Ultimate opportunists - The emergent Enterocytozoon group Microsporidia

This is the final version. Available on open access from Public Library of Science via the DOI in this recordDefraBritish Council Newton FundUniversity of Exete

Preparing eugregarine parasites and their cricket host Acheta domesticus as a model for gregarine infection studies

This is the final version. Available on open access from Elsevier via the DOI in this recordData availability: Data will be made available on request.The increasing global demand for house crickets (Acheta domesticus) necessitates effective health measures. Despite their abundance, the effects of gregarines on these hosts remain underexplored. We present a method for controlled gregarine infection and maintenance of gregarine-free cricket populations. This system, adapted from prior work, is essential for experiments on gregarine infection dynamics, providing insights into parasite evolution and host-parasite interactions within the Apicomplexa group. This protocol includes rearing and maintenance of gregarine-free cricket populations for experimental purposes, gregarine production for oocyst solution and cricket infection, and gregarine infection assessment.European Union Horizon 202

Identification, characterization and heparin binding capacity of a spore-wall, virulence protein from the shrimp microsporidian, Enterocytozoon hepatopenaei (EHP)

This is the final version of the article. Available from the publisher via the DOI in this record.BACKGROUND: The microsporidian Enterocytozoon hepatopenaei (EHP) is a spore-forming, intracellular parasite that causes an economically debilitating disease (hepatopancreatic microsporidiosis or HPM) in cultured shrimp. HPM is characterized by growth retardation and wide size variation that can result in economic loss for shrimp farmers. Currently, the infection mechanism of EHP in shrimp is poorly understood, especially at the level of host-parasite interaction. In other microsporidia, spore wall proteins have been reported to be involved in host cell recognition. For the host, heparin, a glycosaminoglycan (GAG) molecule found on cell surfaces, has been shown to be recognized by many parasites such as Plasmodium spp. and Leishmania spp. RESULTS: We identified and characterized the first spore wall protein of EHP (EhSWP1). EhSWP1 contains three heparin binding motifs (HBMs) at its N-terminus and a Bin-amphiphysin-Rvs-2 (BAR2) domain at its C-terminus. A phylogenetic analysis revealed that EhSWP1 is similar to an uncharacterized spore wall protein from Enterospora canceri. In a cohabitation bioassay using EHP-infected shrimp with naïve shrimp, the expression of EhSWP1 was detected by RT-PCR in the naïve test shrimp at 20 days after the start of cohabitation. Immunofluorescence analysis confirmed that EhSWP1 was localized in the walls of purified, mature spores. Subcellular localization by an immunoelectron assay revealed that EhSWP1 was distributed in both the endospore and exospore layers. An in vitro binding assay, a competition assay and mutagenesis studies revealed that EhSWP1 is a bona fide heparin binding protein. CONCLUSIONS: Based on our results, we hypothesize that EhSWP1 is an important host-parasite interaction protein involved in tethering spores to host-cell-surface heparin during the process of infection.This project was supported by the Agricultural Research Development Agency (ARDA) of Thailand under project CRP5905020530, by the Thailand Research Fund (TRF) under project IRG5980008 and TRG5780032, by the Newton Institutional Links (IL) program to BIOTEC, Thailand and Cefas, UK, and by Mahidol University. PJ would like to thank the Science Achievement Scholarship of Thailand (SAST) for a PhD scholarshi

A recent whole-genome duplication divides populations of a globally-distributed microsporidian

This is the final version of the article. Available from Oxford University Press via the DOI in this record.The Microsporidia are a major group of intracellular fungi and important parasites of animals including insects, fish, and immunocompromised humans. Microsporidian genomes have undergone extreme reductive evolution but there are major differences in genome size and structure within the group: some are prokaryote-like in size and organisation (<3 Mb of gene-dense sequence) whilst others have more typically eukaryotic genome architectures. To gain fine-scale, population-level insight into the evolutionary dynamics of these tiny eukaryotic genomes, we performed the broadest microsporidian population genomic study to date, sequencing geographically isolated strains of Spraguea, a marine microsporidian infecting goosefish worldwide. Our analysis revealed that population structure across the Atlantic Ocean is associated with a conserved difference in ploidy, with American and Canadian isolates sharing an ancestral whole genome duplication that was followed by widespread pseudogenisation and sorting-out of paralogue pairs. Whilst past analyses have suggested de novo gene formation of microsporidian-specific genes, we found evidence for the origin of new genes from noncoding sequence since the divergence of these populations. Some of these genes experience selective constraint, suggesting the evolution of new functions and local host adaptation. Combining our data with published microsporidian genomes, we show that nucleotide composition across the phylum is shaped by a mutational bias favouring A and T nucleotides, which is opposed by an evolutionary force favouring an increase in genomic GC content. This work reveals ongoing dramatic reorganisation of genome structure and the evolution of new gene functions in modern microsporidians despite extensive genomic streamlining in their common ancestor.The authors would like to thank John Brookfield and David Studholme for helpful discussions. This work was supported by a Marie Curie Intra-European postdoctoral fellowship (T.A.W.) and the European Research Council Advanced Investigator Programme and the Wellcome Trust (grant numbers ERC- 2010- AdG-268701 045404 to T.M.E.) It is also supported by a Royal Society University Research Fellowship (B.A.P.W.)

A nested PCR assay to avoid false positive detection of the microsporidian enterocytozoon hepatopenaei (EHP) in environmental samples in shrimp farms

PublishedJournal Article© 2016 Jaroenlak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP) is an important disease of cultivated shrimp. Heavy infections may lead to retarded growth and unprofitable harvests. Existing PCR detection methods target the EHP small subunit ribosomal RNA (SSU rRNA) gene (SSU-PCR). However, we discovered that they can give false positive test results due to cross reactivity of the SSU-PCR primers with DNA from closely related microsporidia that infect other aquatic organisms. This is problematic for investigating and monitoring EHP infection pathways. To overcome this problem, a sensitive and specific nested PCR method was developed for detection of the spore wall protein (SWP) gene of EHP (SWP-PCR). The new SWP-PCR method did not produce false positive results from closely related microsporidia. The first PCR step of the SWP-PCR method was 100 times (104 plasmid copies per reaction vial) more sensitive than that of the existing SSU-PCR method (106 copies) but sensitivity was equal for both in the nested step (10 copies). Since the hepatopancreas of cultivated shrimp is not currently known to be infected with microsporidia other than EHP, the SSU-PCR methods are still valid for analyzing hepatopancreatic samples despite the lower sensitivity than the SWP-PCR method. However, due to its greater specificity and sensitivity, we recommend that the SWP-PCR method be used to screen for EHP in feces, feed and environmental samples for potential EHP carriers.OI acknowledges support from Agricultural Research Development Agency under project CRP5905020530 and Mahidol University. KS received funding from National Research Council Thailand, Division of Plan Administration and Research Budget/2557-79. PJ is supported by the Science Achievement Scholarship of Thailand (SAST). GDS acknowledges support of DG SANCO of the European Commission, and the UK Department of Environment, Food and Rural Affairs under project FB002. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

New Paradigms to Help Solve the Global Aquaculture Disease Crisis.

Published onlineJournal ArticleThis is the final version of the article. Available from Public Library of Science via the DOI in this record.n/aThe authors (GDS, KS) acknowledge funding administered by the British Council under the Newton Fund Researcher Links Programme, for a UK-Thailand bilateral workshop entitled "Scientific, technological and social solutions for sustainable aquaculture in Thailand: a key player in global aquatic food supply," Bangkok, March 2016. Further funding support is acknowledged from the European Commission (EC) and the UK Department for Environment, Food and Rural Affairs (Defra) under contracts C6928 and FB002 (to GDS and DB); from the Royal Society under a University Research Fellowship (to BAPW); and to the Agricultural Research Development Agency (ARDA) and National Research Council of Thailand (NRCT) (to KS, TWF, and OI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Transcriptomic profiling of host-parasite interactions in the microsporidian <i>Trachipleistophora hominis</i>

BACKGROUND: Trachipleistophora hominis was isolated from an HIV/AIDS patient and is a member of a highly successful group of obligate intracellular parasites. METHODS: Here we have investigated the evolution of the parasite and the interplay between host and parasite gene expression using transcriptomics of T. hominis-infected rabbit kidney cells. RESULTS: T. hominis has about 30 % more genes than small-genome microsporidians. Highly expressed genes include those involved in growth, replication, defence against oxidative stress, and a large fraction of uncharacterised genes. Chaperones are also highly expressed and may buffer the deleterious effects of the large number of non-synonymous mutations observed in essential T. hominis genes. Host expression suggests a general cellular shutdown upon infection, but ATP, amino sugar and nucleotide sugar production appear enhanced, potentially providing the parasite with substrates it cannot make itself. Expression divergence of duplicated genes, including transporters used to acquire host metabolites, demonstrates ongoing functional diversification during microsporidian evolution. We identified overlapping transcription at more than 100 loci in the sparse T. hominis genome, demonstrating that this feature is not caused by genome compaction. The detection of additional transposons of insect origin strongly suggests that the natural host for T. hominis is an insect. CONCLUSIONS: Our results reveal that the evolution of contemporary microsporidian genomes is highly dynamic and innovative. Moreover, highly expressed T. hominis genes of unknown function include a cohort that are shared among all microsporidians, indicating that some strongly conserved features of the biology of these enormously successful parasites remain uncharacterised. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1989-z) contains supplementary material, which is available to authorized users

The Anti-interferon Activity of Conserved Viral dUTPase ORF54 is Essential for an Effective MHV-68 Infection

Gammaherpesviruses such as KSHV and EBV establish lifelong persistent infections through latency in lymphocytes. These viruses have evolved several strategies to counteract the various components of the innate and adaptive immune systems. We conducted an unbiased screen using the genetically and biologically related virus, MHV-68, to find viral ORFs involved in the inhibition of type I interferon signaling and identified a conserved viral dUTPase, ORF54. Here we define the contribution of ORF54 in type I interferon inhibition by ectopic expression and through the use of genetically modified MHV-68. ORF54 and an ORF54 lacking dUTPase enzymatic activity efficiently inhibit type I interferon signaling by inducing the degradation of the type I interferon receptor protein IFNAR1. Subsequently, we show in vitro that the lack of ORF54 causes a reduction in lytic replication in the presence of type I interferon signaling. Investigation of the physiological consequence of IFNAR1 degradation and importance of ORF54 during MHV-68 in vivo infection demonstrates that ORF54 has an even greater impact on persistent infection than on lytic replication. MHV-68 lacking ORF54 expression is unable to efficiently establish latent infection in lymphocytes, although it replicates relatively normally in lung tissues. However, infection of IFNAR−/− mice alleviates this phenotype, emphasizing the specific role of ORF54 in type I interferon inhibition. Infection of mice and cells by a recombinant MHV-68 virus harboring a site specific mutation in ORF54 rendering the dUTPase inactive demonstrates that dUTPase enzymatic activity is not required for anti-interferon function of ORF54. Moreover, we find that dUTPase activity is dispensable at all stages of MHV-68 infection analyzed. Overall, our data suggest that ORF54 has evolved anti-interferon activity in addition to its dUTPase enzymatic activity, and that it is actually the anti-interferon role that renders ORF54 critical for establishing an effective persistent infection of MHV-68

Genomic survey of the non-cultivatable opportunistic human pathogen, Enterocytozoon bieneusi

© 2009 The Authors. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS Pathogens 5 (2009): e1000261, doi:10.1371/journal.ppat.1000261.Enterocytozoon bieneusi is the most common microsporidian associated with human disease, particularly in the immunocompromised population. In the setting of HIV infection, it is associated with diarrhea and wasting syndrome. Like all microsporidia, E. bieneusi is an obligate, intracellular parasite, but unlike others, it is in direct contact with the host cell cytoplasm. Studies of E. bieneusi have been greatly limited due to the absence of genomic data and lack of a robust cultivation system. Here, we present the first large-scale genomic dataset for E. bieneusi. Approximately 3.86 Mb of unique sequence was generated by paired end Sanger sequencing, representing about 64% of the estimated 6 Mb genome. A total of 3,804 genes were identified in E. bieneusi, of which 1,702 encode proteins with assigned functions. Of these, 653 are homologs of Encephalitozoon cuniculi proteins. Only one E. bieneusi protein with assigned function had no E. cuniculi homolog. The shared proteins were, in general, evenly distributed among the functional categories, with the exception of a dearth of genes encoding proteins associated with pathways for fatty acid and core carbon metabolism. Short intergenic regions, high gene density, and shortened protein-coding sequences were observed in the E. bieneusi genome, all traits consistent with genomic compaction. Our findings suggest that E. bieneusi is a likely model for extreme genome reduction and host dependence.This research was supported by National Institutes of Health (NIH) grants R21 AI064118 (DEA) and R21 AI52792 (ST). HGM was supported in part by NIH contracts HHSN266200400041C and HHSN2662004037C (Bioinformatics Resource Centers) and by the G. Unger Vetlesen Foundation

The Minimal Proteome in the Reduced Mitochondrion of the Parasitic Protist Giardia intestinalis

The mitosomes of Giardia intestinalis are thought to be mitochondria highly-reduced in response to the oxygen-poor niche. We performed a quantitative proteomic assessment of Giardia mitosomes to increase understanding of the function and evolutionary origin of these enigmatic organelles. Mitosome-enriched fractions were obtained from cell homogenate using Optiprep gradient centrifugation. To distinguish mitosomal proteins from contamination, we used a quantitative shot-gun strategy based on isobaric tagging of peptides with iTRAQ and tandem mass spectrometry. Altogether, 638 proteins were identified in mitosome-enriched fractions. Of these, 139 proteins had iTRAQ ratio similar to that of the six known mitosomal markers. Proteins were selected for expression in Giardia to verify their cellular localizations and the mitosomal localization of 20 proteins was confirmed. These proteins include nine components of the FeS cluster assembly machinery, a novel diflavo-protein with NADPH reductase activity, a novel VAMP-associated protein, and a key component of the outer membrane protein translocase. None of the novel mitosomal proteins was predicted by previous genome analyses. The small proteome of the Giardia mitosome reflects the reduction in mitochondrial metabolism, which is limited to the FeS cluster assembly pathway, and a simplicity in the protein import pathway required for organelle biogenesis