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 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

Decay of the glycolytic pathway and adaptation to intranuclear parasitism within Enterocytozoonidae microsporidia

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this record.Glycolysis and oxidative phosphorylation are the fundamental pathways of ATP generation in eukaryotes. Yet in microsporidia, endoparasitic fungi living at the limits of cellular streamlining, oxidative phosphorylation has been lost: energy is obtained directly from the host or, during the dispersive spore stage, via glycolysis. It was therefore surprising when the first genome from the Enterocytozoonidae – a major family of human and animal-infecting microsporidians - appeared to have lost glycolysis. Here we sequence and analyse genomes from four additional members of this family, shedding new light on their unusual biology. Our survey includes the genome of Enterocytozoon hepatopenaei, a major aquacultural parasite currently causing substantial economic losses in shrimp farming, and Enterospora canceri, a pathogen that lives exclusively inside the nuclei of its crab host. Our analysis of gene content across the clade suggests that Ent. canceri’s adaptation to intranuclear life is underpinned by the expansion of transporter families. We demonstrate that this entire lineage of pathogens has lost glycolysis and, uniquely amongst eukaryotes, lacks any obvious intrinsic means of generating energy. Our study provides an important resource for the investigation of host-pathogen interactions and reductive evolution in one of the most medically and economically important microsporidian lineages.This work was supported by Royal Society University Research Fellowships (B.A.P.W. and T.A.W.) and by a University of Exeter/Cefas strategic alliance studentship (DWB). AP and KS would like to acknowledge support from the Agricultural Research Development Agency (Arda) of Thailand (Grant number 8669), Mahidol University and the National Center for Genetic Engineering and Biotechnology (BIOTEC) of the Thai National Science and Technology Development Agency (NSTDA). OI is supported by Mahidol University. PJ is supported by Science Achievement Scholarship of Thailand (SAST)

Alignments of the SSU-PCR primer sequences and confirmation of cross reactions with closely related microsporidia.

<p>(A) Alignments of the SSU primer sequences (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166320#pone.0166320.t003" target="_blank">Table 3</a>) with homologous SSU regions of other microsporidia (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166320#pone.0166320.t001" target="_blank">Table 1</a>). Black highlights indicate matches with the primer sequences, while asterisks under the sequences indicate regions of 100% identity for all of the aligned sequences. (B) Agarose gel of SSU-PCR amplicons from EHP and other microsporidia. In addition to the pGEM-SSU plasmid (+ve) and water (-ve), total DNA obtained from EHP-infected shrimp (I) and naïve shrimp (U) were used as controls. PCR amplicons and false positive test results are marked with arrowheads and asterisks, respectively. The band at 226 bp show amplicons of residual primers ENR779 from the first PCR step and primers ENF176 from the second nested PCR step.</p

Alignments of the SWP-PCR primer sequences and lack of cross reactions with closely related microsporidia.

<p>(A) Alignments of the SWP primer sequences (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166320#pone.0166320.t003" target="_blank">Table 3</a>) with homologous regions of spore wall protein genes of other microsporidia available in databases (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166320#pone.0166320.t002" target="_blank">Table 2</a>). Black highlights indicate matches with the primer sequences, and asterisks indicate regions of 100% identity for all of the aligned sequences. (B) Agarose gel of SWP-PCR amplicons from EHP and other microsporidia. In addition to the pGEM-SWP plasmid (+ve) and water (-ve), total DNA obtained from EHP-infected shrimp (I) and naïve shrimp (U) were used as controls. PCR amplicons are marked with arrowheads. The 180 bp band is PCR products from residual primers SWP1_R from the first PCR step and primers SWP_2F from the second nested PCR step.</p

Higher sensitivity of first step SWP-PCR compared to first step SSU-PCR.

<p>(A) and (C) show agarose gels of amplicons from the first step PCR reactions, while (B) and (D) show agarose gels of amplicons from the nested step PCR reactions carried out using serial dilutions of the plasmid templates pGEM-SWP and pGEM-SSU, respectively.</p

PCR primers used in this study.

<p>PCR primers used in this study.</p

Small subunit rRNA sequences used for multiple sequence alignment analysis.

<p>Small subunit rRNA sequences used for multiple sequence alignment analysis.</p

Spore wall protein sequences used for multiple sequence alignment analysis.

<p>Spore wall protein sequences used for multiple sequence alignment analysis.</p