Microsporidian Encephalitozoon hellem secretes EhPTP4 to regulate host endoplasmic reticulum-associated degradation

Abstract:

[Objective] Microsporidia are a group of obligate intracellular parasites that can infect humans and nearly all animals. Here, we studied the polar tube protein 4 of Encephalitozoon hellem (EhPTP4) on its subcellular localization and functions as a potential secretory virulence factor in host cells.[Methods] A polyclonal antibody against EhPTP4 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 EhPTP4 fused with HA-EGFP, and the impacts on pathogen proliferation, protein subcellular localization and sequence functions were assessed. RNA sequencing of EhPTP4-transfected cells was conducted to identify differentially expressed genes (DEGs) and pathway responses. The regulatory effects of candidate DEGs were analyzed via RNAi and cell transfection, and the effects were determined with RT-qPCR and Western blotting.[Results] EhPTP4 contains a signal peptide at the N-terminal, a nuclear localization sequence (NLS) and a histidine-rich domain (HRD) at the C-terminal. In the infected and transfected cells, EhPTP4 was secreted into the host nucleus. Transfection and overexpression of EhPTP4 in HEK293 cells significantly promoted pathogen proliferation. RNA-seq of the transfected cells showed that genes involved in endoplasmic reticulum (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 of 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 vital for the modulation by EhPTP4. Moreover, EhPTP4^ΔHRD, a deletion mutant lacking the HRD, could not cause the upregulation of ERAD genes, indicating that the HRD is essential for the function of EhPTP4.[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.