Extracellular vesicles from Trypanosoma cruzi-dendritic cell interaction show modulatory properties and confer resistance to lethal infection as a cell-free based therapy strategy.

Extracellular vesicles (EVs) include a heterogeneous group of particles. Microvesicles, apoptotic bodies and exosomes are the most characterized vesicles. They can be distinguished by their size, morphology, origin and molecular composition. To date, increasing studies demonstrate that EVs mediate intercellular communication. EVs reach considerable interest in the scientific community due to their role in diverse processes including antigen-presentation, stimulation of anti-tumoral immune responses, tolerogenic or inflammatory effects. In pathogens, EV shedding is well described in fungi, bacteria, protozoan and helminths parasites. For Trypanosoma cruzi EV liberation and protein composition was previously described. Dendritic cells (DCs), among other cells, are key players promoting the immune response against pathogens and also maintaining self-tolerance. In previous reports we have demonstrate that T. cruzi downregulates DCs immunogenicity in vitro and in vivo. Here we analyze EVs from the in vitro interaction between blood circulating trypomastigotes (Tp) and bone-marrow-derived DCs. We found that Tp incremented the number and the size of EVs in cultures with DCs. EVs displayed some exosome markers and intracellular RNA. Protein analysis demonstrated that the parasite changes the DC protein-EV profile. We observed that EVs from the interaction of Tp-DCs were easily captured by unstimulated-DCs in comparison with EVs from DCs cultured without the parasite, and also modified the activation status of LPS-stimulated DCs. Noteworthy, we found protection in animals treated with EVs-DCs+Tp and challenged with T. cruzi lethal infection. Our goal is to go deep into the molecular characterization of EVs from the DCs-Tp interaction, in order to identify mediators for therapeutic purposes.

Extracellular non-coding RNA signatures of the metacestode stage of Echinococcus multilocularis.

Extracellular RNAs (ex-RNAs) are secreted by cells through different means that may involve association with proteins, lipoproteins or extracellular vesicles (EV). In the context of parasitism, ex-RNAs represent new and exciting communication intermediaries with promising potential as novel biomarkers. In the last years, it was shown that helminth parasites secrete ex-RNAs, however, most work mainly focused on RNA secretion mediated by EV. Ex-RNA study is of special interest in those helminth infections that still lack biomarkers for early and/or follow-up diagnosis, such as echinococcosis, a neglected zoonotic disease caused by cestodes of the genus Echinococcus. In this work, we have characterised the ex-RNA profile secreted by in vitro grown metacestodes of Echinococcus multilocularis, the casuative agent of alveolar echinococcosis. We have used high throughput RNA-sequencing together with RT-qPCR to characterise the ex-RNA profile secreted towards the extra- and intra-parasite milieus in EV-enriched and EV-depleted fractions. We show that a polarized secretion of small RNAs takes place, with microRNAs mainly secreted to the extra-parasite milieu and rRNA- and tRNA-derived sequences mostly secreted to the intra-parasite milieu. In addition, we show by nanoparticle tracking analyses that viable metacestodes secrete EV mainly into the metacestode inner vesicular fluid (MVF); however, the number of nanoparticles in culture medium and MVF increases > 10-fold when metacestodes show signs of tegument impairment. Interestingly, we confirm the presence of host miRNAs in the intra-parasite milieu, implying their internalization and transport through the tegument towards the MVF. Finally, our assessment of the detection of Echinococcus miRNAs in patient samples by RT-qPCR yielded negative results suggesting the tested miRNAs may not be good biomarkers for this disease. A comprehensive study of the secretion mechanisms throughout the life cycle of these parasites will help to understand parasite interaction with the host and also, improve current diagnostic tools.

A new flatworm species of Temnocephala (Rhabdocoela, Temnocephalidae) ectosymbiont on the freshwater crab Valdivia serrata (Decapoda, Trichodactylidae) from Amazonas, Colombia.

A new species of temnocephalan is described from the branchial chambers of Valdivia serrata in Colombia as Temnocephala ivandarioi sp. nov. The most distinctive characters of the new species are in the cirrus and the epidermal 'excretory' syncytial plates. In the present study, the terminology to describe the cirrus of species of Temnocephala is updated. Comparison between the shape of the cirrus of the temnocephalans associated with trichodactylid crabs is also provided.

Transcytosis of Bacillus subtilis extracellular vesicles through an in vitro intestinal epithelial cell model.

Bacterial EVs have been related to inter-kingdom communication between probiotic/pathogenic bacteria and their hosts. Our aim was to investigate the transcytosis process of B. subtilis EVs using an in vitro intestinal epithelial cell model. In this study, using Confocal Laser Scanning Microscopy, we report that uptake and internalization of CFSE-labeled B. subtilis EVs (115 nm ± 27 nm) by Caco-2 cells are time-dependent. To study the transcytosis process we used a transwell system and EVs were quantified in the lower chamber by Fluorescence and Nanoparticle Tracking Analysis measurements. Intact EVs are transported across a polarized cell monolayer at 60-120 min and increased after 240 min with an estimated average uptake efficiency of 30% and this process is dose-dependent. EVs movement into intestinal epithelial cells was mainly through Z axis and scarcely on X and Y axis. This work demonstrates that EVs could be transported across the gastrointestinal epithelium. We speculate this mechanism could be the first step allowing EVs to reach the bloodstream for further delivery up to extraintestinal tissues and organs. The expression and further encapsulation of bioactive molecules into natural nanoparticles produced by probiotic bacteria could have practical implications in food, nutraceuticals and clinical therapies.

Morphological and molecular characterization of Paragonimus caliensis Little, 1968 (Trematoda: Paragonimidae) from Medellin and Pichinde, Colombia.

Paragonimiasis is a subacute to chronic inflammatory granulomatous lung disease caused by the genus Paragonimus. In Latin America Paragonimus mexicanus Miyazaki & Ishii, 1968 is the only confirmed species to cause human infections. Paragonimus caliensis Little, 1968 is an uncommon species often regarded as a synonym of P. mexicanus. Recently, the study of two types of Paragonimus metacercariae from Costa Rica has provided new molecular and morphological evidence that P. caliensis is a separate species from P. mexicanus. In the present study, molecular, morphological and phylogenetic tools have been used to characterize two populations of Paragonimus located at west of Medellin, Antioquia and at Pichinde, Valle del Cauca (type locality of P. caliensis), Colombia. Adults and metacercariae obtained from Medellin, and metacercariae from Pichinde were analyzed. For morphological observations we used light microscopy and scanning electron microscopy (SEM). Morphology of metacercariae and adults matched with the holotype of P. caliensis. The number and arrangement of sensory papillae in the acetabulum region differs from the morphotypes reported for P. caliensis in Costa Rica. Two morphotypes in branching patterns of ovary and two morphotypes in branching patterns of testes were identified. The main morphological differences between P. caliensis and P. mexicanus corresponded to the size of gonads and their relative positions in the body, and the occasional presence of a cyst wall in P. caliensis metacercariae. The molecular and phylogenetic analyses (using nuclear ribosomal ITS2 and partial cytochrome c oxidase subunit 1 CO1 sequences) confirmed that P. caliensis from the type locality is the same species from Medellin and Costa Rica. Furthermore, these analyses also suggest genetic as well as geographical separation of P. caliensis populations between Colombia and Costa Rica. Currently, P. mexicanus and P. caliensis are sympatric in the Colombian Pacific bioregion, and specific diagnosis based on their egg size is not possible. Therefore, it is necessary to determine the biogeographic distribution ranges of both species and to implement molecular techniques to establish the role of P. caliensis in human paragonimiasis in Colombia.

Cestode parasites release extracellular vesicles with microRNAs and immunodiagnostic protein cargo.

Intercellular communication is crucial in multiple aspects of cell biology. This interaction can be mediated by several mechanisms including extracellular vesicle (EV) transfer. EV secretion by parasites has been reported in protozoans, trematodes and nematodes. Here we report that this mechanism is present in three different species of cestodes, Taenia crassiceps, Mesocestoides corti and Echinococcus multilocularis. To confirm this we determined, in vitro, the presence of EVs in culture supernatants by transmission electron microscopy. Interestingly, while T. crassiceps and M. corti metacestodes secrete membranous structures into the culture media, similar vesicles were observed in the interface of the germinal and laminated layers of E. multilocularis metacestodes and were hardly detected in culture supernatants. We then determined the protein cargo in the EV-enriched secreted fractions of T. crassiceps and M. corti conditioned media by LC-MS/MS. Among the identified proteins, eukaryotic vesicle-enriched proteins were identified as expected, but also proteins used for cestode disease diagnosis, proteins related to neurotransmission, lipid binding proteins as well as host immunoglobulins and complement factors. Finally, we confirmed by capillary electrophoresis the presence of intravesicular RNA for both parasites and detected microRNAs by reverse transcription-PCR. This is the first report of EV secretion in cestode parasites and of an RNA secretion mechanism. These findings will provide valuable data not only for basic cestode biology but also for the rational search for new diagnostic targets.

Microvesicles released from Giardia intestinalis disturb host-pathogen response in vitro.

Giardia intestinalis (G.I), is an anaerobic protozoan and the aetiological agent of giardiasis, a diarrhoea present worldwide and associated with poverty. G.I has a simple life cycle alternating between cyst and trophozoite. Cysts are transmitted orally to the stomach and transform to trophozoites in the intestine by a multifactorial process. Recently, microvesicles (MVs) have been found to be released from a wide range of eukaryotic cells. We have observed a release of MVs during the life cycle of G.I., identifying MVs from active trophozoites and from trophozoites differentiating to the cyst form. The aim of the current work was to investigate the role of MVs from G.I in the pathogenesis of giardiasis. MVs from log phase were able to increase the attachment of G. intestinalis trophozoites to Caco-2 cells. Moreover, MVs from G. intestinalis could be captured by human immature dendritic cells, resulting in increased activation and allostimulation of human dendritic cells. Lipid rafts participate in the MV biogenesis and in the attachment to Caco-2 cells. Nevertheless, proteomic analysis from two types of MVs has shown slight differences at the protein levels. An understanding of biogenesis and content of MVs derived from trophozoites might have important implications in the pathogenesis of the disease.