The Microsporidian Encephalitozoon Hellem Secretes A Host Nucleus-Targeting Protein (Ehhntp1) to Upregulate Endoplasmic Reticulum-Associated Degradation and Promote Protein Ubiquitination
Authors: Han Yinze , Gao Hailong , Han Bing , Xu Jinzhi , Luo Jian , Yu Bin , Wang Chunxia , Pei Boyan , Chen Jiajing , LI Tian , Zhou Zeyang 
Published: March 2021

Abstract
Background: Microsporidia, a group of obligate intracellular parasites that can infect humans and nearly all animals, have lost the pathways for de novo amino acid, lipid and nucleotide synthesis and instead evolved strategies to manipulate host metabolism and immunity. The endoplasmic reticulum (ER) is a vital organelle for producing and processing proteins and lipids and is often hijacked by intracellular pathogens. However, little is known about how microsporidia modulate host ER pathways. Herein, we identified a secreted protein of Encephalitozoon hellem, EhHNTP1, and characterized its subcellular localization and functions in host cells.Methods: A polyclonal antibody against EhHNTP1 was produced to verify the protein subcellular localization in E. hellem-infected cells using indirect immunofluorescence assay (IFA) and Western blotting. HEK293 cells were transfected with wild-type or mutant EhHNTP1 fused with HA-EGFP, and the impacts on pathogen proliferation, protein subcellular localization and sequence functions were assessed. RNA sequencing of EhHNTP1-transfected cells was conducted to identify differentially expressed genes (DEGs) and pathway responses by bioinformatics analysis mainly with R packages. The DEGs in the transfected cells were experimentally confirmed with RT-qPCR and Western blotting. The regulatory effects of candidate DEGs were analyzed via RNA interference and cell transfection, and the effects were determined with RT-qPCR and Western blotting.Results: EhHNTP1 is secreted into the host nucleus, and its translocation depends on a nuclear localization signal sequence (NLS) at the C-terminus from amino acids 239 to 250. Transfection and overexpression of EhHNTP1 in HEK293 cells significantly promoted pathogen proliferation. RNA-seq of the transfected cells showed that genes involved in ER-associated degradation (ERAD), a quality control mechanism that allows for the targeted degradation of proteins in the ER, were prominently upregulated. Upregulation of the ERAD genes PDIA4, HERP, HSPA5 and Derlin3 determined by RNA-seq data was verified using RT-qPCR and Western blotting. Protein ubiquitination in the transfected cells was then assayed and found to be markedly increased, confirming the activation of ERAD. PDIA4 knockdown with RNAi significantly suppressed the expression of HERP, indicating that PDIA4 is a vital ERAD component exploited by EhHNTP1. Moreover, EhHNTP1ΔHRD, a deletion mutant lacking the histidine-rich domain (HRD) in the C-terminus, predominantly suppressed the upregulation of ERAD genes, indicating that the HRD is essential for EhHNTP1 functions.Conclusion: This study is the first report on a microsporidian secretory protein that targets the host nucleus to upregulate the ERAD pathway and subsequently promote protein ubiquitination. Our work provides new insights into microsporidia-host interactions.