Axenic amastigotes of Leishmania species as a suitable model for in vitro studies

Dias-Lopes, Geovane; Zabala-Peñafiel, Anabel; Albuquerque-Melo, Barbara Cristina de; Souza-Silva, Franklin; Canto, Laura Menaguali do; Cysne-Finkelstein, Léa; Alves, Carlos Roberto

doi:10.1016/j.actatropica.2021.105956

Cited by 10 publications

(9 citation statements)

References 120 publications

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“…An important area of Leishmania biology is the study of genes that impact survival of the amastigote stage in the vertebrate host. Many (not all) Leishmania species can differentiate to amastigote-like forms in culture (axenic amastigotes), when grown at conditions resembling those within the parasitophorous vacuole, eg elevated temperature and low pH [39,40]. Here we made use of a clonal derivative of Lbr M2903 (LbrM2903SA2) which had been adapted for growth as axenic amastigotes [23].…”

Section: Resultsmentioning

confidence: 99%

An RNA interference (RNAi) toolkit and its utility for functional genetic analysis ofLeishmania (Viannia)

Lye

Owens

Jang

et al. 2022

Preprint

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RNA interference (RNAi) is a powerful tool whose efficacy against a broad range of targets enables functional genetic tests individually or systematically. However, the RNAi pathway has been lost in evolution by a variety of eukaryotes including most Leishmania sp. RNAi was retained in species of the Leishmania subgenus Viannia, and here we describe the development, optimization, and application of RNAi tools to the study of L. (Viannia) braziliensis. We developed vectors facilitating generation of long-hairpin or stem-loop (StL) RNAi knockdown constructs, using Gateway site-specific recombinase technology. A survey of applications of RNAi in L. braziliensis included genes interspersed within multigene tandem arrays such as QDPR, a potential target or modulator of antifolate sensitivity. Other tests include genes involved in cell differentiation and amastigote proliferation (A600), and essential genes of the intraflagellar transport (IFT) pathway. We tested a range of stem lengths targeting the L. braziliensis hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and reporter firefly luciferase (LUC) genes and found that the efficacy of RNAi increased with stem length, and fell off greatly below about 128 nt. We used the StL length dependency to establish a useful hypomorphic approach not possible with other gene ablation strategies, with shorter IFT140 stems yielding viable cells with compromised flagellar morphology. We showed that co-selection for RNAi against adenine phosphoryl transferase (APRT1) using 4-aminopyrazolpyrimidine (APP) could increase the efficacy of RNAi against reporter constructs, a useful tool that may facilitate improvements in future work. Thus, for many genes, RNAi provides a useful tool for studying Leishmania gene function with some unique advantages.

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Section: Resultsmentioning

confidence: 99%

An RNA interference (RNAi) toolkit and its utility for functional genetic analysis ofLeishmania (Viannia)

Lye

Owens

Jang

et al. 2022

Preprint

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“…Almeida-Souza et al ( 2018), using the same methodology, observed that the IC 50 of Morinda citrifolia fruit juice for the axenic amastigote forms was lower than for the L. amazonensis promastigotes. The genomic microarray of promastigotes and axenic amastigotes shows variation in gene expression that influence stress response and metabolism (Saxena et al, 2007;Srividya et al, 2007;Dias-Lopes et al, 2021). Therefore, the difference obtained in the screening of compounds evaluated against Leishmania could be due to the difference between the life cycle stages of the parasite.…”

Section: Antileishmanial Activity and Cytotoxicitymentioning

confidence: 99%

Monomethylsulochrin isolated from biomass extract of Aspergillus sp. against Leishmania amazonensis: In vitro biological evaluation and molecular docking

Silva-Silva

Moreira

Watanabe

et al. 2022

Front. Cell. Infect. Microbiol.

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Leishmaniasis represents a serious world health problem, with 1 billion people being exposed to infection and a broad spectrum of clinical manifestations with a potentially fatal outcome. Based on the limitations observed in the treatment of leishmaniasis, such as high cost, significant adverse effects, and the potential for drug resistance, the aim of the present study was to evaluate the leishmanicidal activity of the compounds pseurotin A and monomethylsulochrin isolated from the biomass extract of Aspergillus sp. The chromatographic profiles of the extract were determined by high-performance liquid chromatography coupled with a diode-array UV-Vis detector (HPLC-DAD-UV), and the molecular identification of the pseurotin A and monomethylsulochrin were carried out by electrospray ionization mass spectrometry in tandem (LC-ESI-MS-MS) and nuclear magnetic resonance (NMR). Antileishmanial activity was assayed against promastigote and intracellular amastigote of Leishmania amazonensis. As a control, cytotoxicity assays were performed in non-infected BALB/c peritoneal macrophages. Ultrastructural alterations in parasites were evaluated by transmission electron microscopy. Changes in mitochondrial membrane potential were determined by flow cytometry. Only monomethylsulochrin inhibited the promastigote growth (IC50 18.04 ± 1.11 µM), with cytotoxicity to peritoneal macrophages (CC50 5.09 91.63 ± 1.28 µM). Activity against intracellular amastigote forms (IC50 5.09 ± 1.06 µM) revealed an increase in antileishmanial activity when compared with promastigotes. In addition to a statistically significant reduction in the evaluated infection parameters, monomethylsulochrin altered the ultrastructure of the promastigote forms with atypical vacuoles, electron-dense corpuscles in the cytoplasm, changes at the mitochondria outer membrane and abnormal disposition around the kinetoplast. It was showed that monomethylsulochrin leads to a decrease in the mitochondrial membrane potential (25.9%, p = 0.0286). Molecular modeling studies revealed that monomethylsulochrin can act as inhibitor of sterol 14-alpha-demethylase (CYP51), a therapeutic target for human trypanosomiasis and leishmaniasis. Assessed for its drug likeness, monomethylsulochrin follows the Lipinski Rule of five and Ghose, Veber, Egan, and Muegge criteria. Furthermore, monomethylsulochrin can be used as a reference in the development of novel and therapeutically useful antileishmanial agents.

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“…Various Leishmania forms are used experimentally in vitro to develop exploratory screening tests, which are promastigotes, intracellular amastigotes, and axenic amastigotes. These latter forms represent amastigotes that were adapted to grow and develop outside their host cells in a growth medium that mimics the intracellular conditions [25][26][27]. Therefore, it appears complicated to consider only one parasite form in the framework of an exploratory in vitro screening.…”

Section: Challenges Involved In Implementing Exploratory Screening To Identify In Vitro Hit Compounds Against Leishmaniamentioning

confidence: 99%

“…Another challenge in terms of harmonizing work carried out by different research teams is the wide variety of existing options for detection, acquisition, and data processing systems [42]. Thus, cell viability detection was extensively used in the exploratory screening of antileishmanial compounds, especially for primary screenings on extracellular forms [27,. Indeed, there are many colorimetric assays that are usable, simple, inexpensive, and suitable for large-scale screening [43,44].…”

Section: Challenges Involved In Implementing Exploratory Screening To Identify In Vitro Hit Compounds Against Leishmaniamentioning

confidence: 99%

“…This yellow salt is reduced to purple formazan crystals in living cells, allowing for easy determination of parasite viability. Other analogous tests are also described, such as the one using Alamar blue (resazurin) [27,[60][61][62][63][64][65][66][67], an oxidationreduction indicator that changes its color from blue to red in living cells. The use of an analog of MTT, the MTS (3-(4,5-dimethylthiazol-2-yl-5(3-carboxymethylphenyl)-2-(4sulfophenyl)-2H-tetrazolium) [68][69][70], is also described.…”

Section: Challenges Involved In Implementing Exploratory Screening To Identify In Vitro Hit Compounds Against Leishmaniamentioning

confidence: 99%

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Challenges and Tools for In Vitro Leishmania Exploratory Screening in the Drug Development Process: An Updated Review

Cohen

Azas

2021

Pathogens

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Leishmaniases are a group of vector-borne diseases caused by infection with the protozoan parasites Leishmania spp. Some of them, such as Mediterranean visceral leishmaniasis, are zoonotic diseases transmitted from vertebrate to vertebrate by a hematophagous insect, the sand fly. As there is an endemic in more than 90 countries worldwide, this complex and major health problem has different clinical forms depending on the parasite species involved, with the visceral form being the most worrying since it is fatal when left untreated. Nevertheless, currently available antileishmanial therapies are significantly limited (low efficacy, toxicity, adverse side effects, drug-resistance, length of treatment, and cost), so there is an urgent need to discover new compounds with antileishmanial activity, which are ideally inexpensive and orally administrable with few side effects and a novel mechanism of action. Therefore, various powerful approaches were recently applied in many interesting antileishmanial drug development programs. The objective of this review is to focus on the very first step in developing a potential drug and to identify the exploratory methods currently used to screen in vitro hit compounds and the challenges involved, particularly in terms of harmonizing the results of work carried out by different research teams. This review also aims to identify innovative screening tools and methods for more extensive use in the drug development process.

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Axenic amastigotes of Leishmania species as a suitable model for in vitro studies

Cited by 10 publications

References 120 publications

An RNA interference (RNAi) toolkit and its utility for functional genetic analysis ofLeishmania (Viannia)

An RNA interference (RNAi) toolkit and its utility for functional genetic analysis ofLeishmania (Viannia)

Monomethylsulochrin isolated from biomass extract of Aspergillus sp. against Leishmania amazonensis: In vitro biological evaluation and molecular docking

Challenges and Tools for In Vitro Leishmania Exploratory Screening in the Drug Development Process: An Updated Review

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