Dataset Information

Evaluation of the Anti-Leishmanial Effect of Recombinant Clostridium ?-Toxin.

ABSTRACT: Background:Leishmaniasis is an infectious disease common in tropical and subtropical regions caused by the genus Leishmania, which is transmitted by the bite of female sandflies. In this study, we evaluate the anti-leishmanial effect of recombinant Clostridium ?-toxin protein alone and the combination with glucantime through in vitro and in vivo. Materials and Methods:Production, expression, and purification of recombinant ?-toxin were evaluated by SDS-PAGE and Western blotting techniques. The antileishmanial activities of the purified ?-toxin plus and without glucantime were examined in vitro and in vivo. Results:The results indicated successful expression of ?-toxin as a 48 kDa band on SDS-PAGE and Western blot methods. Also, evaluation of ?-toxin IC50 showed the strong fatal effect of it, and glucantime on medium proliferated Leishmania promastigotes at lower concentrations compared with glucantime or ?-toxin alone. Moreover, in vivo surveys showed that at the end of treatment courses, the mean of lesion size diminished in glucantime plus ?-toxin treated mice versus negative control groups (p < 0.001). Also, there was a significant difference in the parasite burden of the spleen and liver of the control versus the test groups (p < 0.001). Conclusion:The results showed recombinant ?-toxin has synergistic effects with glucantime in destroying Leishmania parasites.

SUBMITTER: Namdar F

PROVIDER: S-EPMC7369417 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Evaluation of the Anti-Leishmanial Effect of Recombinant <i>Clostridium</i> α-Toxin.

Namdar Fatemeh F Khanahmad Hossein H Ghayour Zahra Z Mirzaei Farzaneh F Namdar Azam A Aghaei Maryam M Izadi Shahrokh S Khamesipour Faham F Hejazi Seyed Hossein SH

Infection and drug resistance 20200715

<h4>Background</h4>Leishmaniasis is an infectious disease common in tropical and subtropical regions caused by the genus <i>Leishmania</i>, which is transmitted by the bite of female sandflies. In this study, we evaluate the anti-leishmanial effect of recombinant <i>Clostridium</i> α-toxin protein alone and the combination with glucantime through in vitro and in vivo.<h4>Materials and methods</h4>Production, expression, and purification of recombinant α-toxin were evaluated by SDS-PAGE and Weste ...[more]

PMID: 32765010

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Project description:BackgroundLeishmaniasis, one of the most neglected diseases, is a serious public health problem in many countries, including Brazil. Currently available treatments require long-term use and have serious side effects, necessitating the development of new therapeutic interventions. Because translocator protein (TSPO) levels are reduced in Leishmania amazonensis-infected cells and because this protein participates in apoptosis and immunomodulation, TSPO represents a potential target for Leishmania chemotherapy. The present study evaluated PK11195, a ligand of this protein, as an anti-leishmanial agent.ObjectiveTo evaluate the leishmanicidal activity of PK11195 against L. amazonensis in infected CBA mouse macrophages in vitro.MethodsThe viability of axenic L. amazonensis, Leishmania major, and Leishmania braziliensis promastigotes was assessed after 48 h treatment with PK11195 (0.2-400 µM). Additionally, intracellular parasite viability was evaluated to determine IC50 values and the number of viable parasites in infected macrophages treated with PK11195 (50-100 µM). Infected macrophages were then treated with PK11195 (25-100 µM) to determine the percentage of L. amazonensis-infected cells and the number of parasites per infected cell. Electron microscopy was used to investigate morphological changes caused by PK11195. The production of free oxygen radicals, nitric oxide, and pro-inflammatory cytokines was also evaluated in infected macrophages treated with PK11195 and primed or not primed with IFN-γ.FindingsMedian IC50 values for PK11195 were 14.2 µM for L. amazonensis, 8.2 µM for L. major, and 3.5 µM for L. braziliensis. The selective index value for L. amazonensis was 13.7, indicating the safety of PK11195 for future testing in mammals. Time- and dose-dependent reductions in the percentage of infected macrophages, the number of parasites per infected macrophage, and the number of viable intracellular parasites were observed. Electron microscopy revealed some morphological alterations suggestive of autophagy. Interestingly, MCP-1 and superoxide levels were reduced in L. amazonensis-infected macrophages treated with PK11195.Main conclusionsPK11195 causes the killing of amastigotes in vitro by mechanisms independent of inflammatory mediators and causes morphological alterations within Leishmania parasites, suggestive of autophagy, at doses that are non-toxic to macrophages. Thus, this molecule has demonstrated potential as an anti-leishmanial agent.

Project description:All subtypes of Clostridium perfringens (C. perfringens) produce the alpha toxin (CPA), which can cause enteritis or enterotoxemia in lambs, cattle, pigs, and horses, as well as traumatic clostridial myonecrosis in humans and animals. CPA acts on cell membranes, ultimately leading to endocytosis and cell death. Therefore, the neutralization of CPA is crucial for the prevention and treatment of diseases caused by C. perfringens. In this study, utilizing CPA as an antigen, a nanobody (CPA-VHH) with a half-life of 2.9 h, an affinity constant (KD) of 0.9 nmol/L, and good stability below 60 °C was prepared from a natural nanobody library from alpacas. The biological activity analysis of CPA-VHH revealed its ability to effectively neutralize the phospholipase and hemolytic activity of CPA at a 15-fold ratio. In Vero cells, 9.8 μg/mL CPA-VHH neutralized the cytotoxicity of CPA at two times the half-maximal inhibitory concentration (IC50). In a mouse model, 35.7 ng/g body weight (BW) of CPA-VHH neutralized 90% of the lethality caused by a 2× median lethal dose (LD50) of CPA. It was found that CPA-VHH protected 80% of mice within 30 min at 2 × LD50 CPA, but this dropped below 50% after 2 h and to 0% after 4 h. Rescue trials indicated that using CPA-VHH within 30 min post-infection with 2 × LD50 CPA achieved an 80% rescue rate, which decreased to 10% after 2 h. Furthermore, CPA-VHH effectively mitigated the reduction in the expression levels of zonula occludens-1 (ZO-1), Occludin, and Claudin-1, while also attenuating the upregulation of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), and interferon-γ (IFN-γ) induced by CPA infection. Overall, this study has identified a specific nanobody, CPA-VHH, that effectively neutralizes CPA toxins in vitro and in animal models, providing a new tool for inhibiting the pathogenicity resulting from these toxins and laying an important foundation for the development of new anti-C. perfringens toxin-related therapeutic products.

Dataset Information

Evaluation of the Anti-Leishmanial Effect of Recombinant Clostridium ?-Toxin.

Publications

Evaluation of the Anti-Leishmanial Effect of Recombinant <i>Clostridium</i> α-Toxin.

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