization with various species lacking induces robust immunity against a homologous and heterologous virulent challenge, making mutants a putative candidate for a leishmaniasis vaccine. Centrin is a calcium-binding cytoskeletal protein involved in centrosome duplication in higher eukaryotes and spp. lacking centrin are unable to replicate and are non-pathogenic. We developed a -deficient () cell line and confirmed its impaired survival following phagocytosis by macrophages. Upon experimental inoculation into BALB/c mice, failed to induce lesions and parasites were rapidly eliminated. The immune response following inoculation with was characterized by a mixed IFN-γ, IL-4, and IL-10 response and did not confer protection against infection, distinct from , , and centrin-deficient mutants. A prime-boost strategy also did not lead to a protective immune response against homologous challenge. On the contrary, immunization with -deficient () cross-protected against challenge, illustrating the ability of to induce the Th1-dominant protective immunity needed for leishmaniasis control. In conclusion, while deficiency in causes attenuation of virulence, and disrupts the ability to cause disease, it fails to stimulate a protective immune response.
PubMed: 38543944
DOI: 10.3390/vaccines12030310 -
International Journal For Parasitology.... Apr 2024Leishmaniasis and Chagas disease are parasitic infections that affect millions of people worldwide, producing thousands of deaths per year. The current treatments...
Leishmaniasis and Chagas disease are parasitic infections that affect millions of people worldwide, producing thousands of deaths per year. The current treatments against these pathologies are not totally effective and produce some side effects in the patients. Acrylonitrile derivatives are a group of compounds that have shown activity against these two diseases. In this work, four novels synthetic acrylonitriles were evaluated against the intracellular form and extracellular forms of L. amazonensis and T. cruzi. The compounds 2 and 3 demonstrate to have good selectivity indexes against both parasites, specifically the compound 3 against the amastigote form (SI = 6 against L. amazonensis and SI = 7.4 against T. cruzi). In addition, the parasites treated with these two compounds demonstrate to produce a programmed cell death, since they were positive for the events studied related to this type of death, including chromatin condensation, accumulation of reactive oxygen species and alteration of the mitochondrial membrane potential. In conclusion, this work confirms that acrylonitriles is a source of possible new compounds against kinetoplastids, however, more studies are needed to corroborate this activity.
Topics: Humans; Trypanosoma cruzi; Antiprotozoal Agents; Acrylonitrile; Leishmania mexicana; Chagas Disease; Cell Death
PubMed: 38484645
DOI: 10.1016/j.ijpddr.2024.100531 -
Genome Biology and Evolution Mar 2024Isocitrate dehydrogenase is an enzyme converting isocitrate to α-ketoglutarate in the canonical tricarboxylic acid (TCA) cycle. There are three different types of...
Isocitrate dehydrogenase is an enzyme converting isocitrate to α-ketoglutarate in the canonical tricarboxylic acid (TCA) cycle. There are three different types of isocitrate dehydrogenase documented in eukaryotes. Our study points out the complex evolutionary history of isocitrate dehydrogenases across kinetoplastids, where the common ancestor of Trypanosomatidae and Bodonidae was equipped with two isoforms of the isocitrate dehydrogenase enzyme: the NADP+-dependent isocitrate dehydrogenase 1 with possibly dual localization in the cytosol and mitochondrion and NADP+-dependent mitochondrial isocitrate dehydrogenase 2. In the extant trypanosomatids, isocitrate dehydrogenase 1 is present only in a few species suggesting that it was lost upon separation of Trypanosoma spp. and replaced by the mainly NADP+-dependent cytosolic isocitrate dehydrogenase 3 of bacterial origin in all the derived lineages. In this study, we experimentally demonstrate that the omnipresent isocitrate dehydrogenase 2 has a dual localization in both mitochondrion and cytosol in at least four species that possess only this isoform. The apparent lack of the NAD+-dependent isocitrate dehydrogenase activity in trypanosomatid mitochondrion provides further support to the existence of the noncanonical TCA cycle across trypanosomatids and the bidirectional activity of isocitrate dehydrogenase 3 when operating with NADP+ cofactor instead of NAD+. This observation can be extended to all 17 species analyzed in this study, except for Leishmania mexicana, which showed only low isocitrate dehydrogenase activity in the cytosol. The variability in isocitrate oxidation capacity among species may reflect the distinct metabolic strategies and needs for reduced cofactors in particular environments.
Topics: Isocitrate Dehydrogenase; Isocitrates; NADP; NAD; Protein Isoforms
PubMed: 38447055
DOI: 10.1093/gbe/evae042 -
JACS Au Feb 2024Visceral leishmaniasis and Chagas disease are neglected tropical diseases (NTDs) that severely impact the developing world. With current therapies suffering from poor...
Visceral leishmaniasis and Chagas disease are neglected tropical diseases (NTDs) that severely impact the developing world. With current therapies suffering from poor efficacy and safety profiles as well as emerging resistance, new drug leads are direly needed. In this work, 26 alkaloids (9 natural and 17 synthetic) belonging to the benzyltetrahydroisoquinoline (BI) family were evaluated against both the pro/trypomastigote and amastigote forms of the parasites and , the causative agents of these diseases. These alkaloids were synthesized via an efficient and modular enantioselective approach based on Bischler-Napieralski cyclization/Noyori asymmetric transfer hydrogenation to build the tetrahydroisoquinoline core. The bis-benzyltetrahydroisoquinoline (BBI) alkaloids were prepared using an Ullmann coupling of two BI units to form the biaryl ether linkage, which enabled a comprehensive survey of the influence of BI stereochemistry on bioactivity. Preliminary studies into the mechanism of action against demonstrate that these compounds interfere with the cell cycle, potentially through inhibition of kinetoplast division, which may offer opportunities to identify a new target/mechanism of action. Three of the synthesized alkaloids showed promising druglike potential, meeting the Drugs for Neglected Disease (DND) criteria for a hit against Chagas disease.
PubMed: 38425909
DOI: 10.1021/jacsau.4c00007 -
MSphere Mar 2024Over a 6-month span, three patients under 5 years old with cutaneous leishmaniasis presented to the Pediatric Infectious Diseases Clinic at the University of Texas... (Review)
Review
UNLABELLED
Over a 6-month span, three patients under 5 years old with cutaneous leishmaniasis presented to the Pediatric Infectious Diseases Clinic at the University of Texas Southwestern Medical Center/Children's Health Dallas. None had traveled outside of northern Texas/southern Oklahoma; all had infections confirmed by PCR. We provide case descriptions and images to increase the awareness of this disease among United States (US) physicians and scientists. Two patients responded to fluconazole, but the youngest required topical paromomycin. Combining these cases with guidelines and our literature review, we suggest that (i) higher doses (10-12 mg/kg/day) of fluconazole should be considered in young children to maximize likelihood and rapidity of response and (ii) patients should transition to alternate agents if they do not respond to high-dose fluconazole within 6 weeks. Furthermore, and of particular interest to the broad microbiology community, we used samples from these cases as a proof of concept to propose a mechanism to strain-type US-endemic . For our analysis, we sequenced three housekeeping genes and the internal transcribed sequence 2 of the ribosomal RNA gene. We identified genetic changes that not only allow us to distinguish US-based strains from strains found in other areas of the Americas but also establish polymorphisms that differ between US isolates. These techniques will allow documentation of genetic changes in this parasite as its range expands. Hence, our cases of cutaneous leishmaniasis provide significant evolutionary, treatment, and public health implications as climate change increases exposure to formerly tropical diseases in previously non-endemic areas.
IMPORTANCE
Leishmaniasis is a parasitic disease that typically affects tropical regions worldwide. However, the vector that carries is spreading northward into the United States (US). Within a 6-month period, three young cutaneous leishmaniasis patients were seen at the Pediatric Infectious Diseases Clinic at the University of Texas Southwestern Medical Center/Children's Health Dallas. None had traveled outside of northern Texas and southern Oklahoma. We document their presentations, treatments, and outcomes and compare their management to clinical practice guidelines for leishmaniasis. We also analyzed the sequences of three critical genes in isolated from these patients. We found changes that not only distinguish US-based strains from strains found elsewhere but also differ between US isolates. Monitoring these sequences will allow tracking of genetic changes in parasites over time. Our findings have significant US public health implications as people are increasingly likely to be exposed to what were once tropical diseases.
Topics: Child, Preschool; Humans; Communicable Diseases; Fluconazole; Leishmania mexicana; Leishmaniasis, Cutaneous; Texas; United States
PubMed: 38421172
DOI: 10.1128/msphere.00814-23 -
PLoS Pathogens Feb 2024The unicellular parasite Leishmania has a precisely defined cell architecture that is inherited by each subsequent generation, requiring a highly coordinated pattern of...
The unicellular parasite Leishmania has a precisely defined cell architecture that is inherited by each subsequent generation, requiring a highly coordinated pattern of duplication and segregation of organelles and cytoskeletal structures. A framework of nuclear division and morphological changes is known from light microscopy, yet this has limited resolution and the intrinsic organisation of organelles within the cell body and their manner of duplication and inheritance is unknown. Using volume electron microscopy approaches, we have produced three-dimensional reconstructions of different promastigote cell cycle stages to give a spatial and quantitative overview of organelle positioning, division and inheritance. The first morphological indications seen in our dataset that a new cell cycle had begun were the assembly of a new flagellum, the duplication of the contractile vacuole and the increase in volume of the nucleus and kinetoplast. We showed that the progression of the cytokinesis furrow created a specific pattern of membrane indentations, while our analysis of sub-pellicular microtubule organisation indicated that there is likely a preferred site of new microtubule insertion. The daughter cells retained these indentations in their cell body for a period post-abscission. By comparing cultured and sand fly derived promastigotes, we found an increase in the number and overall volume of lipid droplets in the promastigotes from the sand fly, reflecting a change in their metabolism to ensure transmissibility to the mammalian host. Our insights into the cell cycle mechanics of Leishmania will support future molecular cell biology analyses of these parasites.
Topics: Animals; Leishmania mexicana; Cell Cycle; Cell Division; Leishmania; Psychodidae; Parasites; Mammals
PubMed: 38416776
DOI: 10.1371/journal.ppat.1012054 -
PloS One 2024Eosinophils are mainly associated with parasitic infections and allergic manifestations. They produce many biologically active substances that contribute to the...
Eosinophils of patients with localized and diffuse cutaneous leishmaniasis: Differential response to Leishmania mexicana, with insights into mechanisms of damage inflicted upon the parasites by eosinophils.
Eosinophils are mainly associated with parasitic infections and allergic manifestations. They produce many biologically active substances that contribute to the destruction of pathogens through the degranulation of microbicidal components and inflammatory tissue effects. In leishmaniasis, eosinophils have been found within inflammatory infiltrate with protective immunity against the parasite. We analyzed the responses of eosinophils from patients with localized (LCL) and diffuse (DCL) cutaneous leishmaniasis, as well as from healthy subjects, when exposed to Leishmania mexicana. All DCL patients exhibited blood eosinophilia, along with elevated eosinophil counts in non-ulcerated nodules. In contrast, only LCL patients with prolonged disease progression showed eosinophils in their blood and cutaneous ulcers. Eosinophils from DCL patients secreted significantly higher levels of IL-6, IL-8, and IL-13, compared to eosinophils from LCL patients. Additionally, DCL patients displayed higher serum levels of anti-Leishmania IgG antibodies. We also demonstrated that eosinophils from both LCL and DCL patients responded to L. mexicana promastigotes with a robust oxidative burst, which was equally intense in both patient groups and significantly higher than in healthy subjects. Coincubation of eosinophils (from donors with eosinophilia) with L. mexicana promastigotes in vitro revealed various mechanisms of parasite damage associated with different patterns of granule exocytosis: 1) localized degranulation on the parasite surface, 2) the release of cytoplasmic membrane-bound "degranulation sacs" containing granules, 3) release of eosinophil extracellular traps containing DNA and granules with major basic protein. In conclusion, eosinophils damage L. mexicana parasites through the release of granules via diverse mechanisms. However, despite DCL patients having abundant eosinophils in their blood and tissues, their apparent inability to provide protection may be linked to the release of cytokines and chemokines that promote a Th2 immune response and disease progression in these patients.
Topics: Animals; Humans; Leishmaniasis, Diffuse Cutaneous; Leishmania mexicana; Parasites; Eosinophils; Leishmaniasis, Cutaneous; Disease Progression; Eosinophilia
PubMed: 38359037
DOI: 10.1371/journal.pone.0296887 -
The Journal of Biological Chemistry Mar 2024Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Because of their complex life cycle involving both vertebrate...
Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Because of their complex life cycle involving both vertebrate and insect hosts, and >1 billion years of evolutionarily distance, the cell biology of trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is divergent when compared with other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique α-helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family and is similar to the structure of WASP homology-2 (WH2) domain, a small actin-binding motif found in many other cytoskeletal regulators. The WH2-like motif contributes to actin monomer binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Moreover, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, and the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers molecular principles by which profilin regulates actin dynamics in trypanosomatids.
Topics: Animals; Humans; Actin Cytoskeleton; Actins; Amino Acid Motifs; Binding Sites; Conserved Sequence; Crystallization; Crystallography, X-Ray; Leishmania major; Parasites; Profilins; Protein Binding; Protein Domains
PubMed: 38340794
DOI: 10.1016/j.jbc.2024.105740