The Antimicrobial Peptide Mastoparan X Protects Against Enterohemorrhagic Escherichia coli O157:H7 Infection, Inhibits Inflammation, and Enhances the Intestinal Epithelial Barrier

Abstract

Escherichia coli can cause intestinal diseases in humans and livestock, destroy the intestinal barrier, exacerbate systemic inflammation, and seriously threaten human health and animal husbandry development. The aim of this study was to investigate whether the antimicrobial peptide mastoparan X (MPX) was effective against E. coli infection. BALB/c mice infected with E. coli by intraperitoneal injection, which represents a sepsis model. In this study, MPX exhibited no toxicity in IPEC-J2 cells and notably suppressed the levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), myeloperoxidase (MPO), and lactate dehydrogenase (LDH) released by E. coli. In addition, MPX improved the expression of ZO-1, occludin, and claudin and enhanced the wound healing of IPEC-J2 cells. The therapeutic effect of MPX was evaluated in a murine model, revealing that it protected mice from lethal E. coli infection. Furthermore, MPX increased the length of villi and reduced the infiltration of inflammatory cells into the jejunum. SEM and TEM analyses showed that MPX effectively ameliorated the jejunum damage caused by E. coli and increased the number and length of microvilli. In addition, MPX decreased the expression of IL-2, IL-6, TNF-α, p-p38, and p-p65 in the jejunum and colon. Moreover, MPX increased the expression of ZO-1, occludin, and MUC2 in the jejunum and colon, improved the function of the intestinal barrier, and promoted the absorption of nutrients. This study suggests that MPX is an effective therapeutic agent for E. coli infection and other intestinal diseases, laying the foundation for the development of new drugs for bacterial infections.

Keywords: Enterohemorrhagic Escherichia coli O157:H7; antimicrobial peptide mastoparan X; inflammation; intestinal barrier; mice.

Figure 1

MPX does not induce cytotoxicity and alleviates inflammation in IPEC-J2 cells. (A) Cell viability was measured using the CCK-8 assay, IPEC-J2 cells were cultured with different concentrations (2-512 µg/mL) of MPX for 24 h. (B) The release of LDH from IPEC-J2 cells after treatment with different concentrations (2-512 µg/mL) of MPX for 24 h. (C) MPX decreased the E. coli-induced release of LDH from IPEC-J2 cells (MOI = 10) at different times. (D,E) The mRNA expression of IL-6 and TNF-α after MPX treatment. (F) The expression of p-p38, p-p65 and TLR4 in IPEC-J2 cells assessed by confocal laser microscopy. Error bars represent mean ± SEM, n = 3. Statistical significance was defined as follows: #P < 0.05; ##P < 0.01; ###P < 0.001 E. coli vs control; *P < 0.05; **P < 0.01; ***P < 0.001 MPX treatment vs E. coli.

Figure 2

MPX enhances IPEC-J2 cell barrier function. (A) The E. coli-induced mRNA expression of ZO-1, occludin and claudin-1 in IPEC-J2 cells after treatment with MPX. (B) IPEC-J2 cells were incubated with medium alone or MPX (10 μg/mL) in a wound healing assay. Images were obtained at 0 h, 48 h and 96 h, and the wound width was measured at 48 h. (C) The E. coli-induced protein level of occludin in IPEC-J2 cells after pretreatment with MPX was determined by western blotting. (D) The effect of MPX on the expression of occludin in IPEC-J2 cells was assessed by confocal laser microscopy. (E) MPX increases TEER of differentiated IPEC-J2 cells monolayers subjected to E. coli stimulus. Error bars represent mean ± SEM, n = 3. Statistical significance was defined as follows: #P < 0.05; ##P < 0.01; ###P < 0.001 E. coli vs control; *P < 0.05; **P < 0.01; ***P < 0.001 MPX treatment vs E. coli.

Figure 3

MPX protects mice against infection with a lethal dose of E. coli. (A) The survival rate of mice infected with E. coli after MPX treatment. (B) The clinical symptom score of mice infected with E. coli after MPX treatment. (C) The weight of mice infected with E. coli after MPX treatment. (D-F) The weight of the liver, spleen and lung of mice infected with E. coli after MPX treatment. (G) The number of bacteria in the liver, spleen and lung of mice infected with E. coli after MPX treatment. (H) The number of bacteria in the feces of mice infected with E. coli after MPX treatment. Control group, mice injected with sterile saline; E. coli group, mice injected with E. coli; MPX group, mice treated with intraperitoneal injection MPX and injected with E. coli. Error bars represent mean ± SEM, n = 5. Statistical significance was defined as follows: #P < 0.05; ##P < 0.01; ###P < 0.001 E. coli vs control; *P < 0.05; **P < 0.01; ***P < 0.001 MPX and Enro treatment vs E. coli.

Figure 4

MPX inhibits inflammatory cytokine expression and improves intestinal morphology. (A) The levels of inflammatory cytokines (IL-2, IL-6 and TNF-α) and MPO in mouse serum were detected using ELISA. (B) The jejunum of mice was stained with H&E (bars, 100 μm); images were obtained at 200× magnification. (C) The length of jejunal villi, the depth of crypts, and the ratios of villi length to crypt depth were detected by ipwin32 software. Error bars represent mean ± SEM, n = 5. Statistical significance was defined as follows: #P < 0.05; ##P < 0.01; ###P < 0.001 E. coli vs control; *P < 0.05; **P < 0.01; ***P < 0.001 MPX and Enro treatment vs E. coli.

Figure 5

MPX improves the intestinal morphology of the jejunum and the microvilli of intestinal epithelial cells. (A) Morphological changes in the jejunum villi were observed by SEM (upper, 200×; lower, 30,000×). (B) Morphological changes in the microvilli and tight junction proteins in intestinal epithelial cells were observed by TEM (upper, 1500×; lower, 3000×).

Figure 6

MPX suppresses intestinal inflammation by inhibiting the activation of the MAPK and P65 signaling pathways. (A) The mRNA expression of IL-2, IL-6, and TNF-α in the jejunum and colon after MPX treatment was measured by real-time PCR. (B) The expression of p-p38, p-pJNK and p-pERK in the jejunum after MPX treatment was determined by immunohistochemistry (bars, 100 μm). (C) The effect of MPX on the expression of p-p65 in the colon was assessed by immunofluorescence. Error bars represent mean ± SEM, n = 5. Statistical significance was defined as follows: #P < 0.05; ##P < 0.01; ###P < 0.001 E. coli vs control; *P < 0.05; **P < 0.01; ***P < 0.001 MPX and Enro treatment vs E. coli.

Figure 7

MPX improves the expression of tight junction proteins and mucin in the jejunum and colon. (A) The mRNA expression of claudin-1, ZO-1, occludin and MUC2 in the jejunum and colon. (B) The protein expression of claudin-1, occludin, ZO-1 and MUC2 (red) and DAPI (blue) in the jejunum. (C) The protein expression of claudin-1, occludin, ZO-1 and MUC2 (red) and DAPI (blue). Scale bar = 10 μm.

Figure 8

MPX regulates epithelial cells and in vivo signaling pathways.