Biology subjects

Sotillo, J.

Publications and source records attributed to Sotillo, J..

4 recordsLinked to original sources

bioRxiv2018

Comprehensive analysis of human hookworm secreted proteins using a proteogenomic approach

The human hookworm Necator americanus infects more than 400 million people worldwide, contributing substantially to the poverty in these regions. Adult stage N. americanus live in the small intestine of the human host where they inject excretory/secretory (ES) products into the mucosa. ES products have been characterized at the proteome level for a number of animal hookworm species, but until now, the difficulty in obtaining sufficient live N. americanus has been an obstacle in characterizing the secretome of this important human pathogen. Herein we describe the ES proteome of N. americanus and utilize this information to conduct the first proteogenomic analysis of a parasitic helminth, significantly improving the available genome and thereby generating a robust description of the parasite secretome. The genome annotation resulted in a a revised prediction of 3,425 fewer genes than initially reported, accompanied by a significant increase in the number of exons and introns, total gene length and the percentage of the genome covered by genes. Almost 200 ES proteins were identified by LC-MS/MS with SCP/TAPS proteins, hypothetical proteins and proteases among the most abundant families. These proteins were compared to commonly used model species of human parasitic infections, including Ancylostoma caninum, Nippostrongylus brasiliensis and Heligmosomoides polygyrus. Our findings provide valuable information on important families of proteins with both known and unknown functions that could be instrumental in host-parasite interactions, including protein families that might be key for parasite survival in the onslaught of robust immune responses, as well as vaccine and drug targets.

microbiology↗

bioRxiv2018

Programmed mutation of liver fluke granulin using CRISPR/Cas9 attenuates virulence of infection-induced hepatobiliary morbidity

Infections with several flatworm parasites represent group 1 biological carcinogens, i.e. definite causes of cancer. Infection with the food-borne liver fluke Opisthorchis viverrini causes cholangiocarcinoma (CCA). Whereas the causative agent for most cancers, including CCA in the West, remains obscure, the principal risk factor for CCA in Thailand is opisthorchiasis. We exploited this established link to explore the role of the secreted parasite growth factor termed liver fluke granulin (Ov-GRN-1) in pre-malignant lesions of the biliary tract. We targeted the Ov-grn-1 gene for programmed knockout and investigated gene-edited parasites in vitro and in experimentally infected hamsters. Both adult and juvenile stages of the liver fluke were transfected with a plasmid encoding a guide RNA sequence specific for exon 1 of Ov-grn-1 and the Cas9 nuclease. Deep sequencing of amplicon libraries from genomic DNA from gene-edited parasites exhibited programmed, Cas9-catalyzed mutations within the Ov-grn-1 locus, and tandem analyses by RT-PCR and western blot revealed rapid depletion of Ov-grn-1 transcripts and protein. Newly excysted juvenile flukes that had undergone editing of Ov-grn-1 colonized the biliary tract, grew and developed over a period of 60 days, were active and motile, and induced a clinically relevant pathophysiological tissue phenotype of attenuated biliary hyperplasia and fibrosis in comparison to infection with wild type flukes. This is the first report of gene knock-out using CRISPR/Cas9 in a parasitic flatworm, demonstrating the activity and utility of the process for functional genomics in these pathogens. The striking clinical phenotype highlights the role in virulence that liver fluke growth factors play in biliary tract morbidity during chronic opisthorchiasis.

molecular biology↗

bioRxiv2017

Revisiting The Ancylostoma caninum Secretome Provides New Information On Hookworm-Host Interactions

Hookworm infection is a major tropical parasitic disease affecting almost 500 million people worldwide. These soil-transmitted helminths can survive for many years in the intestine of the host, where they feed on blood, causing iron deficiency anaemia and other complications. To avoid the hosts immune response the parasite releases excretory/secretory products (ESPs), a complex mixture of glycans, lipids and proteins that represent the major host-parasite interface. Using a combination of separation techniques such as SDS-PAGE and OFFGEL electrophoresis, in combination with state-of-the-art mass spectrometry we have reanalysed the dog hookworm, Ancylostoma caninum, ESPs (AcES). We identified 315 proteins present in the AcES, compared with just 105 identified in previous studies. The most highly represented family of proteins is the SCP/TAPs (90 of the 315 proteins), and the most abundant constituents of AcES are homologues of the tissue inhibitors of metalloproteases (TIMP) family. We identified putative vaccine candidates and proteins that could have immunomodulatory effects for treating inflammatory diseases. This study provides novel information about the proteins involved in host-hookworm interactions, and constitutes a comprehensive dataset for the development of vaccines and the discovery of new immunoregulatory biologics.

microbiology↗

bioRxiv2017

Characterisation Of Trichuris muris Secreted Proteins And Extracellular Vesicles Provides New Insights Into Host-Parasite Communication

Whipworms are parasitic nematodes that live in the gut of more than 500 million people worldwide. Due to the difficulty in obtaining parasite material, the mouse whipworm Trichuris muris has been extensively used as a model to study human whipworm infections. These nematodes secrete a multitude of compounds that interact with host tissues where they orchestrate a parasitic existence. Herein we provide the first comprehensive characterisation of the excretory/secretory products of T. muris. We identify 148 proteins secreted by T. muris and show for the first time that the mouse whipworm secretes exosome-like extracellular vesicles (EVs) that can interact with host cells. We use an Optiprep(R) gradient to purify the EVs, highlighting the suitability of this method for purifying EVs secreted by a parasitic nematode. We also characterise the proteomic and genomic content of the EVs, identifying >350 proteins, 56 miRNAs (22 novel) and 475 full-length mRNA transcripts mapping to T. muris gene models. Many of the miRNAs putatively mapped to mouse genes involved in regulation of inflammation, implying a role in parasite-driven immunomodulation. In addition, for the first time to our knowledge, we use colonic organoids to demonstrate the internalisation of parasite EVs by host cells. Understanding how parasites interact with their host is crucial to develop new control measures. This first characterisation of the proteins and EVs secreted by T. muris provides important information on whipworm-host communication and forms the basis for future studies.

microbiology↗