Microsporidian EnP1 alters host cell H2B monoubiquitination and prevents ferroptosis facilitating microsporidia survival

Abstract

Microsporidia are intracellular eukaryotic pathogens that pose a substantial threat to immunocompromised hosts. The way these pathogens manipulate host cells during infection remains poorly understood. Using a proximity biotinylation strategy we established that microsporidian EnP1 is a nucleus-targeted effector that modifies the host cell environment. EnP1's translocation to the host nucleus is meditated by nuclear localization signals (NLSs). In the nucleus, EnP1 interacts with host histone H2B. This interaction disrupts H2B monoubiquitination (H2Bub), subsequently impacting p53 expression. Crucially, this inhibition of p53 weakens its control over the downstream target gene SLC7A11, enhancing the host cell's resilience against ferroptosis during microsporidian infection. This favorable condition promotes the proliferation of microsporidia within the host cell. These findings shed light on the molecular mechanisms by which microsporidia modify their host cells to facilitate their survival.

Keywords: EnP1; ferroptosis; histone H2B; microsporidia; nucleus-targeted effector.

Fig. 1.

EnP1 is identified as a potential nucleus-targeted effector of microsporidia by proximity labeling approach. (A) Schematic representing plasmid transfection and nuclear accumulation of 3HA-TurboID-NLS proteins and the IFA detection of the heterologous expression of 3HA-TurboID-NLS in HEK293T cells using αHA monoclonal antibody (mAb). (B) Schematic representing nuclear accumulation of biotinylated proteins after supplementing with biotin, and the IFA detection of the biotinylated proteins in HEK293T cells using Alexa Fluor 488-conjugated streptavidin. (A) and (B), Negative control: Cells transfected with pcDNA3.1 vector. (Scale bars, 20 μm.) (C) Schematic representation of the biotin-based proximity labeling approach that identified nucleus-targeted effectors of microsporidia. (D) Immunoblot detection of biotinylated proteins in host cell nucleus purified by streptavidin beads. M, molecular mass marker lane. (E) Mass spectrometry revealed that both EnP1 and EnP2 were identified. Both proteins exhibit characteristics of potential effectors targeted to the nucleus, possessing both the SP and the nuclear localization signal (NLS). The diagram illustrated the probability for Signal and NLS predictors. EnP1 harbors two NLS sequences, with probability of 0.56 and 0.82, respectively. (F) and (G) Immunoblot analysis of EnP1 and EnP2 heterologous expression in HEK293T cells. (H) IFA of the EnP1 and EnP2 heterologous expression in HEK293T cells. (Scale bars, 20 μm.)

Fig. 2.

EnP1 is secreted into the host nucleus during infection and promoted microsporidian proliferation. (A) EnP1 amino acid sequence analysis. SP sequences are highlighted in red and NLS sequences were highlighted in yellow (NLS^156–221) and green (NLS^312–327). (B) YSST assay of EnP1 SP. CMD-W plates were used to select yeast strain YTK12 carrying the pSUC2 vector. YPRAA plates were used to indicate invertase secretion. (C) Immunoblot of secreted EnP1 in cytosol protein extractions (CPE) and nuclear protein extractions (NPE) of infected cells using EnP1 mPcAb. (D) IFA demonstrating localization of EnP1 in infected cells using EnP1 mPcAb. N, host cell nucleus; PV, parasitophorous vacuole. (Scale bars, 5 μm.) (E) Immunoblot of EnP1ΔNLS^156–221 (lane 1), EnP1ΔNLS^312–327 (lane 2), and EnP1ΔNLS (lane 3) in CPE and NPE respectively. (F) IFA of the localization of the exogenously expressed EnP1ΔNLS^156–221, EnP1ΔNLS^312–327, and EnP1ΔNLS in HEK293T cells stained with αFlag antibody. (Scale bars, 20 μm.) (G) Parasite load of microsporidia in HEK293T cells expressing EnP1 and EnP1ΔNLS at 48 hpi. Pathogen copy number was determined by quantitative PCR. (H and I) The effect of EnP1 and EnP1ΔNLS expression in HFF cells on Eh infection was assessed by counting PVs number on each slide (H) and the total area of PVs within the infected cells (I) at 48 hpi. *P < 0.05, ***P < 0.001, ns = not significant. Tubulin and H2B were used as the control of CPE and NPE respectively.

Fig. 3.

EnP1 exhibits specific binding affinity toward H2B, particularly with H2B1K. (A) Silver staining of immunoprecipitation (IP) samples precipitated by αHA magnetic beads from EnP1-HA expressing host cells. Negative control: Cells transfected with an empty vector. Arrowheads indicate the bands of interest. (B) IFA colocalization of EnP1 and H2B in the nucleus of HEK293T cells which were cotransfected with pcDNA3.1::EnP1-Flag and pcDNA3.1-3HA::H2B. αFlag (green) and αHA (red) mAb were used according to the procedures outlined in the Materials and Methods section. The merged image demonstrates the colocalization of EnP1 and H2B in the cell nucleus. (Scale bars, 20 μm.) The boxed region represents magnified images. (Scale bars, 5 μm) (C) Immunoblot of EnP1 in samples precipitated by H2B mAb from cells expressing EnP1. (D) His-tag pull-down demonstrating detection of H2B from the NPE of cells expressing H2B-HA using EnP1-His conjugated Ni-NTA agarose beads. αHA mAb was used to detect H2B-HA. Negative control: BSA-conjugated Ni-NTA agarose beads. (E) Far-WB of the interaction between EnP1 and H2B. Purified rH2B-Flag-His was subjected to SDS-PAGE, transferred to a PVDF membrane (lane 1), and then purified rEnP1-HA-His was incubated with the PVDF membrane, and the EnP1 that bound to H2B was detected by an αHA antibody (lane 2). Negative control: BSA (lane 3). (F) Immunoblot of EnP1 or EnP1S157A in IP samples precipitated by αHA magnetic beads from host cells expressing Flag-EnP1 and HA-H2B or Flag-EnP1S157A and HA-H2B. Immunoblot with αHA (H2B) and αFlag antibodies (EnP1 or EnP1S157A). (G) Quantitative analysis of the interaction between EnP1 and H2B family using Far-WB and recombinant proteins (expressed in E. coli). The purified rH2B variants (rH2B1A, rH2B1B, rH2B1D, rH2B1O, and rH2B1K) fused with Flag tag were loaded on the SDS-page gel and transferred to a PVDF membrane. The purified rEnP1-HA-His was then incubated with the PVDF membrane and the EnP1 that bound to H2B was detected by an αHA antibody. Negative control: BSA. H2B variants were detected by αFlag antibody.

Fig. 4.

EnP1 promotes microsporidian proliferation by suppressing H2B monoubiquitination. (A) H2Bub detection in host cell nucleus following EnP1 expression. Negative control: Cells expressing GFP. (B) Relative band intensities quantification using ImageJ based on three independent experiments, including the one shown in (A). (C) H2Bub in host cell nucleus after Eh infection. Negative control: Uninfected cells. (D) Relative band intensities quantification using ImageJ based on three independent experiments, including the one shown in (C). (E) Immunoblot of H2Bub in siRNF20 treated host cells. Negative control: Untreated cells and control siRNA. (F) Immunoblot of H2Bub in RNF20 (RNF20OE) expressed cells. (G) Immunoblot of H2Bub in the nucleus of host cells expressing H2BK120R. Negative control: Cells expressing wild-type H2B (H2BWT). (H–J) Effect of RNF20 knockdown (H), RNF20 overexpression (RNF20OE) (I), or H2BK120R expression (J) in cells on microsporidian proliferation within host cells. Pathogen copy number was determined by quantitative PCR. (K) Immunoblot of H2Bub in host cells expressing EnP1 and GFP with or without RNF20 overexpression. (L) Immunoblot of RNF20 in IP samples precipitated by αFlag magnetic beads from host cells expressing Flag-EnP1 and HA-RNF20. (M) Far-WB of the binding of H2BK120R and EnP1. Negative control: H2BWT. (N) Immunoblot of RNF20 in IP sample precipitated by αH2B antibodies from host cells expressing both Flag-EnP1 and HA-RNF20. Negative control: IP sample precipitated from cells expressing RNF20-HA. (O) Relative band intensities quantification using ImageJ based on three independent experiments, including the one shown in (N). (P) Schematic representation of EnP1 regulating host H2Bub. Immunoblot with αHA (RNF20) and αFlag (EnP1) antibodies in (L) and (N). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5.

EnP1 suppresses host ferroptosis by enhancing the expression of SLC7A11. (A) The heat map of clustering between the EnP1 expression group and control group demonstrates that the samples are well clustered and significantly different from each other. Upregulated and downregulated genes are colored in red and blue, respectively. (B) Differential gene KEGG enrichment analysis focused on pathways related to the expression of EnP1. (C) Expression difference volcano plot, which demonstrates that SLC7A11 was significantly up-regulated in the EnP1 group. (D) qRT-PCR analysis of the change in SLC7A11 expression in host cells expressing EnP1. Negative control: Cells expressing GFP. (E) Immunoblot of SLC7A11 expression in host cells expressing either EnP1 or GFP. (F) qRT-PCR analysis of the SLC7A11 expression in host cells infected with Eh. Negative control: Noninfected cells. (G) Immunoblot analysis of SLC7A11 expression in host cells postinfection with Eh compared to noninfected cells. (H) Immunoblot of the relative protein levels of EnP1 and SLC7A11 at different time points (0, 24, 48, 72 hpi) during host cell infection with Eh. (I) Immunoblot of SLC7A11 in host cells transfected with siSLC7A11. Negative controls: Untreated cells and control siRNA. (J) Effect of SLC7A11 knockdown on the proliferation of microsporidia in host cells expressing EnP1 or GFP. Pathogen copy number was determined by quantitative PCR. (K) Host cells expressing EnP1 or GFP were treated with increasing concentrations of erastin and their cell viability was measured using CCK-8. (L) Host cells expressing EnP1 or GFP were treated with increasing concentrations of erastin and the LDH cytotoxicity was assayed. (M) Measurement of the ratio of GSH to GSSG in host cells expressing EnP1 and control host cells after induction with 20 μM and 25 μM erastin. (N) Quantification of the number of microsporidia within host cells after treatment with 10 μM Fer-1. Pathogen copy number was determined by quantitative PCR. (O) Quantification of the number of microsporidia within host cells after treatment with 20 μM erastin. The pathogen copy number was determined by quantitative PCR. (P) Schematic representation of EnP1 suppressing host ferroptosis by enhancing the expression of SLC7A11. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 6.

EnP1 negatively regulates p53 expression by inhibiting H2Bub. (A) Schematic representation of the p53 tumor suppressor enhancing ferroptosis by transcriptional suppression of SLC7A11 in tumor cells (40). (B) Immunoblot of p53 protein expression in cells expressing EnP1 or GFP. (C) Immunoblot of p53 protein expression in Eh cells and uninfected cells. (D) Detection of TP53 transcription level in host cells transfected with siRNF20 and siNC. (E) Immunoblot of p53 protein expression in host cells transfected with siRNF20 and siNC. (F) Detection of TP53 transcription level in cells overexpressing RNF20 (RNF20OE) and untreated cells. (G) Immunoblot of p53 protein expression in cells overexpressing RNF20 (RNF20OE) and untreated cells. (H) Detection of SLC7A11 transcription level in host cells transfected with sip53. (I) Immunoblot of SLC7A11 protein expression in host cells transfected with sip53. Negative controls of (H) and (I): Untreated cells and control siRNA. (J) Detection of SLC7A11 transcription level in cells overexpressing p53 (p53OE) and untreated cells. (K) Immunoblot of SLC7A11 protein expression in cells overexpressing p53 (p53OE) and untreated cells. (L) Cell viability of host cells expressing GFP, EnP1, and EnP1 with p53OE was measured using CCK-8 after induction with 20 μM and 25 μM erastin. (M) The LDH cytotoxicity of host cells expressing GFP, EnP1, and EnP1 with p53OE was assayed after induction with 20 μM and 25 μM erastin. (N) Measurement of the ratio of GSH to GSSG in host cells expressing GFP, EnP1, and EnP1 with p53OE after induction with 20 μM and 25 μM erastin. (O) Effect of p53 knockdown on microsporidian proliferation within these host cells. Pathogen copy number was determined by quantitative PCR. (P) Effect of p53 overexpression (p53OE) on microsporidian proliferation within these host cells. Pathogen copy number was determined by quantitative PCR. (Q) Schematic representation of EnP1 regulating host ferroptosis. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 7.

The model of EnP1 modules host ferroptosis. Schematic diagram of the mechanism by which the microsporidia effector protein EnP1 enters the nucleus to regulate host cell functions and promote the proliferation of microsporidia.