Transcriptome Analysis Reveals the Multiple Functions of pBD2 in IPEC-J2 Cells against E. coli

Abstract

Defensins play an important role in fighting bacteria, and are a good candidate for bactericidal agents. However, the function and mechanism of defensins in regulating host responses against bacteria is unclear. In this study, transcriptome analysis was used to study the comprehensive functions of pBD2 in IPEC-J2 cells against E. coli. In total, 230 differentially expressed genes (DEGs) were identified in IPEC-J2 cells between the control and E. coli groups, and were found by KEGG analysis to be involved in many signaling pathways related to immunity. Furthermore, 812 DEGs were observed between E. coli and E. coli +pBD2 groups, involved in the ribosome, oxidative phosphorylation, and certain disease pathways. Among these, 94 overlapping DEGs were in the two DEG groups, and 85 DEGs were reverse expression, which is involved in microRNA in cancer, while PTEN and CDC6 were key genes according to PPI net analysis. The results of qRT-PCR verified those of RNA-seq. The results indicated that pBD2 plays an important role against E. coli by acting on the genes related to immune response, cell cycle, ribosomes, oxidative phosphorylation, etc. The results provide new insights into the potential function and mechanism of pBD2 against E. coli. Meanwhile, this study provides a certain theoretical basis for research and the development of novel peptide drugs.

Keywords: E. coli infection; defensins; functions; immune response; transcriptome analysis.

Figure 1

Analysis of purified pBD2 by SDS-PAGE. Lanes 1, 2, 3, 4, 5, 6, 8 indicate the purified protein from different collected tubes; Lane 7 indicates the protein marker.

Figure 2

Morphological changes of E. coli treated with pBD2, viewed by scanning electron microscopy. (A,B) indicate the E. coli treated with 0 and 20 μg/mL pBD2 for 1 h, respectively; (C,D) indicated the E. coli treated with 0 and 20 μg/mL pBD2 for 4 h, respectively.

Figure 3

KEGG pathway analysis of DEGs between the control and E. coli groups.

Figure 4

PPI analysis of DEGs in the control and E. coli groups.

Figure 5

KEGG pathway analysis of DEGs in the E. coli and E. coli +pBD2 groups.

Figure 6

PPI analysis of the upregulated DEGs in the E. coli and E. coli +pBD2 groups.

Figure 7

PPI analysis of the downregulated DEGs in the E. coli and E. coli +pBD2 groups.

Figure 8

Overview of the DEGs after different treatments. (A) Venn diagrams of known DEGs based on comparisons of different groups. (B) Hierarchical clustering of the overlapping DEGs among different groups. The red indicates upregulated genes and the blue indicates downregulated genes.

Figure 9

KEGG enrichment analysis of the DEGs overlapping between the two DEGs groups.

Figure 10

PPI analysis of the DEGs overlapping between the two DEG groups.

Figure 11

The verification of RNA-Seq results by qRT-PCR, between E. coli and E. coli +pBD2 groups. The samples were analyzed in triplicate by qRT-PCR, and fold-changes in gene expression were calculated by 2^−ΔΔCT methods with TUBA1B (tubulin alpha 1b) as a reference gene. MX1: Interferon-induced GTP-binding protein Mx1; EGFR: Epidermal growth factor receptor; EIF2AK2: Eukaryotic translation initiation factor 2 alpha kinase 2; PIK3R1: Phosphoinositide-3-kinase regulatory subunit alpha; PTEN: Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase; OAS2: 2′-5′-oligoadenylate synthetase 2; ATP6V0B: ATPase H+ transporting V0 subunit b; RPL30: Ribosomal protein L30; NDUFA2: NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2; NR4A1: Nuclear receptor subfamily 4, group A, member 1.