Dolores Bernal

The transcriptional response to alkaline pH in Saccharomyces cerevisiae: evidence for calcium‐mediated signalling

The short‐time transcriptional response of yeast cells to a mild increase in external pH (7.6) has been investigated using DNA microarrays. A total of 150 genes increased their mRNA level at least twofold within 45 min. Alkalinization resulted in the repression of 232 genes. The response of four upregulated genes, ENA1 (encoding a Na+‐ATPase also induced by saline stress) and PHO84, PHO89 and PHO12 (encoding genes upregulated by phosphate starvation), was characterized further. The alkaline response of ENA1 was not affected by mutation of relevant genes involved in osmotic or oxidative signalling, but was decreased in calcineurin and rim101 mutants. Mapping of the ENA1 promoter revealed two pH‐responsive regions. The response of the upstream region was fully abolished by the drug FK506 or mutation of CRZ1 (a transcription factor activated by calcium/calcineurin), whereas the response of the downstream region was essentially calcium independent. PHO84 and PHO12 responses were unaffected in crz1 cells, but required the presence of Pho2 and Pho4. In contrast, part of the alkali‐induced expression of PHO89 was maintained in pho4 or pho2 cells, but was fully abolished in a crz1 strain or in the presence of FK506. Heterologous promoters carrying the minimal calcineurin‐dependent response elements found in ENA1 or FKS2 were able to drive alkaline pH‐induced expression. These results demonstrate that the transcriptional response to alkaline pH involves different signalling mechanisms, and that calcium signalling is a relevant component of this response.

Extracellular vesicles from parasitic helminths contain specific excretory/secretory proteins and are internalized in intestinal host cells.

The study of host-parasite interactions has increased considerably in the last decades, with many studies focusing on the identification of parasite molecules (i.e. surface or excretory/secretory proteins (ESP)) as potential targets for new specific treatments and/or diagnostic tools. In parallel, in the last few years there have been significant advances in the field of extracellular vesicles research. Among these vesicles, exosomes of endocytic origin, with a characteristic size ranging from 30–100 nm, carry several atypical secreted proteins in different organisms, including parasitic protozoa. Here, we present experimental evidence for the existence of exosome-like vesicles in parasitic helminths, specifically the trematodes Echinostoma caproni and Fasciola hepatica. These microvesicles are actively released by the parasites and are taken up by host cells. Trematode extracellular vesicles contain most of the proteins previously identified as components of ESP, as confirmed by proteomic, immunogold labeling and electron microscopy studies. In addition to parasitic proteins, we also identify host proteins in these structures. The existence of extracellular vesicles explains the secretion of atypical proteins in trematodes, and the demonstration of their uptake by host cells suggests an important role for these structures in host-parasite communication, as described for other infectious agents.

Identification of enolase as a plasminogen-binding protein in excretory–secretory products of Fasciola hepatica

We have followed a combined proteomic approach to identify proteins of Fasciola hepatica that could be involved in host–parasite interactions. Using two‐dimensional gel electrophoresis, far Western immunoblot and mass spectrometry analyses, we have identified the enolase enzyme, present in the excretory/secretory materials of F. hepatica, as a human plasminogen‐binding protein. This enzyme has an apparent molecular weight of 47 kDa with pI ranging from 6.2 to 7.2. These results suggest that enolase could act as a plasminogen receptor.

Extracellular vesicles in parasitic diseases

Parasitic diseases affect billions of people and are considered a major public health issue. Close to 400 species are estimated to parasitize humans, of which around 90 are responsible for great clinical burden and mortality rates. Unfortunately, they are largely neglected as they are mainly endemic to poor regions. Of relevance to this review, there is accumulating evidence of the release of extracellular vesicles (EVs) in parasitic diseases, acting both in parasite–parasite inter-communication as well as in parasite–host interactions. EVs participate in the dissemination of the pathogen and play a role in the regulation of the host immune systems. Production of EVs from parasites or parasitized cells has been described for a number of parasitic infections. In this review, we provide the most relevant findings of the involvement of EVs in intercellular communication, modulation of immune responses, involvement in pathology, and their potential as new diagnostic tools and therapeutic agents in some of the major human parasitic pathogens.

Leucine Aminopeptidase Is an Immunodominant Antigen of Fasciola hepatica Excretory and Secretory Products in Human Infections

ABSTRACT The liver fluke Fasciola hepatica parasitizes humans and ruminant livestock worldwide, and it is now being considered a reemerging zoonotic disease, especially in areas in which it is endemic, such as South America. This study investigates the immune response to excretory and secretory products produced by F. hepatica in a group of patients from the Peruvian Altiplano, where the disease is highly endemic. Using a proteomic approach and immunoblotting techniques, we have identified the enzymes leucine aminopeptidase (LAP) and phosphoenolpyruvate carboxykinase as immunodominant antigens recognized by sera from fasciolosis patients. An indirect enzyme-linked immunosorbent assay using recombinant LAP as the antigen was developed to check sera from individuals of this region. Our results demonstrate that LAP produces a specific and strong reaction, suggesting its potential use in the serologic diagnosis of F. hepatica infections in humans.

The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis

Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterization of the total secretome of this zoonotic parasite. Fasciola secretes at least two subpopulations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialized cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic data sets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular “machinery” required for EV biogenesis is constitutively expressed across the intramammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack.

The Role of Extracellular Vesicles in Modulating the Host Immune Response during Parasitic Infections.

Parasites are the cause of major diseases affecting billions of people. As the inflictions caused by these parasites affect mainly developing countries, they are considered as neglected diseases. These parasitic infections are often chronic and lead to significant immunomodulation of the host immune response by the parasite, which could benefit both the parasite and the host and are the result of millions of years of co-evolution. The description of parasite extracellular vesicles (EVs) in protozoa and helminths suggests that they may play an important role in host–parasite communication. In this review, recent studies on parasitic (protozoa and helminths) EVs are presented and their potential use as novel therapeutical approaches is discussed.

The Transcriptome Analysis of Strongyloides stercoralis L3i Larvae Reveals Targets for Intervention in a Neglected Disease

Background Strongyloidiasis is one of the most neglected diseases distributed worldwide with endemic areas in developed countries, where chronic infections are life threatening. Despite its impact, very little is known about the molecular biology of the parasite involved and its interplay with its hosts. Next generation sequencing technologies now provide unique opportunities to rapidly address these questions. Principal Findings Here we present the first transcriptome of the third larval stage of S. stercoralis using 454 sequencing coupled with semi-automated bioinformatic analyses. 253,266 raw sequence reads were assembled into 11,250 contiguous sequences, most of which were novel. 8037 putative proteins were characterized based on homology, gene ontology and/or biochemical pathways. Comparison of the transcriptome of S. strongyloides with those of other nematodes, including S. ratti, revealed similarities in transcription of molecules inferred to have key roles in parasite-host interactions. Enzymatic proteins, like kinases and proteases, were abundant. 1213 putative excretory/secretory proteins were compiled using a new pipeline which included non-classical secretory proteins. Potential drug targets were also identified. Conclusions Overall, the present dataset should provide a solid foundation for future fundamental genomic, proteomic and metabolomic explorations of S. stercoralis, as well as a basis for applied outcomes, such as the development of novel methods of intervention against this neglected parasite.

The revised microRNA complement of Fasciola hepatica reveals a plethora of overlooked microRNAs and evidence for enrichment of immuno-regulatory microRNAs in extracellular vesicles

MicroRNAs (miRNAs) are gene regulators that have recently been shown to down-regulate the immune response via extracellular vesicles in the mammalian host of helminthic parasites. Using the miRNA prediction pipeline miRCandRef, we expanded the current miRNA set of the liver fluke Fasciola hepatica (Platyhelminthes, Trematoda) from 16 to 54 miRNAs (42 conserved and 13 novel). Comparing the cellular expression levels with extracellular vesicles, we found all miRNAs expressed and enriched for miRNAs with immuno-regulatory function, tissue growth and cancer. Our findings support the hypothesis that miRNAs are the molecular mediators of the previously demonstrated immune modulatory function of extracellular vesicles.

Proteomic analysis of Strongyloides stercoralis L3 larvae

Strongyloidiasis can be perpetuated by autoinfection with the filariform larvae L3, causing asymptomatic chronic infections and creating a population of carriers, affecting not only developing countries. So far, very little is known about the proteins that interact with the human host, and few proteins from the infective Strongyloides stercoralis L3 have been characterized. Here, we report results obtained from a proteomic analysis of the proteins from S. stercoralis L3 larvae obtained from patients. Since the genome of S. stercoralis is not yet available, we used proteomic analysis to identify 26 different proteins, 13 of them released by short digestion with trypsin, which could represent surface-associated proteins. The present work extends our knowledge of host-parasite interactions by identifying proteins that could be of interest in the development of diagnostic tools, vaccines, or treatments for a neglected disease like strongyloidiasis.