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. Author manuscript; available in PMC: 2019 Feb 15.
Published in final edited form as: Vet Parasitol. 2017 Dec 20;251:27–33. doi: 10.1016/j.vetpar.2017.12.015

Nuclear Delivery of Parasite Cdg2_FLc_0220 RNA Transcript to Epithelial Cells during Cryptosporidium parvum Infection Modulates Host Gene Transcription

Guang-Hui Zhao 1,2, Ai-Yu Gong 2, Yang Wang 2, Xin-Tian Zhang 2, Min Li 2, Nicholas W Mathy 2, Xian-Ming Chen 2,*
PMCID: PMC5808497  NIHMSID: NIHMS931838  PMID: 29426472

Abstract

Intestinal infection by the zoonotic protozoan, Cryptosporidium parvum, causes significant alterations in the gene expression profile in host epithelial cells. The molecular mechanisms of how C. parvum may modulate host cell gene transcription and the pathological significance of such alterations are largely unclear. Previous studies demonstrate that a panel of parasite RNA transcripts are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of intestinal cryptosporidiosis, in this study, we analyzed the impact of host delivery of C. parvum Cdg2_FLc_0220 RNA transcript on host gene expression profile. We found that alterations in host gene expression profile following C. parvum infection were partially associated with the nuclear delivery of Cdg2_FLc_0220. Specifically, we identified a total of 46 overlapping upregulated genes and 8 overlapping downregulated genes in infected cells and cells transfected with Full-Cdg2_FLc_0220. Trans-suppression of the DAZ interacting zinc finger protein 1 like (DZIP1L) gene, the top overlapping downregulated gene in host cells following C. parvum infection and cells transfected with Full-Cdg2_FLc_0220, was mediated by G9a, independent of PRDM1. Cdg2_FLc_0220-mediated trans-suppression of the DZIP1L gene was independent of H3K9 and H3K27 methylation. Data from this study provide additional evidence that delivery of C. parvum Cdg2_FLc_0220 RNA transcript in infected epithelial cells modulates the transcription of host genes, contributing to the alterations in the gene expression profile in host epithelial cells during C. parvum infection.

Keywords: Cryptosporidium, Intestinal epithelium, DZIP1L, Gene transcription, Epithelial homeostasis

1. Introduction

Cryptosporidium is an important protozoan diarrheal pathogen in animals and humans (Checkley et al., 2015, Certad et al., 2017). Persistent watery diarrhea caused by Cryptosporidium infection has been reported as potentially fetal in youth, the in immunocompromised (e.g., HIV/AIDS), and transplant recipients (Borad and Ward, 2010; Fishman et al., 2011; Acikgoz et al., 2012). Investigations on diarrheal etiologies in children also showed that Cryptosporidium is responsible for 15–25% of diarrheal cases (Chen et al., 2002; Checkley et al., 2015). Although asymptomatic infection of Cryptosporidium was observed in the majority of cases in humans and animals, colonization of this parasite can damage the intestinal barriers, affecting nutrition absorption, possibly causing persistent retardation of growth and impairing the immune response of host (Guerrant et al., 1999; Mondal et al., 2009; Squire and Ryan, 2017). More importantly, uneliminated oocysts excreted from these ignored hosts into environment could spread the infection to other hosts and would be an important source of waterborne outbreak of cryptosporidiosis (Checkley et al., 2015). At least 163 outbreaks of waterborne diseases were caused by Cryptosporidium infection (Karanis et al., 2007), and this protist has been listed as an indicator for water quality in the USA, UK, Australia and China. However, only one drug, nitazoxanide, has been approved by the American Food and Drug Administration (FDA) to treat cryptosporidiosis, and is efficacious in only 56–96% of immunocompetent hosts, but lacks efficacy in cryptosporidiosis patients with advanced AIDS (Rossignol et al., 2001; Amadi et al., 2002, 2009; Checkley et al., 2015).

Considering the close relationship between the severity of cryptosporidiosis and host status (e.g., immunity, nutrition, and age), exploring the mechanism of interaction between Cryptosporidium and host is key to developing resolution strategies for controlling Cryptosporidium. After internalization and residence in mature parasitophorous vacuoles at the apex of the host cells (Marcial and Madara, 1986), a direct connection (the feeder organelle) is formed between Cryptosporidium and host cell cytoplasm at the host cell-parasite interfaces, and is an important structure for regulating transportation of nutrition, molecular effectors and drugs (Marcial and Madara, 1986; Tzipori and Griffiths, 1998; Perkins et al., 1999; Huang et al., 2004; O’Hara and Chen, 2011; Wang et al., 2017a, b). Genomic and transcriptomic analyses showed that various protein-coding genes encoded in the Cryptosporidium parvum genome were released and involved in host-parasite interaction, and parasite intracellular development (Abrahamsen et al., 2004; Huang et al., 2004; Puiu et al., 2004; Wastling et al., 2009; Certad et al., 2017). A comprehensive transcriptomic analysis of the intracellular stages of C. parvum revealed a cascade of gene expression consistent with unique biologies for each developmental stage following parasitization of intestinal epithelial cells; many of these putative developmental stage-specific genes are of unknown function (Mauzy et al., 2012). In 2011, one hundred eighteen “orphan” RNA transcripts were identified in the sporozoites of C. parvum (Yamagishi et al., 2011). Our previous study indicated that several of them could be selectively delivered into the nuclei of infected host epithelial cells (Wang et al., 2017a). Further study revealed that nuclear delivery of parasite Cdg7_FLc_0990 RNA (GeneBank ID: FX115678.1) (Yamagishi et al., 2011) into infected intestinal epithelial cells suppresses transcription of the LRP5, SLC7A8, and IL33 genes through histone modification-mediated epigenetic mechanisms (Wang et al., 2017b). The parasite Cdg2_FLc_0220 RNA, a transcript from a hypothetical protein gene (GeneBank ID: FX115592.1) located at the Chromosome 2 (Yamagishi et al., 2011), is delivered into the nuclei of infected host epithelial cells (Wang et al., 2017a). In the present study, the distinct role of nuclear delivery of Cdg2_FLc_0220 RNA in modulating transcription of host genes, such as the DAZ interacting zinc finger protein 1-like (DZIP1L), in intestinal epithelial cells infected with C. parvum was addressed.

2. Materials and methods

2.1 Parasites and in vitro infection model

C. parvum oocysts used in this study were the Iowa isolate purchased from Bunch Grass Farm. Oocysts were firstly treated with 20% sodium hypochlorite at 4°C for 20 min, and then washed twice with Phosphate Buffered Saline (PBS) and RPMI-1640. Viable oocysts were resuspended in RPMI-1640, and respectively used to infect human carcinoma intestinal epithelial HCT-8 cells (ATCC) and non-carcinoma small intestinal epithelial FHs 74 Int cells (INT) (ATCC) with a ratio of oocysts to host cells at 5:1 to 10:1 in the serum free mediums to establish in vitro infection models. Stable HCT-8-G9a−/− cells were generated through transfection of cells with the G9a-CRISPR/Cas9 KO(h) and G9a-HDR plasmids (Santa Cruz), as previously reported (Ming et al., 2017). Cells were washed with PBS to remove oocyst walls, oocysts and free sporozoites, and changed with complete culture medium at 4 h post infection.

2.2 Overexpression of C. parvum Cdg2_FLc_0220

Total RNA was isolated from C. parvum oocyst using Tri-reagent solution (invitrogen) and chloroform/isopropanol. The genomic DNA was removed from total RNA samples by DNase Treatment & Removal (ambion), and treated RNA was reverse transcribed into cDNA with M-MLV (invitrogen). C. parvum Cdg2_FLc_0220 was amplified using primers listed in Table 1, sub-cloned into the pcDNA3.1 vector (invitrogen) and transformed into Escherichia coli DH5α to construct the overexpression plasmid of Cdg2_FLc_0220 (named as Full-Cdg2_FLc_0220). INT (1×105) and HCT-8 (2×106) cells were respectively seeded into 24-well plates. After cultured for 24 h at 37°C and 5% CO2, each well with cells was transfected with 1 μg Full-Cdg2_FLc_0220 or empty vector pcDNA3.1 using the Lipofectamine 2000 Reagent (Invitrogen) and Opti-MEM (Gibco). Cells were changed with complete mediums and collected at 24 h and 48 h after transfection for further study.

2.3 Microarray analysis

The Agilent SurePrint G3 Human Gene Expression Microarray and service to process the samples were applied to genome-wide analysis. Briefly, INT cells were grown to 80% confluence and exposed to C. parvum infection or Full-Cdg2_FLc_0220transfection, respectively. Total RNA of harvested cells was isolated with the RNeasy Mini kit (Qiagen). A mixture of equal amounts of total RNAs from each group was used as the reference pool. A total of 2 μg RNA from each sample was then labeled with the Agilent Gene Expression Hybridization Kit (Agilent). After hybridization, the slides were scanned with the Agilent Microarray Scanner (Agilent). The Feature Extraction software (version10.7.1.1, Agilent Technologies) was used to analyze array images to get raw data and Genesrping software was employed to finish the basic analysis with the raw data. Quantified positive signals were then extracted and analyzed by the LC Sciences, in accordance with MIAME guidelines. Protein coding genes differentially expressed after C. parvum infection and overexpression of Cdg2_FLc_0220 were screened by fold change > 1.3 and P < 0.05. The heatmaps for genes differentially expressed in both arrays after infection and overexpression were depicted by using MeV 4.9.0.

2.4 Total RNA extraction from whole cells and nuclear extracts and cDNA synthesis

Cells were directly cleaved by using Tri-reagent solution (invitrogen) to extract total RNA from whole cells, and cell pellets were collected by using trypsin-EDTA (Gemini Bio Products) and washed with PBS for isolation nuclear extracts. Cell pellets were then treated by 500 μl nuclear buffer A (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40) for 10 min at 4°C, centrifuged at 1000 rpm for 2 min at 4°C, and resuspended in 500 μl nuclear buffer B (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2). Nuclear extracts were collected by centrifugation at 2300 rpm for 2 min at 4°C, and treated by Tri-reagent solution (invitrogen). Both total RNA samples of whole cells and nuclear extracts were extracted and reversely transcribed into cDNA as 2.2.

2.5 Real-time PCR

Realtime PCR was performed using the SYBR Green polymerase chain reaction master mix (Applied Biosystems) in ABI-Prism 7900HT (Applied Biosystems, Foster City, CA, USA) with primers listed in Table S1. Experiments were performed in triplicate and the values were normalized to GAPDH (for whole cell analysis) or U2 (for nuclear delivery analysis) and expressed as 2−ΔΔCt.

2.6 Western Blot

Whole cell protein lysates were prepared using M-PER® Mammalian Protein Extraction Reagent (Thermo scientific) in the presence of protease inhibitors and measured using Bio-Rad DC Protein Assay Reagent (Bio-Rad). Proteins (20 μg) were loaded into 10% SDS–polyacrylamide electrophoresis gels and transferred to nitrocellulose membrane. The following antibodies were used for blotting: anti-DZIP1L (Santa Cruz Biotechnology), anti-H3 (Cell Signaling), anti-Cyclophilin A (Cell Signaling), and anti-GAPDH (Santa Cruz Biotechnology).

2.7 Formaldehyde cross-linking RNA Immunoprecipitation (RIP)

Cell pellets were collected for HCT-8 cells infected with C. parvum oocysts at 24 h post infection (pi) and washed with PBS. The cross-linking reaction was performed with 0.3% of formaldehyde at 37°C for 10 min and quenched by 0.25 M glycine at room temperature for 5 min. Nuclear extracts from resultant cell pellets were isolated by using 500 μl nuclear buffer A and 500 μl nuclear buffer B as above in the presence of protease inhibitors. Pellets were resuspended in 100 μl nuclear buffer C (10 mM Tris-HCl pH 7.4, 400 mM NaCl, 1 mM EDTA, 1 mM DTT) and 400 μl WCE buffer (20 mM HEPES, pH 7.4, 0.2 M NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM EDTA, 1 mM EGTA and 1 mM DTT) with protease and RNA inhibitors, sonicated and measured for nuclear protein levels. A total of 500 μg protein for each sample was immunoprecipitated with 2 μg normal mouse or rabbit IgG (Santa Cruz Biotechnology), and polyclonal antibodies of mouse PRDM1 (Santa Cruz Biotechnology) and rabbit G9α (Millipore) by using Magna Protein A+G Magnetic Beads (Millipore). Formaldehyde cross-linking RIP binding complex were reversed by incubation at 65 C for 4 h with rotation and the RNA level of Cdg2_FLc_0220 was determined by using Realtime PCR in the CFX Connect Real-time detection system (Bio-Rad) and normalized to the control IgG and the U2 gene of the input (10% of the starting sample).

2.8 Chromatin Immunoprecipitation (ChIP)

Cell pellets were collected for HCT-8 cells infected with C. parvum and transfected with the Full-Cdg2_FLc_0220 after 24 h, washed with PBS, cross-linked with 1% formaldehyde at 37°C for 10 min and quenched by 0.25 M glycine at room temperature for 5 min. Cell protein isolation and genomic DNA fragmentation were performed by using SDS lysis buffer (1% SDS, 10 mM EDTA and 50 mM Tris-HCl pH 8.1) and sonication. 500 μg protein for each sample was used for ChIP analysis with Salmon Sperm DNA/Protein A Agarose (Millipore) and 2 μg normal mouse or rabbit IgG (Santa Cruz Biotechnology), rabbit polyclonal to Histone H3 (tri methyl K9) (Abcam), mouse monoclonal to Histone H3 (tri methyl K27) (Abcam), mouse polyclonal antibody to PRDM1 (Santa Cruz Biotechnology), and rabbit polyclonal antibody to G9α (Millipore) at 4°C overnight. Formaldehyde cross-linking complex was reversed at 65°C overnight. The methylation level for each selective DNA site was quantified by using Realtime PCR with primers listed in Table S1, and the value was normalized to the input (1% of the starting chromatin).

2.9 Statistical analysis

Data was analyzed using the program Graphpad Prism 5.0 and expressed as mean ± standard error of the mean (SEM). The student t test was used to compare differences between three independent experiments, and differences were considered statistically significant when p value < 0.05.

3. Results

3.1 Nuclear delivery of parasite Cdg2_FLc_0220 RNA during C. parvum infection

In our previous studies, we identified that several C. parvum RNA transcripts were selectively delivered into the nuclei of infected host epithelial cells (Wang et al., 2017a). One of such transcripts is Cdg2_FLc_0220, a transcript with a sequence of 1189 nt from a hypothetical protein gene (GeneBank ID: FX115592.1) located at the Chromosome 2 (Yamagishi et al., 2011) (Fig. 1A). After exposure to C. parvum infection for 24 h and 48 h, INT and HCT-8 cells were collected and nuclear extracts isolated. A significant level of Cdg2_FLc_0220 was detected in the nuclear extracts of INT cells (Fig. 1B) and HCT-8 cells (Fig. 1C) after exposure to C. parvum. We used the nuclear extracts because it is not technically feasible to separate the internalized intracellular parasites from the cytoplasmic components of infected cells. The purity of our nuclear extract preparation was confirmed by Western blot for the nuclear protein marker (H3) and a cytoplasmic protein marker (Cyclophilin A); nuclear extracts from cells exposed to heat-inactive C. parvum oocysts or parasite lysis showed no detectable level of Cdg2_FLc_0220 (data not shown). Moreover, we further measured its nuclear content in cells expressing Cdg2_FLc_0220. The full-length of Cdg2_FLc_0220 was amplified by PCR using primers listed in Table S1, sequenced and cloned into the plasmid pcDNA3.1. We then transfected INT and HCT-8 cells with the Full-Cdg2_FLc_0220 construct for 24 h and 48 h. A significantly higher level of Cdg2_FLc_0220 was detected in the nuclear extracts of INT and HCT-8 cells expressing Cdg2_FLc_0220, compared with that in cells transfected with the empty vector (Fig. 1D and 1F).

Fig. 1.

Fig. 1

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Delivery of parasite Cdg2_FLc_0220 RNA transcript into the nuclei of intestinal epithelial cells following C. parvum infection. (A) Genomic location and sequence of the Cdg2_FLc_0220 gene (GeneBank ID: FX115592.1) in C. parvum. (B) and (C) Delivery of Cdg2_FLc_0220 RNA transcript into the nuclei of cultured human intestinal epithelial cells following C. parvum infection in vitro. INT and HCT-8 cell lines were cultured and exposed to C. parvum oocyst infection for 24 h and 48 h. Nuclear extracts of INT (B) and HCT-8 (C) cell cultures were isolated and contents of Cdg2_FLc_0220 were quantified by real-time PCR, normalized by the expression level of U2 RNA. (D) and (E) Overexpression of Cdg2_FLc_0220 in INT and HCT-8 cells resulted in nuclear delivery of Cdg2_FLc_0220 in the cells. Cells were transfected with the Full-Cdg2_FLc_0220 vector for 24 h and 48 h. Nuclear extracts of INT (D) and HCT-8 (E) cell cultures were isolated and contents of Cdg2_FLc_0220 were quantified by real-time PCR, normalized by the expression level of U2 RNA. Cells transfected with the empty vector were used as the control. Data represent three independent experiments. ** P<0.01 and ***P<0.001, ANOVA versus non-infected or empty vector controls.

3.2 Alterations in host gene expression profile following C. parvum infection is partially associated with the nuclear delivery of Cdg2_FLc_0220

Since C. parvum Cdg2_FLc_0220 could be delivered into host cell nuclei, we examined the potential influence of Cdg2_FLc_0220 on the host gene expression profile. INT cells exposed to C. parvum for infection for 48 h or transfected with the Full-Cdg2_FLc_0220 for 48 h were collected and genome-wide transcriptome analysis was performed using the Agilent SurePrint G3 Human Gene Expression Microarray (G4851B). Non-infected cells and cells transfected with the empty vector pcDNA3.1 for 48 h were used as the controls. All array data were deposited at GEO database (accession number: GSE94128; for reviewer access: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=cxybmaqajfajhyf&acc=GSE94128). Given the fact that the impact of C. parvum infection on host cell gene transcription is generally very mild compared to other pathogens (Deng et al., 2004; Yang et al., 2009; Zhou et al., 2009), we chose fold changes >1.3 combined with a P < 0.05 as the threshold for data analysis. We identified that 1776 genes were upregulated and 1966 genes were downregulated in infected cells compared with the non-infected cells (Fig. 2A, Table S2 and S3). Compared with cells transfected with the empty vector, 287 genes were upregulated and 97 genes were downregulated in cells transfected with the Full-Cdg2_FLc_0220 (Fig. 2A, Table S4 and S5). There were 46 overlapping upregulated genes and 8 overlapping downregulated genes in infected cells and cells transfected with Full-Cdg2_FLc_0220 (Fig. 2A and B, Table S6). Heatmaps represent these overlapping genes either upregulated or downregulated (Fig. 2B).

Fig. 2.

Fig. 2

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Alterations in host gene expression profiles in cells following C. parvum infection and overexpressing Cdg2_FLc_0220. (A) Genome-wide transcriptome analysis in INT cells following C. parvum infection and cells after transfection of the Full-Cdg2_FLc_0220 for 48 h, taking the fold changes >1.3 combined with a p < 0.05 as the threshold. The total numbers of genes whose expression is significantly altered after infection or transfection are shown. (B) Heatmaps representing the overlapping genes either upregulated or downregulated in cells following Full-Cdg2_FLc_0220 transfection and C. parvum infection. Expression levels of RNAs were presented as the fold changes normalized to the mean value of the non-infected control. (C) Downregulation of the DZLP1L gene in INT cells following C. parvum infection or in cells following Full-Cdg2_FLc_0220 transfection as measured by using real-time PCR. INT cells following C. parvum infection and cells after transfection of the Full-Cdg2_FLc_0220 for 48 h, followed by real-time PCR analysis of DZLP1L. (D) Downregulation of the DZLP1L gene at the mRNA and protein levels in cells following C. parvum infection or Full-Cdg2_FLc_0220 transfection was further validated in HCT-8 cells. HCT-8 cells following C. parvum infection and cells after transfection of the Full-Cdg2_FLc_0220 for 48 h were collected, followed by real-time PCR and Western blot for DZLP1L. Data represent three independent experiments. *P<0.05 and ** P<0.01, ANOVA versus non-infected or empty vector controls.

These overlapping upregulated genes include interleukins (e, g., IL6, IL1B, IL1A) and their receptors (e. g., IL13RA2), chemokine ligands (e. g., CXCL10, CXCL2, CXCL3, CXCL1, CXCL8), complement components (e, g., C3, C1QTNF1) and other inflammatory mediators (e, g., TNFSF14, TRAF2, GKN1, ELF3, NFKB2, NFKBIZ), indicating a similar inflammatory response in cells following C. parvum infection or transfection with the parasite Cdg2_FLc_0220 RNA. Of the eight overlapping downregulated genes, the top one is the DZIP1L gene. Downregulation of DZIP1L at the RNA level in INT cells following C. parvum infection or transfection with Full-Cdg2_FLc_0220 was confirmed by real-time PCR (Fig. 2C). Furthermore, downregulation of DZIP1L at both the RNA and protein levels was also validated in HCT-8 cells infected with C. parvum and transfected with Full-Cdg2_FLc_0220 (Fig. 2D).

3.3 Trans-suppression of the DZIP1L gene in host cells following C. parvum infection is mediated by G9a, independent of PRDM1

We then focused on the downregulation of the DZIP1L gene to explore the molecular mechanisms underlying Cdg2_FLc_0220-medicated trans-suppression of host genes in infected cells. Our previous studies demonstrated that G9a, an important histone methyltransferase mediating transcriptional repression of genes (Artal-Martinez de Narvajas et al., 2013; Tong et al., 2013; Fan et al., 2015), is involved in parasite RNA-mediated trans-suppression of several host genes during C. parvum infection (Ming et al., 2017; Wang et al., 2017a, b). We then questioned whether G9a is recruited to the DZIP1L gene locus in HCT-8 cells after C. parvum infection or overexpression of Cdg2_FLc_0220. We designed PCR primer sets (Set 1 to 4) covering the different promoter regions of the DZIP1L gene locus for ChIP analysis (Fig. 3A). Of four selected regions within the promoter of DZIP1L gene locus, three (Met1, Met3, Met4) regions showed an increase in G9a recruitment in cells following infection for 24 h (Fig. 3A). Interestingly, two regions (Met3 and Met4) also showed an increase in recruitment of G9a in cells after transfection of Full- Cdg2_FLc_0220 (Fig. 3A). To further explore the role of G9a for C. parvum infection, we generated the stable G9a knockout (G9a−/−) HCT-8 cells using the G9a-CRISPR/Cas9 KO(h) and G9a-HDR plasmids (Santa Cruz). In contrast to the downregulation of the DZIP1L gene in HCT-8 cells following infection, we detected a significant increase of DZIP1L RNA level in the G9a−/− HCT-8 cells following C. parvum infection (Fig. 3B). These results suggested that the downregulated expression of DZIP1L was associated with G9a. To test the physical relationship between G9a and Cdg2_FLc_0220 in the infected cells, we performed the RIP analysis. Anti-G9a antibody was used to pull down the G9a complex in the nuclear extracts of HCT-8 cells infected with C. parvum for 24 h. A significant amount of Cdg2_FLc_0220 was detected in the immunoprecipitated G9a complex from the nuclear extracts of infected HCT-8 cells (Fig. 4A), indicating the assembly of Cdg2_FLc_0220 in the G9a complex in the nuclei of infected cells.

Fig. 3.

Fig. 3

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Trans-suppression of DZLP1L gene in HCT-8 cells following C. parvum infection is associated with promoter recruitment of G9a. (A) Schematic map of the transcribed region and promoter of the DZIP1L gene (upper), and recruitment of the G9a protein into the promoter regions of the DZIP1L gene by the CHIP technology after C. parvum infection or overexpression of Cdg2_FLc_0220 in HCT-8 cells. (B) mRNA level of DZIP1L gene in wild and G9a−/− HCT-8 cells following C. parvum infection. *P<0.05, ** P<0.01, and ***P<0.001, t test versus non-infected cells or cells transfected with the empty vector.

Fig. 4.

Fig. 4

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Assembly of Cdg2_FLc_0220 to the G9a- and PRDM1-complexes, but not the enrichment of PRDM1 within the promoter of the DZIP1L gene, in HCT-8 cells following C. parvum infection or transfection of the Full-Cdg2_FLc_0220. (A and B) HCT-8 cells were exposed to C. parvum infection for 24 h, followed by RNA immunoprecipitation analysis using anti-G9a (A) and anti-PRDM1 (B), respectively, followed by real-time PCR analysis of Cdg2_FLc_0220. Non-specific IgG was used as the control. Data represent means ± SEM from three independent experiments. *P<0.05 and ***P<0.001, ANOVA versus non-infected controls. (C and D) Recruitment of the PRDM1 within the promoter of the DZIP1L gene in HCT-8 cells after C. parvum infection and transfection of the Full-Cdg2_FLc_0220. HCT-8 cells were exposed to C. parvum infection for 24 h or transfected with the Full-Cdg2_FLc_0220 for 24 h. Non-infected cells or cells transfected with the empty vector were used as the control. The recruitment of the PRDM1 within promoter of the DZIP1L gene in cells after C. parvum infection (C) and transfection of the Full-Cdg2_FLc_0220 (D) were measured by ChIP analysis, using anti-PRDM1 and the PCR primer sets as designed. Data represent means ± SEM from three independent experiments. ** P<0.01, and ***P<0.001, ANOVA versus non-infected.

The PR domain zinc finger protein 1 (PRDM1, also known as BLIMP-1), a G9a-interacting protein (John and Garrett-Sinha, 2009) and an RNA binding protein using the PR zinc finger domain to interact with RNA molecule (John and Garrett-Sinha, 2009), has been shown to be involved in the transcriptional repression of genes through recruitment of G9a (Gyory et al., 2004). We then determined whether PRDM1 is involved in Cdg2_FLc_0220-mediated suppression of DZIP1L. We first measured the physical relationship between PRDM1 and Cdg2_FLc_0220 in the infected cells using the RIP analysis. A significant amount of Cdg2_FLc_0220 was detected in the immunoprecipitated PRDM1 complex in the nuclear extracts from HCT-8 cells following infection for 24 h (Fig. 4B). However, ChIP analysis failed to detect any recruitment of the PRDM1 complex to the promoter region of the DZIP1L gene in HCT-8 cells following C. parvum infection (Fig. 4C) or transfection with the Full-Cdg2_FLc_0220 (Fig. 4D), suggesting that PRDM1 would not be directly involved in Cdg2_FLc_0220-mediated transcriptional repression of DZIP1L in cells following infection.

3.4 Cdg2_FLc_0220-mediated trans-suppression of the DZIP1L gene during C. parvum infection is independent of H3K9 and H3K27 methylations

Given the importance of G9a as a histone methyltransferase to enhance histone methylation to induce epigenetic gene suppression, coupled with the recruitment of G9a to the DZIP1L gene promoter in infected cells, we speculated that H3K9 and H3K27 methylations associated with the DZIP1L gene locus may occur in cells following C. parvum infection. HCT-8 cells infected with C. parvum and transfected with Cdg2_FLc_0220 for 24 h were collected, followed by ChIP analysis using anti-H3K9me3 and anti-H3K27me3, respectively. In contrast to our speculation, de-enrichment of H3K9 and H3K27 methylations was detected in the DZIP1L gene promoter in cells following C. parvum infection (Figs. 5A, B) or overexpression of Cdg2_FLc_0220 (Figs. 5C, D), indicating no association of DZIP1L repression with the methylation of H3K9 and H3K27.

Fig. 5.

Fig. 5

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Enrichments of H3K9me3 and H3K27me3 within the promoter of the DZIP1L gene in HCT-8 cells after C. parvum infection and transfection of the Full-Cdg2_FLc_0220. HCT-8 cells were exposed to C. parvum infection for 24 h or transfected with the Full-Cdg2_FLc_0220 for 24 h. Non-infected cells or cells transfected with the empty vector were used as the control. The enrichments of H3K9me3 (A and C) and H3K27me3 (B and D) within promoter of the DZIP1L gene in cells after C. parvum infection (A and B) and transfection of the Full-Cdg2_FLc_0220 (C and D) were measured by ChIP analysis, using anti-H3K9me3 or anti-H3K27me3 and the PCR primer sets as designed. Data represent means ± SEM from three independent experiments. *P<0.05, ** P<0.01, and ***P<0.001, ANOVA versus non-infected or empty vector controls.

4. Discussion

Intestinal infection by Cryptosporidium causes significant alterations in the gene expression profile in host epithelial cells. Whereas alterations in the expression levels of many genes are due to host responses to infection, such as inflammatory and defense genes, increasing evidence suggests that Cryptosporidium may modulate host gene transcription to its own benefit (Deng, et al., 2004; Ming et al., 2017; Wang et al., 2017a and 2017b). A well-known example is the suppression of host DEFB1 gene in intestinal epithelial cells following infection, which may help the parasite to evade host defense against infection (Zaalouk et al., 2004). Neverthenless, the molecular mechanisms of how C. parvum may modulate host cell gene transcription and the pathological significance of such alterations are largely unclear. Previous studies demonstrated that a panel of C. parvum RNA transcripts are delivered into infected host cells and may modulate host gene transcription (Wang et al., 2017a and 2017b). Data from this study provide additional evidence that Cdg2_FLc_0220, one of the parasite RNAs that are selectively delivered into the nuclei of infected cells, may modulate the transcription of host genes, contributing to the alterations in the gene expression profile in host epithelial cells during C. parvum infection.

Consistent with findings from previous studies (Ming et al., 2017; Wang et al., 2017a, b), nuclear delivery of Cdg2_FLc_0220 may only account for the alterations for a small fraction of host genes induced by C. parvum infection. Specifically, we identified a total of 46 overlapping upregulated genes and 8 overlapping downregulated genes in infected cells and cells transfected with Full-Cdg2_FLc_0220, whereas a total of 1776 genes were upregulated and 1966 genes were downregulated in infected cells compared with the non-infected cells. Of note, most of these overlapping upregulated genes are inflammatory mediators, indicating a general inflammatory response in cells following C. parvum infection or transfection with the parasite Cdg2_FLc_0220 RNA. In contrast, we speculate that these overlapping downregulated genes, such as the DZIP1L gene, may be modulated by parasite Cdg2_FLc_0220 RNA.

How nuclear delivery of Cdg2_FLc_0220 RNA may cause trans-suppression of the DZIP1L gene is still unclear. Previous studies demonstrated that promoter recruitment of G9a (a key methyltransferase for H3K9) (Shinkai and Tachibana, 2011) and PRDM1 (a G9a-interacting protein) (John and Garrett-Sinha, 2009; Gyory et al., 2004) is involved in the trans-suppression of the host CDH3 gene by C. parvum Cdg7_FLc_1000 RNA in infected cells (Ming et al., 2017). Trans-suppression of host LRP5, SLC7A8, and interleukin 33 genes mediated by nuclear delivery of Cdg_FLc_0990 RNA in infected cells also requires the promoter recruitment of G9a/PRDM1 complex (Wang et al., 2017a, b). Interestingly, an increased recruitment of G9a, but not PRDM1, was detected in the promoter region of the DZIP1L gene locus in cells following infection or transfection of the Full-Cdg2_FLc_0220. This suggests to us a G9a-dependent mechanism for Cdg2_FLc_0220-mediated trans-suppression of the DZIP1L gene in host cells. This is further evident by the completely restoration of DZIP1L expression in the G9a−/− cells following C. parvum or transfection with the Full-Cdg2_FLc_0220.

As a key methyltransferase for H3K9, G9a has been reported to suppress gene transcription through induction of H3K9 methylation (Shinkai and Tachibana, 2011). In contrast with our speculation, a decrease of both H3K9me3 and H3K27me3 was detected within the promoter region of the DZIP1L gene locus in cells following infection or transfection of the Full-Cdg2_FLc_0220. Therefore, G9a may mediate trans-suppression of DZIP1L associated with the nuclear delivery of Cdg2_FLc_0220 through other mechanisms, rather than induction of H3K9 methylation. The DZIP1L gene encodes the DZIP1L protein, which localizes to centrioles and to the distal ends of basal bodies, and interacts with septin2, a protein implicated in maintenance of the periciliary diffusion barrier at the ciliary transition zone (Glazer et al., 2010). Differing from the ciliated columnar epithelium in the upper respiratory tract, intestinal epithelium is non-ciliated and appears to lack primary cilia (single cilia) (Saqui-Salces et al., 2012). Therefore, the significance of ZDIP1L downregulation in the pathogenesis of intestinal cryptosporidiosis is unclear and merits further investigation.

Supplementary Material

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Highlights.

  • The parasite Cdg2_FLc_0220 RNA transcript is delivered into the nuclei of infected host epithelial cells following C. parvum infection.

  • Alterations in host gene expression profile following C. parvum infection are partially associated with the nuclear delivery of Cdg2_FLc_0220.

  • Trans-suppression of the DAZ interacting zinc finger protein 1 like (DZIP1L) gene, the top overlapping downregulated gene in host cells following C. parvum infection, is mediated by G9a, independent of PRDM1.

  • Cdg2_FLc_0220-mediated trans-suppression of the DZIP1L gene is independent of H3K9 and H3K27 methylation.

Acknowledgments

We thank Dr. Zhenping Ming (Wuhan University, China) for helpful and stimulating discussions, and Barbara L. Bittner (Creighton University) for her assistance in writing the manuscript. This work was supported by funding from the National Institutes of Health (AI116323 and AI136877) and by revenue from Nebraska’s excise tax on cigarettes awarded to Creighton University through the Nebraska Department of Health & Human Services (DHHS) (LB595). Dr. Guang-Hui Zhao was a visiting scholar supported by the China Scholarship Council. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the State of Nebraska, or DHHS.

Footnotes

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NIHMS931838-supplement-4.xlsx (189.2KB, xlsx)
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NIHMS931838-supplement-5.xlsx (215.4KB, xlsx)
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NIHMS931838-supplement-6.xlsx (15.6KB, xlsx)

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