Publisher Correction: Exploitation of the Leishmania exosomal pathway by Leishmania RNA virus 1.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Exploitation of the Leishmania exosomal pathway by Leishmania RNA virus 1.

Leishmania are ancient eukaryotes that have retained the exosome pathway through evolution. Leishmania RNA virus 1 (LRV1)-infected Leishmania species are associated with a particularly aggressive mucocutaneous disease triggered in response to the double-stranded RNA (dsRNA) virus. However, it is unclear how LRV1 is exposed to the mammalian host cells. In higher eukaryotes, some viruses are known to utilize the host exosome pathway for their formation and cell-to-cell spread. As a result, exosomes derived from infected cells contain viral material or particles. Herein, we investigated whether LRV1 exploits the Leishmania exosome pathway to reach the extracellular environment. Biochemical and electron microscopy analyses of exosomes derived from LRV1-infected Leishmania revealed that most dsRNA LRV1 co-fractionated with exosomes, and that a portion of viral particles was surrounded by these vesicles. Transfer assays of LRV1-containing exosome preparations showed that a significant amount of parasites were rapidly and transiently infected by LRV1. Remarkably, these freshly infected parasites generated more severe lesions in mice than non-infected ones. Moreover, mice co-infected with parasites and LRV1-containing exosomes also developed a more severe disease. Overall, this work provides evidence that Leishmania exosomes function as viral envelopes, thereby facilitating LRV1 transmission and increasing infectivity in the mammalian host.

Leishmania exosomes and other virulence factors: Impact on innate immune response and macrophage functions.

Leishmania parasites are the causative agents of the leishmaniases, a collection of vector-borne diseases that range from simple cutaneous to fatal visceral forms. Employing potent immune modulation mechanisms, Leishmania is able to render the host macrophage inactive and persist inside its phagolysosome. In the last few years, the role of exosomes in Leishmania-host interactions has been increasingly investigated. For instance, it was reported that Leishmania exosome release is augmented following temperature shift, a condition mimicking parasite's entry into its mammalian host. Leishmania exosomes were found to strongly affect macrophage cell signaling and functions, similarly to whole parasites. Importantly, these vesicles were shown to be pro-inflammatory, capable to recruit neutrophils at their inoculation site exacerbating the pathology. In this review, we provide the most recent insights on the role of exosomes and other virulence factors, especially the surface protease GP63, in Leishmania-host interactions, deepening our knowledge on leishmaniasis and paving the way for the development of new therapeutics.

Exosome Secretion by the Parasitic Protozoan Leishmania within the Sand Fly Midgut.

Despite several studies describing the secretion of exosomes by Leishmania in vitro, observation of their formation and release in vivo has remained a major challenge. Herein, we show that Leishmania constitutively secretes exosomes within the lumen of the sand fly midgut through a mechanism homologous to the mammalian pathway. Through egestion experiments, we demonstrate that Leishmania exosomes are part of the sand fly inoculum and are co-egested with the parasite during the insect's bite, possibly influencing the host infectious process. Indeed, co-inoculation of mice footpads with L. major plus midgut-isolated or in-vitro-isolated L. major exosomes resulted in a significant increase in footpad swelling. Notably, co-injections produced exacerbated lesions through overinduction of inflammatory cytokines, in particular IL-17a. Our data indicate that Leishmania exosomes are an integral part of the parasite's infectious life cycle, and we propose to add these vesicles to the repertoire of virulence factors associated with vector-transmitted infections.

Adaptation of Leishmania donovani to cutaneous and visceral environments: in vivo selection and proteomic analysis.

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa. Two main forms are found in the Old World, self-limited cutaneous leishmaniasis and potentially fatal visceral leishmaniasis, with parasite dissemination to liver, bone marrow, and spleen. The Leishmania donovani species complex is the causative agent of visceral leishmaniasis worldwide, but atypical L. donovani strains can cause cutaneous leishmaniasis. We hypothesized that L. donovani can adapt to survive in response to restrictions imposed by the host environment. To assess this, we performed in vivo selection in BALB/c mice with a cutaneous L. donovani clinical isolate to select for parasites with increased capacity to survive in visceral organs. We then performed whole cell proteomic analysis and compared this visceral-selected strain to the original cutaneous clinical isolate and to a visceral leishmaniasis clinical isolate. Overall, there were no major shifts in proteomic profiles; however, translation, biosynthetic processes, antioxidant protection, and signaling were elevated in visceral strains. Conversely, transport and trafficking were elevated in the cutaneous strain. Overall, these results provide new insight into the adaptability of Leishmania parasites to the host environment and on the factors that mediate their ability to survive in different organs.

Leishmania virulence factors: focus on the metalloprotease GP63.

Parasites of Leishmania genus have developed elegant strategies permitting them to evade the innate immune response upon their initial interaction with macrophages. Their capacity to dodge the induction of macrophages microbicidal functions was found to correlate with the alteration of several signalling pathways regulating those latter. In this review, the role of the Leishmania GP63 as a critical virulence factor influencing macrophage physiology will be discussed.

Enucleated L929 cells support invasion, differentiation, and multiplication of Trypanosoma cruzi parasites.

Cell infection with Trypanosoma cruzi, the agent of Chagas' disease, begins with the uptake of infective trypomastigotes within phagosomes and their release into the cytosol, where they transform into replicating amastigotes; the latter, in turn, differentiate into cytolytically released and infective trypomastigotes. We ask here if the T. cruzi infection program can develop in enucleated host cells. Monolayers of L929 cells, enucleated by centrifugation in the presence of cytochalasin B and kept at 34 degrees C to extend the survival of cytoplasts, were infected with parasites of the CL strain. Percent infection, morphology, stage-specific markers, and numbers of parasites per cell were evaluated in nucleated and enucleated cells, both of which were present in the same preparations. Parasite uptake, differentiation and multiplication of amastigotes, development of epimastigote- and trypomastigote-like forms, and initial cytolytic release of parasites were all documented for cytoplasts and nucleated cells. Although the doubling times were similar, parasite loads at 48 and 72 h were significantly lower in the cytoplasts than in nucleated cells. Similar results were obtained with the highly virulent strain Y as well as with strains CL-14 and G, which exhibit low virulence for mice. Cytoplasts could also be infected with the CL strain 24 or 48 h after enucleation. Thus, infection of cells by T. cruzi can take place in enucleated host cells, i.e., in the absence of modulation of chromosomal and nucleolar gene transcription and of RNA modification and processing in the nucleus.