The Microsporidian Encephalitozoon Hellem Secretes A Host Nucleus-Targeting Protein (Ehhntp1) to Upregulate Endoplasmic Reticulum-Associated Degradation and Promote Protein Ubiquitination

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 identied 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 immunouorescence 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 conrmed 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 signicantly 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 veried using RT-qPCR and Western blotting. Protein ubiquitination in the transfected cells was then assayed and found to be markedly increased, conrming the activation of ERAD. PDIA4 knockdown with RNAi signicantly suppressed the expression of HERP, indicating that PDIA4 is a vital ERAD component exploited by EhHNTP1. Moreover, EhHNTP1 ΔHRD , 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 rst 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.


Introduction
Microsporidia are a group of obligate intracellular parasites with a broad range of hosts from invertebrates to vertebrates, including humans. More than 200 genera and 1400 species have been identi ed [1]. Encephalitozoon is recognized as a common mammal-infecting species [1,2] that causes comprehensive immune responses [3]. Having emerged as important opportunistic pathogens in humans, microsporidia have been identi ed in patients with AIDS, organ transplant recipients, aged individuals and children [4,5,6,7]. Microsporidia have undergone extreme genomic compaction and reduction and lost canonical mitochondria and the genes for many metabolic pathways, such as the tricarboxylic acid cycle, and the de novo synthesis of nucleotides and amino acids [8,9,10]. Instead, microsporidia evolved strategies to manipulate pathways and rely on host nutrients, as well as escape host immunity [3,11].
Thus, ERAD maintains protein quality by degrading proteins that fail to attain their native conformation due to mutations, errors in transcription or translation, or ine cient assembly into their native oligomeric complexes. ERAD has also been found to play important roles during pathogen infection. Certain types of pathogens, such as viruses, bacteria and protozoa, preferably hijack the host ERAD machinery to support their requirements [18,19,20,21], which generally includes the following three steps. First, proteolytic secretory and membrane proteins involved in the immune response, including MHC class and CD4, are exploited by pathogens to evade host immunity [22,23]. Second, ERAD is hijacked as transportation machinery from the ER to the cytosol for the invasion of pathogens [24]. Third, ERAD is utilized by pathogens to favor their nutritional requirements for virulence [25].
To subvert and manipulate host pathways, pathogens usually secrete proteins as virulent factors into host organelles [26,27,28,29,30]. For example, pathogen proteins secreted into the host nucleus play important roles in regulating immunity, proliferation and apoptosis [31,32,33]. Here, for the rst time, we report that a secreted protein of E. hellem, EhHNTP1, is delivered into the host nucleus and disturbs the host ERAD pathway and protein degradation.

Preparation of E. hellem spores
Con uent monolayer RK13 cells were infected with E. hellem spores and maintained in MEM with penicillin-streptomycin supplemented with 10% FBS. Spores were collected from the culture medium, puri ed with 75% Percoll by centrifugation at 1000 rpm for 10 min, and washed three times with sterile distilled water by centrifugation at 1000 rpm for 5 min. The puri ed spores were then counted and stored in sterile distilled water at 4°C.

Preparation of recombinant EhHNTP1 and antiserum
The coding sequence (CDS) of EhHNTP1 was ampli ed from E. hellem genomic DNA (gDNA) using PrimeSTAR Max Premix DNA polymerase with the forward primer 5'-CCATGGCTGATATCGGATCCGAATTCATGTCAACGTTTGTGGGTGC-3' and the reverse primer 5'-GTGCTCGAGTGCGGCCGCAAGCTTTCTTTATAGACGGTAAGTGC-3'. The PCR products were inserted into the pET32a (+) vector, which contains a hexa-histidine tag (His), using homologous recombinase (Yeasen) according to the instruction manual. The recombinant plasmid pET32-EhHNTP1-6×His was veri ed by sequencing. Escherichia coli Transetta (DE3) cells were transformed with the plasmid and cultured at 37°C overnight in 5 mL of Luria-Bertani (LB) medium (10% tryptone, 5% yeast extract, 10% sodium chloride) with 100 µg/mL ampicillin, inoculated into 400 mL of LB medium and cultured to an OD 600 = 0.6. EhHNTP1 was then induced for 20 h at 16°C by the addition of 0.5 mM isopropyl-Dthiogalactoside to the culture medium. Subsequently, the bacterial cells were harvested by centrifugation HFF cells infected with E. hellem for ve days were harvested to extract nuclear proteins using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime). The extracted proteins were boiled at 100°C upon the addition of protein loading buffer, separated by SDS-PAGE and transferred to polyvinylidene di uoride (PVDF) membranes. The membranes were blocked with western blocking buffer (2 g of skim milk in 40 mL of Tris-buffered saline supplemented with 0.05% Tween 20 (TBST)) for 2 h at room temperature (RT). Then, the membranes were incubated with anti-EhHNTP1 diluted 1:150 in blocking buffer for 2 hours at RT and washed three times with TBST. The blots were then incubated with goat antimouse IgG conjugated to horseradish peroxidase (HRP) or goat anti-rabbit IgG conjugated to HRP. After three washes, the membranes were exposed with an ECL Western blot detection kit (Thermo Fisher Scienti c) and imaged with an Azure Biosystems C300 imaging system.

EhHNTP1 transfection of HEK293 cells
The CDSs for EhHNTP1, EhHNTP1 Δ1-16 , EhHNTP1 Δ239-278 and EhHNTP1 ΔHRD were ampli ed from E. hellem gDNA with speci c primers (Supplementary Table 1). DNA fragments conjugated to an HA tag and EGFP were inserted into the pCDNA3.0 plasmid and transformed into E. coli DH5α for replication. The replicated plasmid DNA was extracted using an Endofree Minimal Plasmid Kit (Tiangen) and transfected into HEK293 cells using a Lipofectamine 3000 kit (Invitrogen). After 48 hours, the transfected cells were harvested to analyze protein expression and subcellular localization.

Indirect immuno uorescence assay (IFA)
Infected HEK293 cells were xed with 4% paraformaldehyde for 10 min, washed three times with PBS, and permeabilized in 0.1% Triton X-100 for 30 min. The cells were then blocked in PBST containing 10% goat serum and 5% BSA for 1 h at 37°C and washed three times with PBS. Samples were incubated with anti-EhHNTP1 (diluted 1:150 in blocking buffer) or negative serum (diluted 1:150 in blocking buffer) for 1 h at RT, washed three times, and then incubated with secondary anti-mouse IgG conjugated to Alexa Fluor 488 (diluted 1:1000 in PBS) for 1 hour. Both host cells and pathogenic nuclei were stained with 4,6diamidino-2-phenylindole (DAPI) for 30 min at RT and washed three times. The samples were observed and photographed using an Olympus FV1200 laser scanning confocal microscope.

RNA-seq analysis of EhHNTP1-transfected HEK293 cells
The transcriptomic responses of HEK293 cells transfected with HA-EGFP and EhHNTP1-HA-EGFP were investigated with RNA-seq, with three replicates of each group. RNA samples were extracted and assessed using a Nanodrop 2100 and Agilent 2000 system. Libraries were constructed and sequenced on the Illumina NovaSeq platform. Clean sequencing reads were mapped to the human genome (ftp://ftp.ensembl.org/pub/release-95/) using HISAT2 [34] and assembled with StringTie [35]. Fragments per kilobase per million (FPKM) values were calculated and used to compare gene expression levels. Differentially expressed genes (DEGs) between the EhHNTP1-HA-EGFP-and HA-EGFP-transfected cells were analyzed using DEseq2 [36]. The DEGs were ltered with the thresholds of a P-value < 0.01 and a fold change ≥ 2, and DEGs found in at least in two replicates were considered credible. KEGG enrichment analysis of the DEGs was performed using clusterPro ler [37], which is an R package for comparing biological themes among gene clusters. Pathways enriched in at least two genes and a P-value < 0.05 were considered signi cant.

Real time quantitative PCR (RT-qPCR)
Total RNA was extracted from RK13 cells transfected with EhHNTP1 using the E.Z.N.A.® Total RNA Kit II (OMEGA, China) according to the manufacturer's instructions. cDNA was synthesized with 1 µg of total RNA using the Hifair® III 1st Strand cDNA Synthesis Kit (gDNA digester plus) (Yeasen). RT-qPCR was performed using the primers for candidate genes shown in Supplementary Table 2. The transcription level was elevated by the 2 −ΔΔt values with three replicates. All statistical t-tests were conducted using GraphPad Prism 6.0 for two-tailed comparison tests, the results of which with a P-value < 0.05 were considered signi cant.
To assess the growth of E. hellem in cell culture, quantitative PCR was performed with β-tubulin primers (F: TGAAGATGAGCAATCCAGGGTA, R: TAGCAATCAGGGGTGCAAAT). The gDNA of EhHNTP1-and EGFP-transfected HEK293 cells infected with E. hellem was extracted using an E.Z.N.A.™ Tissue DNA kit (OMEGA, China) according to the manufacturer's instructions.

Sequence features of EhHNTP1
EhHNTP1, whose locus name is EHEL_071080 (GenBank accession: AFM98634.1), was annotated as a hypothetical protein and is composed of 278 amino acids. Predictions with SignalP 5.0 [38] and NLS Mapper [39] showed that EhHNTP1 contains a signal peptide (SP) from amino acids 1 to 16 and a nuclear localization signal sequence (NLS) from amino acids 239 to 250, respectively, indicating that EhHNTP1 is a secreted protein that probably targets the host nucleus (Fig. 1). In addition, the C-terminal region of EhHNTP1 encodes a histidine-rich domain (HRD) from amino acids 258 to 266, which was suggested to be functional in transcriptional regulation in the nucleus. Multiple sequence alignment analysis demonstrated that EhHNTP1 is highly conserved with homologs among Encephalitozoon species (Fig. 1), suggesting important functions of this protein in Encephalitozoon.

The expression pro le and subcellular localization of EhHNTP1
To characterize the subcellular localization of EhHNTP1 in infected cells, recombinant EhHNTP1 was expressed in E. coli and puri ed for the immunization of mice and production of a mouse polyclonal antibody (anti-EhHNTP1) (Fig. 2a), which was then used to examine the localization of EhHNTP1-infected cells using an IFA. EhHNTP1 was found to translocate into the nuclei of infected cells (Fig. 2b), suggesting that EhHNTP1 is a secreted protein targeting the host nucleus. Therefore, we renamed it a host nucleus-targeting protein (EhHNTP1). Moreover, in parasitophorous vacuoles (PVs), EhHNTP1 was detected in only meronts but not spores, indicating that this protein is mainly synthesized and secreted by meronts and likely plays important roles during proliferation (Fig. 2b).

The NLS is required for the translocation of EhHNTP1 into the host nucleus
The NLS is an essential sequence for the targeting of some proteins to the nucleus [40,41,42]. The predicted NLS of EhHNTP1 is probably necessary for nuclear localization. Therefore, EhHNTP1 deletion mutants lacking the SP (EhHNTP1 Δ1-16 -HA-EGFP) and NLS (EhHNTP1 Δ239-278 -HA-EGFP) were constructed and expressed in HEK293 cells. As detected by IFA and Western blotting, EhHNTP1-HA-EGFP and EhHNTP1 Δ1-16 -HA-EGFP were found in the nucleus (Fig. 3, Supplementary Fig. 2), while EhHNTP1 Δ239-278 -HA-EGFP was present in only the cytoplasm (Fig. 3), suggesting that the NLS is indispensable for the nuclear targeting of EhHNTP1.

EhHNTP1 promotes E. hellem proliferation
The expression pro le of EhHNTP1 was investigated in E. hellem-infected HEK293 cells using RT-qPCR. The results showed that EhHNTP1 was expressed from 12 hours postinfection (hpi) and highly expressed from 36 hpi (Fig. 4a), at which time the pathogens were at the meront stage. In HEK293 cells transfected with EhHNTP1-HA-EGFP and HA-EGFP (control), the pathogen load was determined and indicated by the copy number of E. hellem β-tubulin. The overexpression of EhHNTP1 signi cantly promoted the proliferation of E. hellem (Fig. 4b). These results indicate that EhHNTP1 is vital to pathogen growth.

EhHNTP1 activates the host ERAD response
Being secreted into the host nucleus, EhHNTP1 probably regulates host gene expression. Thus, the gene expression levels of HEK293 cells transfected with EhHNTP1-HA-EGFP and HA-EGFP (control) were determined with RNA-seq ( Supplementary Fig. 3). As shown by the results, a total of 82 DEGs, including 41 upregulated genes and 41 downregulated genes, were detected (Supplementary Table 3). KEGG enrichment analysis of the DEGs showed that the ERAD pathway was signi cantly enriched in the DEGs, including four upregulated genes, PDIA4, HERP, HSPA5 and DERL3, which are vital components in ERAD (Fig. 5). The upregulation of these genes was veri ed by RT-qPCR and Western blotting (Fig. 6a and 6b). These results indicated that EhHNTP1 is secreted into the host nucleus and dysregulates the ERAD pathway. ERAD is vital for protein homeostasis due to the degradation of misfolded and unfolded proteins in the ER. In this process, substrate proteins undergo recognition, dislocation, ubiquitination, translocation from the ER, and then degradation by the proteasome. Hence, we checked protein ubiquitination using Western blotting with anti-ubiquitin and found it to be increased in EhHNTP1transfected HEK293 cells (Fig. 6c).
Protein disul de isomerase (PDI), most abundant ER protein, is responsible for the formation, breakage and rearrangement of protein disul de bonds and also helps with the binding of misfolded proteins for subsequent degradation [43,44]. PDIA4 is one of the largest members of the PDI family and acts as an inducer of the ER stress response by forming a chaperone complex with other proteins that binds unfolded protein substrates [45]. In EhHNTP1-transfected HEK293 cells, both PDIA4 expression and protein ubiquitination were increased (Fig. 6d and 6e), suggesting that EhHNTP1 induces host ERAD via PDIA4. For further validation, HEK293 cells were transfected with PDIA4-FLAG, and protein ubiquitination was found to be increased upon the overexpression of PDIA4 (Fig. 6e). Moreover, the knockdown of PDIA4 with RNAi suppressed HERP (Fig. 6f), which is also an important component of the ERAD pathway, revealing that PDIA4 is a key factor hijacked by microsporidia to modulate host ERAD via EhHNTP1.

HRD is essential for the regulatory function of EhHNTP1
The HRD is conserved among homologs of EhHNTP1 in Encephalitozoon species, indicating its important function for the protein. The HRD identi ed in vertebrate cyclin T1 was proven to markedly enhance the binding of positive transcription elongation factors to the C-terminal domain (CTD) of the RPB1 subunit of human RNA polymerase II, leading to the hyperphosphorylation of the CTD, which is essential for transcriptional elongation and mRNA processing [46]. This suggests that the localization of EhHNTP1 in the host nucleus probably promotes host gene expression with an HRD-dependent mechanism. Therefore, we constructed EhHNTP1 ΔHRD -HA-EGFP-transfected HEK293 cells and checked the localization of the mutant and ERAD responses. EhHNTP1 ΔHRD -HA-EGFP showed markedly increased aggregation and formed signi cantly larger multimers in the nucleus than EhHNTP1-HA-EGFP (Fig. 7a). As determined by RT-qPCR, the expression levels of PDIA4, HSPA5 and HERP were signi cantly decreased in EhHNTP1 ΔHRD -HA-EGFP-transfected cells (Fig. 7b), indicating that the HRD is essential for EhHNTP1 in activating host ERAD.

Discussion
ERAD is a principal quality control mechanism responsible for targeting native, misfolded and unfolded proteins for dislocation across the ER membrane and proteasomal degradation and plays vital roles in multiple cellular processes and functions [13,47]. Thus, by modulating ERAD, pathogens broadly affect host cell physiology. Studies have revealed that some intracellular pathogens, including viruses and bacteria, may dysregulate ERAD to enhance their chances of survival in the host [20,25,47]. For example, Orientia tsutsugamushi, an obligate intracellular bacterial pathogen that is auxotrophic for aromatic amino acids and histidine, can induce the host unfolded protein response (UPR) and promote ERAD to seize amino acids during early-stage growth [25]. This implies that microsporidia may obtain host amino acids with the same strategy because these pathogens lost the genes for the de novo synthesis of amino acids. On the other hand, hijacking ERAD for proteolytic suppression of immune proteins is also a strategy for certain pathogens [18]. The observed increases in protein ubiquitination and degradation (Fig. 6c) suggest that the dysregulation of ERAD may help with the immune evasion of microsporidia. Therefore, determining the ubiquitylated proteins in transfected cells would help further understanding of the functions and regulation of EhHNTP1. The UPR is a cytoprotective process that promotes ERAD [17,44,47,48], suggesting that EhHNTP1 may promote the ERAD machinery by activating the UPR. Through our RNA-seq data, however, we found no DEGs involved in the UPR (Supplementary Table 3), indicating that EhHNTP1 probably dysregulates ERAD through other pathways.
Our study revealed that EhHNTP1 is a secreted protein that targets the host nucleus. A previous study, however, showed that this protein localizes on the tip of the polar tube and interacts with host transferrin receptor 1 (TfR1) on the membrane, demonstrating its important roles during infection [49]. Therefore, EhHNTP1 is likely a multifunctional protein that plays roles in mediating invasion of the polar tube and targeting the host nucleus to modulate host gene expression. Both studies provide insights into the new functions of this secreted protein conserved in Encephalitozoon whose functions, however, require indepth study. Regarding the functions of EhHNTP1 in the host nucleus, it is necessary to determine how EhHNTP1 upregulates ERAD genes, what proteins it interacts with, what proteins are degraded, and what downstream pathways are regulated.
In summary, we rst characterized a microsporidian protein targeting the host nucleus that upregulates the expression of genes involved in ERAD and subsequently increases protein ubiquitination (Fig. 8). This work provides a new viewpoint for an in-depth understanding of the mechanisms with which microsporidia and hosts interact.

Declarations Acknowledgments
The authors would like to thank all the authors who published the manuscripts included in this work.
Authors' contributions TL, ZYZ and BH conceived and designed the experiments; YZH, BH, JZX, JL, BY, CXW, BYP and JJC contributed experimental reagents, materials and analytical tools; YZH and HLG performed the experiments; YZH and TL analyzed the data; TL and YZH developed the manuscript; and TL, BH, YZH and ZYZ reviewed the manuscript. All authors read and approved the nal manuscript.

Funding
This work was supported by grants from the National Natural Science Foundation of China [31772678, 31770159 and 31472151] and the Natural Science Foundation of Chongqing, China (cstc2019yszx-jcyjX0010).

Availability of data and materials
Data are available in Additional le 1: Tables S1-S2 and Additional le 2: Table S3.
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