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  1. Article ; Online: Deletion of the lipid droplet protein kinase gene affects lipid droplets biogenesis, parasite infectivity, and resistance to trivalent antimony in Leishmania infantum.

    Ribeiro, Juliana Martins / Silva, Paula Alves / Costa-Silva, Héllida Marina / Santi, Ana Maria Murta / Murta, Silvane Maria Fonseca

    PLoS neglected tropical diseases

    2024  Volume 18, Issue 1, Page(s) e0011880

    Abstract: The Lipid Droplet Protein Kinase (LDK) facilitates lipid droplet (LD) biogenesis, organelles involved in various metabolic and signaling functions in trypanosomatids. As LDK's function has not been previously explored in Leishmania spp., we utilized ... ...

    Abstract The Lipid Droplet Protein Kinase (LDK) facilitates lipid droplet (LD) biogenesis, organelles involved in various metabolic and signaling functions in trypanosomatids. As LDK's function has not been previously explored in Leishmania spp., we utilized CRISPR/Cas9 technology to generate LDK-knockout lines of Leishmania infantum to investigate its role in this parasite. Our findings demonstrate that LDK is not an essential gene for L. infantum, as its deletion did not impede parasite survival. Furthermore, removing LDK did not impact the growth of promastigote forms of L. infantum lacking LDK. However, a noticeable reduction in LDs occurred during the stationary phase of parasite growth following LDK deletion. In the presence of myriocin, a LD inducer, LDK-knockout parasites displayed reduced LD abundance during both logarithmic and stationary growth phases compared to control parasites. Moreover, an infection analysis involving THP-1 cells revealed that 72 h post-infection, LDK-knockout L. infantum lines exhibited fewer infected macrophages and intracellular amastigotes than control parasites. LDK-knockout L. infantum lines also displayed 1.7 to 1.8 -fold greater resistance to trivalent antimony than control parasites. There were no observed alterations in susceptibility to amphotericin B, miltefosine, or menadione in LDK-knockout L. infantum lines. Our results suggest that LDK plays a crucial role in the biogenesis and/or maintenance of LDs in L. infantum, as well as in parasite infectivity and resistance to trivalent antimony.
    MeSH term(s) Animals ; Leishmania infantum/physiology ; Parasites ; Antimony/pharmacology ; Lipid Droplets ; Parasitic Diseases ; Protein Kinases
    Chemical Substances Antimony (9IT35J3UV3) ; Protein Kinases (EC 2.7.-)
    Language English
    Publishing date 2024-01-18
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2429704-5
    ISSN 1935-2735 ; 1935-2735
    ISSN (online) 1935-2735
    ISSN 1935-2735
    DOI 10.1371/journal.pntd.0011880
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Downregulation of FeSOD-A expression in Leishmania infantum alters trivalent antimony and miltefosine susceptibility.

    Santi, Ana Maria Murta / Silva, Paula Alves / Santos, Isabella Fernandes Martins / Murta, Silvane Maria Fonseca

    Parasites & vectors

    2021  Volume 14, Issue 1, Page(s) 366

    Abstract: Background: Superoxide dismutase (SOD), a central component of the antioxidant defence system of most organisms, removes excess superoxide anions by converting them to oxygen and hydrogen peroxide. As iron (Fe) SOD is absent in the human host, this ... ...

    Abstract Background: Superoxide dismutase (SOD), a central component of the antioxidant defence system of most organisms, removes excess superoxide anions by converting them to oxygen and hydrogen peroxide. As iron (Fe) SOD is absent in the human host, this enzyme is a promising molecular target for drug development against trypanosomatids.
    Results: We obtained Leishmania infantum mutant clones with lower FeSOD-A expression and investigated their phenotypes. Our attempts to delete this enzyme-coding gene using three different methodologies (conventional allelic replacement or two different CRISPR/methods) failed, as FeSOD-A gene copies were probably retained by aneuploidy or gene amplification. Promastigote forms of WT and mutant parasites were used in quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and western blot analyses, and these parasite forms were also used to assess drug susceptibility. RT-qPCR and western blot analyses revealed that FeSOD-A transcript and protein levels were lower in FeSOD-A
    Conclusions: The unsuccessful attempts to delete FeSOD-A suggest that this gene is essential in L. infantum. This enzyme plays an important role in the defence against oxidative stress and infectivity in THP-1 macrophages. FeSOD-A-deficient L. infantum parasites deregulate their metabolic pathways related to antimony and miltefosine resistance.
    MeSH term(s) Antimony/pharmacology ; Antiprotozoal Agents/pharmacology ; Down-Regulation ; Leishmania infantum/drug effects ; Leishmania infantum/enzymology ; Leishmania infantum/genetics ; Mutation ; Oxidative Stress/drug effects ; Phosphorylcholine/analogs & derivatives ; Phosphorylcholine/pharmacology ; Superoxide Dismutase/genetics
    Chemical Substances Antiprotozoal Agents ; Phosphorylcholine (107-73-3) ; miltefosine (53EY29W7EC) ; Antimony (9IT35J3UV3) ; Superoxide Dismutase (EC 1.15.1.1)
    Language English
    Publishing date 2021-07-15
    Publishing country England
    Document type Journal Article
    ZDB-ID 2409480-8
    ISSN 1756-3305 ; 1756-3305
    ISSN (online) 1756-3305
    ISSN 1756-3305
    DOI 10.1186/s13071-021-04838-8
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article: Downregulation of FeSOD-A expression in Leishmania infantum alters trivalent antimony and miltefosine susceptibility

    Santi, Ana Maria Murta / Silva, Paula Alves / Santos, Isabella Fernandes Martins / Murta, Silvane Maria Fonseca

    Parasites & vectors. 2021 Dec., v. 14, no. 1

    2021  

    Abstract: BACKGROUND: Superoxide dismutase (SOD), a central component of the antioxidant defence system of most organisms, removes excess superoxide anions by converting them to oxygen and hydrogen peroxide. As iron (Fe) SOD is absent in the human host, this ... ...

    Abstract BACKGROUND: Superoxide dismutase (SOD), a central component of the antioxidant defence system of most organisms, removes excess superoxide anions by converting them to oxygen and hydrogen peroxide. As iron (Fe) SOD is absent in the human host, this enzyme is a promising molecular target for drug development against trypanosomatids. RESULTS: We obtained Leishmania infantum mutant clones with lower FeSOD-A expression and investigated their phenotypes. Our attempts to delete this enzyme-coding gene using three different methodologies (conventional allelic replacement or two different CRISPR/methods) failed, as FeSOD-A gene copies were probably retained by aneuploidy or gene amplification. Promastigote forms of WT and mutant parasites were used in quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and western blot analyses, and these parasite forms were also used to assess drug susceptibility. RT-qPCR and western blot analyses revealed that FeSOD-A transcript and protein levels were lower in FeSOD-A⁻/⁻/⁺ L. infantum mutant clones than in the wild-type (WT) parasite. The decrease in FeSOD-A expression in L. infantum did not interfere with the parasite growth or susceptibility to amphotericin B. Surprisingly, FeSOD-A⁻/⁻/⁺ L. infantum mutant clones were 1.5- to 2.0-fold more resistant to trivalent antimony and 2.4- to 2.7-fold more resistant to miltefosine. To investigate whether the decrease in FeSOD-A expression was compensated by other enzymes, the transcript levels of five FeSODs and six enzymes from the antioxidant defence system were assessed by RT-qPCR. The transcript level of the enzyme ascorbate peroxidase increased in both the FeSOD-A⁻/⁻/⁺ mutants tested. The FeSOD-A⁻/⁻/⁺ mutant parasites were 1.4- to 1.75-fold less tolerant to oxidative stress generated by menadione. Infection analysis using THP-1 macrophages showed that 72 h post-infection, the number of infected macrophages and their intracellular multiplication rate were lower in the FeSOD-A⁻/⁻/⁺ mutant clones than in the WT parasite. CONCLUSIONS: The unsuccessful attempts to delete FeSOD-A suggest that this gene is essential in L. infantum. This enzyme plays an important role in the defence against oxidative stress and infectivity in THP-1 macrophages. FeSOD-A-deficient L. infantum parasites deregulate their metabolic pathways related to antimony and miltefosine resistance.
    Keywords Leishmania infantum ; Western blotting ; amphotericin B ; aneuploidy ; antimony ; antioxidant activity ; ascorbate peroxidase ; drug development ; gene amplification ; genes ; humans ; hydrogen peroxide ; macrophages ; menadione ; mutants ; oxidative stress ; oxygen ; parasites ; pathogenicity ; reverse transcriptase polymerase chain reaction ; superoxide dismutase
    Language English
    Dates of publication 2021-12
    Size p. 366.
    Publishing place BioMed Central
    Document type Article
    ZDB-ID 2409480-8
    ISSN 1756-3305
    ISSN 1756-3305
    DOI 10.1186/s13071-021-04838-8
    Database NAL-Catalogue (AGRICOLA)

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  4. Article ; Online: Disruption of multiple copies of the Prostaglandin F2alpha synthase gene affects oxidative stress response and infectivity in Trypanosoma cruzi.

    Santi, Ana Maria Murta / Ribeiro, Juliana Martins / Reis-Cunha, João Luís / Burle-Caldas, Gabriela de Assis / Santos, Isabella Fernandes Martins / Silva, Paula Alves / Resende, Daniela de Melo / Bartholomeu, Daniella Castanheira / Teixeira, Santuza Maria Ribeiro / Murta, Silvane Maria Fonseca

    PLoS neglected tropical diseases

    2022  Volume 16, Issue 10, Page(s) e0010845

    Abstract: Chagas disease, caused by the protozoan Trypanosoma cruzi, is a serious chronic parasitic disease, currently treated with Nifurtimox (NFX) and Benznidazole (BZ). In addition to high toxicity, these drugs have low healing efficacy, especially in the ... ...

    Abstract Chagas disease, caused by the protozoan Trypanosoma cruzi, is a serious chronic parasitic disease, currently treated with Nifurtimox (NFX) and Benznidazole (BZ). In addition to high toxicity, these drugs have low healing efficacy, especially in the chronic phase of the disease. The existence of drug-resistant T. cruzi strains and the occurrence of cross-resistance between BZ and NFX have also been described. In this context, it is urgent to study the metabolism of these drugs in T. cruzi, to better understand the mechanisms of resistance. Prostaglandin F2α synthase (PGFS) is an enzyme that has been correlated with parasite resistance to BZ, but the mechanism by which resistance occurs is still unclear. Our results show that the genome of the CL Brener clone of T. cruzi, contains five PGFS sequences and three potential pseudogenes. Using CRISPR/Cas9 we generated knockout cell lines in which all PGFS sequences were disrupted, as shown by PCR and western blotting analyses. The PGFS deletion did not alter the growth of the parasites or their susceptibility to BZ and NFX when compared to wild-type (WT) parasites. Interestingly, NTR-1 transcripts were shown to be upregulated in ΔPGFS mutants. Furthermore, the ΔPGFS parasites were 1.6 to 1.7-fold less tolerant to oxidative stress generated by menadione, presented lower levels of lipid bodies than the control parasites during the stationary phase, and were less infective than control parasites.
    MeSH term(s) Humans ; Trypanosoma cruzi ; Nifurtimox/therapeutic use ; Dinoprost/therapeutic use ; Trypanocidal Agents/therapeutic use ; Vitamin K 3/therapeutic use ; Chagas Disease/parasitology ; Oxidative Stress
    Chemical Substances Nifurtimox (M84I3K7C2O) ; Dinoprost (B7IN85G1HY) ; Trypanocidal Agents ; Vitamin K 3 (723JX6CXY5) ; benzonidazole (YC42NRJ1ZD)
    Language English
    Publishing date 2022-10-19
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2429704-5
    ISSN 1935-2735 ; 1935-2735
    ISSN (online) 1935-2735
    ISSN 1935-2735
    DOI 10.1371/journal.pntd.0010845
    Database MEDical Literature Analysis and Retrieval System OnLINE

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