Fig 2: The expression of METTL3 gene is increased and that of FTO gene is decreased in the AD group. Student’s t-test was used to detect differences between the two groups, and data are presented as mean ± SEM; n = 5 per group, *P < 0.05, **P < 0.01.

Fig 3: m6A methylation-mediated intestinal epithelial anti-C. parvum defense in human intestinal epithelium. (A) Increase of global m6A RNA methylation in HCT-8 cells following C. parvum infection. Cells were exposed to C. parvum infection for 24h and m6A RNA methylation was measured by m6A RNA methylation quantitation assay. (B) Decrease of ALKBH5 and FTO expression levels in HCT-8 cells following C. parvum infection. Cells were exposed to C. parvum infection for 24h and expression levels of ALKBH5 and FTO was measured by real-time PCR. (C) Knockdown of ALKBH5 via siRNA in HCT-8 cells. Cells were treated with the siRNA to ALKBH5 for 24h and knockdown of ALKBH5 was confirmed by real-time PCR. Cells transfected with non-specific control siRNA were used as the control. (D) Knockdown ALKBH5 in HCT-8 cells decreased C. parvum infection burden. HCT-8 cells were first treated with the siRNA-ALKBH5 and then exposed to C. parvum infection for 24h. Infection burden of C. parvum was quantified by measuring parasite cpHsp70 or cp18s using real-time PCR. Data represent three independent experiments. *p<.05 vs the non-infected control (in A–D).

Fig 4: m6A methylation modulates intestinal epithelial innate defense against C. parvum infection. (A) Knockdown of Alkbh5 or Fto in IEC4.1 cells. Cells were transfected with the CRISPR/Cas9 KO(h) for Alkbh5 or Fto and the HDR plasmids. Stable transfected cells were cloned and confirmed by real-time PCR and Western blot analysis. Gapdh was also blotted for control. (B) Knockdown of Alkbh5 or Fto in IEC4.1 cells decreased the infection burden of C. parvum infection. Knockdown of Alkbh5 or Fto increased m6A RNA methylation in IEC4.1 cells, as measured by m6A RNA methylation quantitation assay and dot blot. IEC4.1 cells and cells deficient with Alkbh5 or Fto were then exposed to C. parvum infection for 24 h. IEC4.1 cells transfected with the empty vector, marked as IEC4.1-WT (wild type) were used as the control. Infection burden of C. parvum was quantified by measuring parasite cpHsp70 or cp18s using real-time PCR. (C) Knock-in of Alkbh5 or Fto in IEC4.1 cells. Cells were transfected with the CRISPR/Cas9 KO(h) for active Alkbh5 (Alkbh5-KI) or Fto (Fto-KI) vectors. Stable transfected cells were cloned and confirmed by Western blot analysis. (D) Overexpression of Alkbh5, but not Fto, increased the infection burden of C. parvum in IEC4.1 cells. Cells stably expressing Alkbh5 or Fto were exposed to C. parvum infection for 24 h and infection burden of C. parvum was quantified. (E) Knockdown of Alkbh5 via siRNA decreased the infection burden of C. parvum in IEC4.1 cells. Cells were treated with the siRNA to Alkbh5 for 24 h and exposed to C. parvum infection for additional 24 h. Cells treated with the non-specific scrambled siRNA were used as the control and infection burden of C. parvum was quantified. (F) Knockdown of Alkbh5 via siRNA decreased the infection burden of C. parvum in 2D intestinal epithelial monolayers. Monolayers were cultured and treated with the siRNA to Alkbh5 for 24 h and exposed to C. parvum infection for additional 24 h. Cells treated with the non-specific scrambled siRNA were used as the control and infection burden of C. parvum was quantified. Data represent three independent experiments. *p<.05 vs cells transfected with the empty-vector control (as IEC4.1-WT in A, B, D) or cells treated with the control-siRNA (in E, F).

Fig 5: Downregulation of Alkbh5 and Fto in intestinal epithelial cells C. parvum infection. (A) Heatmaps showing expression profile of key genes involved in the m6A RNA methylation machinery in IEC4.1 cells following C. parvum infection. Cells were exposed to C. parvum infection for 24 h followed by genome-wide array analysis. (B) Dynamics of Alkbh5 and Fto downregulation in IEC4.1 cells following C. parvum infection. IEC4.1 were exposed to C. parvum infection for 4-48 h and RNA expression levels of Alkbh5 and Fto were validated by using real-time quantitative PCR. Expression levels of Cxcl2 (as a positive control), Mettl3, Mettl14 and Wtap were also measured. (C) Decreased abundance of Alkbh5 and Fto proteins in IEC4.1 cells following C. parvum infection. IEC4.1 were exposed to C. parvum infection for 48-72 h and expression levels of Alkbh5 and Fto at the protein level were validated using Western blot. ß-Actin was also blotted for internal control. Representative gels were shown. (D) Downregulation of Alkbh5 and Fto in murine intestinal epithelium following C. parvum infection in vivo. Neonates of mice at 5 days of age received C. parvum administration by oral gavage and intestinal ileum epithelium were isolated after infection for 24h. Expression levels of expression levels of Alkbh5 and Fto were measured. (E) Downregulation of Alkbh5 and Fto in 2D murine intestinal epithelial monolayers following C. parvum infection ex vivo. Data represent three independent experiments. *p<.05 vs the non-infected control.