Genome-wide mapping reveals single-origin chromosome replication in Leishmania, a eukaryotic microbe

Background DNA replication initiates on defined genome sites, termed origins. Origin usage appears to follow common rules in the eukaryotic organisms examined to date: all chromosomes are replicated from multiple origins, which display variations in firing efficiency and are selected from a larger pool of potential origins. To ask if these features of DNA replication are true of all eukaryotes, we describe genome-wide origin mapping in the parasite Leishmania. Results Origin mapping in Leishmania suggests a striking divergence in origin usage relative to characterized eukaryotes, since each chromosome appears to be replicated from a single origin. By comparing two species of Leishmania, we find evidence that such origin singularity is maintained in the face of chromosome fusion or fission events during evolution. Mapping Leishmania origins suggests that all origins fire with equal efficiency, and that the genomic sites occupied by origins differ from related non-origins sites. Finally, we provide evidence that origin location in Leishmania displays striking conservation with Trypanosoma brucei, despite the latter parasite replicating its chromosomes from multiple, variable strength origins. Conclusions The demonstration of chromosome replication for a single origin in Leishmania, a microbial eukaryote, has implications for the evolution of origin multiplicity and associated controls, and may explain the pervasive aneuploidy that characterizes Leishmania chromosome architecture

The first detection of Leishmania major in naturally infected Sergentomyia minuta in Portugal

Phlebotomine sandflies of the genus Sergentomyia are widely distributed throughout the Old World. It has been suggested that Sergentomyia spp are involved in the transmission of Leishmania in India and Africa, whereas Phlebotomus spp are thought to be the sole vectors of Leishmania in the Old World. In this study, Leishmania major DNA was detected in one Sergentomyia minuta specimen that was collected in the southern region of Portugal. This study challenges the dogma that Leishmania is exclusively transmitted by species of the genus Phlebotomus in the Old World.EU/FEDER [PTDC/CVT/112371/2009]; EU [FP7-261504 EDENext]info:eu-repo/semantics/publishedVersio

Genetic structure and evolution of the Leishmania genus in Africa and Eurasia: what does MLSA tell us

Leishmaniasis is a complex parasitic disease from a taxonomic, clinical and epidemiological point of view. The role of genetic exchanges has been questioned for over twenty years and their recent experimental demonstration along with the identification of interspecific hybrids in natura has revived this debate. After arguing that genetic exchanges were exceptional and did not contribute to Leishmania evolution, it is currently proposed that interspecific exchanges could be a major driving force for rapid adaptation to new reservoirs and vectors, expansion into new parasitic cycles and adaptation to new life conditions. To assess the existence of gene flows between species during evolution we used MLSA-based (MultiLocus Sequence Analysis) approach to analyze 222 Leishmania strains from Africa and Eurasia to accurately represent the genetic diversity of this genus. We observed a remarkable congruence of the phylogenetic signal and identified seven genetic clusters that include mainly independent lineages which are accumulating divergences without any sign of recent interspecific recombination. From a taxonomic point of view, the strong genetic structuration of the different species does not question the current classification, except for species that cause visceral forms of leishmaniasis (L. donovani, L. infantum and L. archibaldi). Although these taxa cause specific clinical forms of the disease and are maintained through different parasitic cycles, they are not clearly distinct and form a continuum, in line with the concept of species complex already suggested for this group thirty years ago. These results should have practical consequences concerning the molecular identification of parasites and the subsequent therapeutic management of the disease

Molecular mechanisms of drug resistance in natural Leishmania populations vary with genetic background

The evolution of drug-resistance in pathogens is a major global health threat. Elucidating the molecular basis of pathogen drug-resistance has been the focus of many studies but rarely is it known whether a drug-resistance mechanism identified is universal for the studied pathogen; it has seldom been clarified whether drug-resistance mechanisms vary with the pathogen's genotype. Nevertheless this is of critical importance in gaining an understanding of the complexity of this global threat and in underpinning epidemiological surveillance of pathogen drug resistance in the field. This study aimed to assess the molecular and phenotypic heterogeneity that emerges in natural parasite populations under drug treatment pressure. We studied lines of the protozoan parasite Leishmania (L.) donovani with differential susceptibility to antimonial drugs; the lines being derived from clinical isolates belonging to two distinct genetic populations that circulate in the leishmaniasis endemic region of Nepal. Parasite pathways known to be affected by antimonial drugs were characterised on five experimental levels in the lines of the two populations. Characterisation of DNA sequence, gene expression, protein expression and thiol levels revealed a number of molecular features that mark antimonial-resistant parasites in only one of the two populations studied. A final series of in vitro stress phenotyping experiments confirmed this heterogeneity amongst drug-resistant parasites from the two populations. These data provide evidence that the molecular changes associated with antimonial-resistance in natural Leishmania populations depend on the genetic background of the Leishmania population, which has resulted in a divergent set of resistance markers in the Leishmania populations. This heterogeneity of parasite adaptations provides severe challenges for the control of drug resistance in the field and the design of molecular surveillance tools for widespread applicability

Insights on adaptive and innate immunity in canine leishmaniosis

Canine leishmaniosis (CanL) is caused by the parasite Leishmania infantum and is a systemic disease, which can present with variable clinical signs, and clinicopathological abnormalities. Clinical manifestations can range from subclinical infection to very severe systemic disease. Leishmaniosis is categorized as a neglected tropical disease and the complex immune responses associated with Leishmania species makes therapeutic treatments and vaccine development challenging for both dogs and humans. In this review, we summarize innate and adaptive immune responses associated with L. infantum infection in dogs, and we discuss the problems associated with the disease as well as potential solutions and the future direction of required research to help control the parasite

Detection of Leishmania DNA in wild foxes and associated ticks in Patagonia, Argentina, 2000 km south of its known distribution area

Indexación: Web of Science; PubMedBackground: Zoonotic Visceral Leishmaniasis (ZVL) is a vector-borne disease affecting humans and other mammals and caused by the protozoan parasite Leishmania (Leishmania) infantum (syn. L. chagasi), belonging to the L. donovani complex. The regions in Northern Argentina (above 32 degrees S) are its southern distribution limit in South America. Results: We detected Leishmania sp. DNA (most likely belonging to the L. donovani complex) in 37.5 % of 32 grey foxes (Pseudalopex griseus) captured in Argentinean Patagonia (48 degrees S and 50 degrees S). Eleven monosexual pools of Amblyomma tigrinum ticks from eight different foxes (six grey foxes and two culpeo foxes P. culpaeus) were also positive. The southernmost known distribution limit for L. infantum, and the southernmost reported capture of a phlebotominae, had previously been 2000 and 750 km north of our study area, respectively. Conclusions: This finding is significant because it markedly extends the distribution area of leishmaniasis; supports the existence of a sylvatic cycle in the absence of dogs; and has implications in transmission, indicating that either sand fly distribution is broader than currently thought or non-sand fly Leishmania maintenance is possible. Additional molecular, parasitological, epidemiological and entomological studies are still needed.http://parasitesandvectors.biomedcentral.com/articles/10.1186/s13071-016-1515-

Changes to cholesterol trafficking in macrophages by Leishmania parasites infection

Leishmania spp. are protozoan parasites that are transmitted by sandfly vectors during blood sucking to vertebrate hosts and cause a spectrum of diseases called leishmaniases. It has been demonstrated that host cholesterol plays an important role during Leishmania infection. Nevertheless, little is known about the intracellular distribution of this lipid early after internalization of the parasite. Here, pulse‐chase experiments with radiolabeled cholesteryl esterified to fatty acids bound to low‐density lipoproteins indicated that retention of this source of cholesterol is increased in parasite‐containing subcellular fractions, while uptake is unaffected. This is correlated with a reduction or absence of detectable NPC1 (Niemann–Pick disease, type C1), a protein responsible for cholesterol efflux from endocytic compartments, in the Leishmania mexicana habitat and infected cells. Filipin staining revealed a halo around parasites within parasitophorous vacuoles (PV) likely representing free cholesterol accumulation. Labeling of host cell membranous cholesterol by fluorescent cholesterol species before infection revealed that this pool is also trafficked to the PV but becomes incorporated into the parasites’ membranes and seems not to contribute to the halo detected by filipin. This cholesterol sequestration happened early after infection and was functionally significant as it correlated with the upregulation of mRNA‐encoding proteins required for cholesterol biosynthesis. Thus, sequestration of cholesterol by Leishmania amastigotes early after infection provides a basis to understand perturbation of cholesterol‐dependent processes in macrophages that were shown previously by others to be necessary for their proper function in innate and adaptive immune responses

Immunization with antigenic extracts of Leishmania associated with Montanide ISA 763 adjuvant induces partial protection in BALB/c mice against Leishmania (Leishmania) amazonensis infection

Background/Purpose: A proper adjuvant has a relevant role in vaccine formulations to generate an effective immune response. In this study, total Leishmania antigen (TLA) formulated with Montanide ISA 763 or R848 as adjuvants were evaluated as a first generation Leishmania vaccine in a murine model. Methods: Immunization protocols were tested in BALB/c mice with a subcutaneous prime/boost regimen with an interval of 3 weeks. Mice immunized with unadjuvanted TLA and phosphate-buffered saline (PBS) served as control groups. On Day 21 and Day 36 of the protocol, we evaluated the humoral immune response induced by each formulation. Fifteen days after the boost, the immunized mice were challenged with 1 × 105 promastigotes of Leishmania (Leishmania) amazonensis in the right footpad (RFP). The progress of the infection was followed for 10 weeks; at the end of this period, histopathological studies were performed in the RFP. Results: Vaccines formulated with Montanide ISA 763 generated an increase in the production of immunoglobulin G (IgG; p < 0.05) compared with the control group. There were no statistically significant differences in IgG1 production between the study groups. However, immunization with TLA-Montanide ISA 763 resulted in an increase in IgG2a compared to the unadjuvanted control (p < 0.001). Also noteworthy was the fact that a significant reduction in swelling and histopathological damage of the RFP was recorded with the Montanide ISA 763 formulation. Conclusion: We conclude that the immunization of BALB/c mice with a vaccine formulated with TLA and Montanide ISA 763 generated a protective immune response against L. (L.) amazonensis, characterized by an intense production of IgG2a.Fil: Cargnelutti, Diego Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo; ArgentinaFil: Salomón, María Cristina. Universidad Nacional de Cuyo; ArgentinaFil: Celedon, Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Garcia Bustos, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Morea, Gastón. Universidad Nacional de Cuyo; ArgentinaFil: Cuello Carrión, Fernando Darío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo; ArgentinaFil: Scodeller, Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo; Argentin

Analysis by RNA-seq of transcriptomic changes elicited by heat shock in Leishmania major

Besides their medical relevance, Leishmania is an adequate model for studying post-transcriptional mechanisms of gene expression. In this microorganism, mRNA degradation/stabilization mechanisms together with translational control and post-translational modifications of proteins are the major drivers of gene expression. Leishmania parasites develop as promastigotes in sandflies and as amastigotes in mammalians, and during host transmission, the parasite experiences a sudden temperature increase. Here, changes in the transcriptome of Leishmania major promastigotes after a moderate heat shock were analysed by RNA-seq. Several of the up-regulated transcripts code for heat shock proteins, other for proteins previously reported to be amastigote-specific and many for hypothetical proteins. Many of the transcripts experiencing a decrease in their steady-state levels code for transporters, proteins involved in RNA metabolism or translational factors. In addition, putative long noncoding RNAs were identified among the differentially expressed transcripts. Finally, temperature-dependent changes in the selection of the spliced leader addition sites were inferred from the RNA-seq data, and particular cases were further validated by RT-PCR and Northern blotting. This study provides new insights into the post-transcriptional mechanisms by which Leishmania modulate gene expressionThis work was supported by grants (to B.A. and J.M.R.) from Ministerio de Economía, Industria y Competitividad, project number SAF2017-86965-R (co-funded with FEDER funds), and by the Network of Tropical Diseases Research RICET (RD16/0027/0008), co-funded with FEDER funds. The CBMSO receives institutional grants from the Fundación Ramón Areces and from the Fundación Banco de Santande

Molecular variation in Leishmania parasites from sandflies species of a zoonotic cutaneous leishmaniasis in northeast of Iran

Background & objectives: In the well-known zoonotic cutaneous leishmaniasis (ZCL) focus in Turkmen Sahara, border of Iran and Turkmenistan, ZCL has increased among humans in the past five years. The present study was undertaken to incriminate vectors of ZCL in the region, and to find molecular variation in Leishmania parasites. Methods: The sandflies were sampled using CDC light-traps and sticky papers. All the sandflies were identified using morphological characters of the head and abdominal terminalia. DNA was extracted from the dissected thorax and attached anterior abdomen of individual female sandfly. Leishmania detection and identification of sandflies were performed using PCR, digestion of BsuRI restriction enzyme and sequencing of ITS-rDNA gene and also by semi-nested PCR to amplify minicircle kinetoplast (k) DNA of Leishmania. Results: Leishmania infections were detected in 26 out of 206 female sandflies. Of the infected sandflies, 18 were Phlebotomus papatasi while eight were P. caucasicus/P. mongolensis. Two infections of L. turnica were detected, one in P. papatasi and other in P. caucasicus/P. mongolensis and the rest of the sandflies were found infected with L. major. Conclusion: Our finding showed that L. major had low diversity with only one common haplotype (GenBank Access No. EF413075). The novel haplotypes were discovered in L. major (GenBank Access No. KF152937) and in L. turanica (GenBank Access No. EF413079) in low frequency. These Leishmania parasites are circulating to maintain infections in the P. papatasi and P. caucasicus/P. mongolensis in Turkmen Sahara