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Temporal analysis of the autophagic and apoptotic phenotypes in Leishmania parasites.

ABSTRACT: The leishmaniases are worldwide neglected tropical diseases caused by parasitic protozoa of the Leishmania genus. Different stimuli induce Leishmania cell death, but the proteins involved remain poorly understood. Furthermore, confusion often appears between cell death and the cell survival process autophagy, whose phenotype is not clearly defined. In this article, we present a comprehensive and temporal analysis of the cellular events occurring during miltefosine-induced cell death and autophagy in L. major. We also provide a list of features in order to clearly identify apoptotic cells, autophagic cells and to distinguish both processes. Furthermore, we demonstrate that autophagy is followed by apoptosis in the absence of nutrients. Finally, we show that cells treated with the generic kinase inhibitor staurosporine express apoptotic as well as autophagic markers and therefore cannot be used as an apoptosis inducer in Leishmania. These descriptions lead to a better recognition and understanding of apoptosis and autophagy, enabling their targeting in the development of new anti-leishmanial drugs. These researches also make it possible to better understand these processes in general, through the study of an ancestral eukaryote.

SUBMITTER: Basmaciyan L

PROVIDER: S-EPMC6167523 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Temporal analysis of the autophagic and apoptotic phenotypes in Leishmania parasites.

Basmaciyan Louise L Berry Laurence L Gros Julie J Azas Nadine N Casanova Magali M

Microbial cell (Graz, Austria) 20180801 9

The leishmaniases are worldwide neglected tropical diseases caused by parasitic protozoa of the Leishmania genus. Different stimuli induce Leishmania cell death, but the proteins involved remain poorly understood. Furthermore, confusion often appears between cell death and the cell survival process autophagy, whose phenotype is not clearly defined. In this article, we present a comprehensive and temporal analysis of the cellular events occurring during miltefosine-induced cell deat ...[more]

PMID: 30280103

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Project description:BackgroundPatients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence.Methodology/principal findingsThis study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice.Conclusions/significanceWe propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis.

Project description:BackgroundCutaneous leishmaniasis is caused by different protozoan parasites of the genus Leishmania. Leishmania RNA virus (LRV) was identified as the first Leishmania infecting virus in 1998. Different studies showed the presence and role of the LRV in Leishmania parasites causing cutaneous leishmaniasis (CL). However, there is limited data on the pooled prevalence of LRV in Leishmania parasites causing CL. Therefore, the aim of this systematic review and meta-analysis was to determine the pooled prevalence of LRV in Leishmania parasite isolates and/or lesion biopsies in patients with CL from the available literature globally.MethodologyWe retrieved the studies from different electronic databases. The studies were screened and identified based on the inclusion and exclusion criteria. We excluded studies exclusively done in experimental animals and in vitro studies. The review was conducted in line with PRISMA guidelines. The meta-analysis was performed with Stata software version 14 with metan command. The forest plot with random-effect model was used to estimate the pooled prevalence with 95% confidence interval. Inverse variance index (I2) was used to assess the heterogeneity among the included articles.Principal findingsA total of 1215 samples from 25 studies were included. Of these, 40.1% (487/1215) were positive for LRV. The overall pooled prevalence of LRV globally was 37.22% (95% CI: 27.54% - 46.90%). The pooled prevalence of LRV in the New World (NW) and Old World (OW) regions was 34.18% and 45.77%, respectively. Leishmania guyanensis, L. braziliensis, L. major, and L. tropica were the most studied species for the detection of LRV. The prevalence of LRV from Leishmania isolates and lesion biopsies was 42.9% (349/813) and 34.3% (138/402), respectively.ConclusionThis systematic study revealed that there is high prevalence of LRV in Leishmania parasites isolated from patients with CL. More comprehensive studies would be required to investigate the presence of the LRV in other Leishmania species such as L. aethiopica to fully understand the role of LRV in different clinical manifestations and disease pathology presented in CL patients.

Project description:BackgroundLeishmaniasis is endemic in Tunisia and presents with different clinical forms, caused by the species Leishmania infantum, Leishmania major, and Leishmania tropica. The life cycle of Leishmania is complex and involves several phlebotomine sand fly vectors and mammalian reservoir hosts. The aim of this work is the development and evaluation of a high-resolution melting PCR (PCR-HRM) tool to detect and identify Leishmania parasites in wild and domestic hosts, constituting confirmed (dogs and Meriones rodents) or potential (hedgehogs) reservoirs in Tunisia.MethodsUsing in vitro-cultured Leishmania isolates, PCR-HRM reactions were developed targeting the 7SL RNA and HSP70 genes. Animals were captured or sampled in El Kef Governorate, North West Tunisia. DNA was extracted from the liver, spleen, kidney, and heart from hedgehogs (Atelerix algirus) (n = 3) and rodents (Meriones shawi) (n = 7) and from whole blood of dogs (n = 12) that did not present any symptoms of canine leishmaniasis. In total, 52 DNA samples were processed by PCR-HRM using both pairs of primers.ResultsThe results showed melting curves enabling discrimination of the three Leishmania species present in Tunisia, and were further confirmed by Sanger sequencing. Application of PCR-HRM assays on reservoir host samples showed that overall among the examined samples, 45 were positive, while seven were negative, with no Leishmania infection. Meriones shawi were found infected with L. major, while dogs were infected with L. infantum. However, co-infections with L. major/L. infantum species were detected in four Meriones specimens and in all tested hedgehogs. In addition, multiple infections with the three Leishmania species were found in one hedgehog specimen. Sequence analyses of PCR-HRM products corroborated the Leishmania species found in analyzed samples.ConclusionsThe results of PCR-HRM assays applied to field specimens further support the possibility of hedgehogs as reservoir hosts of Leishmania. In addition, we showed their usefulness in the diagnosis of canine leishmaniasis, specifically in asymptomatic dogs, which will ensure a better evaluation of infection extent, thus improving elaboration of control programs. This PCR-HRM method is a robust and reliable tool for molecular detection and identification of Leishmania and can be easily implemented in epidemiological surveys in endemic regions.

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Temporal analysis of the autophagic and apoptotic phenotypes in Leishmania parasites.

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Temporal analysis of the autophagic and apoptotic phenotypes in <i>Leishmania</i> parasites.

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