CBP22, a Novel Bacteriocin Isolated from Clostridium butyricum ZJU-F1, Protects against LPS-Induced Intestinal Injury through Maintaining the Tight Junction Complex

Abstract

A novel bacteriocin secreted by Clostridium butyricum ZJU-F1 was isolated using ammonium sulfate fractionation, cation exchange chromatography, affinity chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). The bacteriocin, named CBP22, contained 22 amino acids with the sequence PSAWQITKCAGSIAWALGSGIF. Analysis of its structure and physicochemical properties indicated that CBP22 had a molecular weight of 2264.63 Da and a +1 net charge. CBP22 showed activity against E. col K88, E. coli ATCC25922, and S. aureus ATCC26923. The effects and potential mechanisms of bacteriocin CBP22 on the innate immune response were investigated with a lipopolysaccharide- (LPS-) induced mouse model. The results showed that pretreatment with CBP22 prevented LPS-induced impairment in epithelial tissues and significantly reduced serum levels of IgG, IgA, IgM, TNF-α, and sIgA. Moreover, CBP22 treatment increased the expression of the zonula occludens and reduced permeability as well as apoptosis in the jejunum in LPS-treated mice. In summary, CBP22 inhibits the intestinal injury and prevents the gut barrier dysfunction induced by LPS, suggesting the potential use of CBP22 for treating intestinal damage.

Figure 1

Experimental design and scheme of animal treatments.

Figure 2

Isolation and identification of CBP22. (a) Cation exchange chromatography of the supernatant from C. butyricum culture medium at a wavelength of 215 nm and SDS-PAGE analysis. M: mark; lane 1: before purification; lane 2: after purification. (b) Affinity chromatography of products from (a) at a wavelength of 215 nm and SDS-PAGE analysis. M: mark; lane 1: before purification; lane 2: after purification. (c) Secondary structure prediction of CBP22. (d) Helical wheel plots of CBP22. (e) Hemolytic activity of CBP22 against porcine red blood cells. The hemoglobin release was monitored at 576 nm. The data are expressed as the mean ± SEM, n = 3 biological replicates; bars with different small capital letters are statistically different from one another.

Figure 3

The protective effects of CBP22 on jejunal morphology in mice treated with LPS. (a) H&E staining of the jejunal mucosa, scale bar = 100 μm. (b) Mucosal damage grading. (c) Villus heights in the jejunum. (d) Crypt depths in the jejunum. The data are expressed as the mean ± SEM, n = 10 biological replicates; bars with different small capital letters are statistically different from one another.

Figure 4

The effect of CBP22 on jejunum cell apoptosis in mice. (a) TUNEL staining of jejunal epithelial tissues, brown signals, scale bar = 50 μm. (b) Apoptosis index of jejunal epithelial cells. The data are expressed as the mean ± SEM, n = 10 biological replicates; bars with different small capital letters are statistically different from one another.

Figure 5

Effects of CBP22 on immune function in LPS-stimulated mice. (a–d) The concentration of IgA (a), IgG (b), IgM (c), and TNF-α (d) in serum. (e) Real-time PCR analysis of TNF-α mRNA expression in the jejunum. (f) Concentration of sIgA in the ileum. The q-PCR results are presented relative to those of GAPDH. The data are expressed as the mean ± SEM, n = 10 biological replicates; bars with different small capital letters are statistically different from one another.

Figure 6

The protective effects of CBP22 on the intestinal barrier. (a) Intestinal permeability is determined by serum FD4 concentration. (b–d) RT-PCR analysis. The mRNA expression of tight junctions and results are presented relative to those of GAPDH, (b) ZO-1, (c) ZO-2, (d) occludin, and (e) claudin-1. The data are expressed as the mean ± SEM, n = 10 biological replicates; bars with different small capital letters are statistically different from one another.