Abstract
Our previous studies demonstrated that the micro integral membrane protein (MIMP) located within the integral membrane protein of Lactobacillus plantarum CGMCC 1258 protected the intestinal barrier from injury. To further analyze the protective effects conferred to intestinal epithelial cells by MIMP, we established transient MIMP-expressing NCM460 cells (NCM460/MIMP) as a means of assessing their susceptibility to infection. We constructed a recombinant eukaryotic expression vector using pcDNA3.1(-) and the MIMP gene. The recombinant vector was then transduced into NCM460 cells and the anti-infective properties of the transient MIMP-expressing NCM460/MIMP cells assessed. Flow cytometric analysis revealed that 82.16% ±12.22% of NCM460/MIMP cells expressed MIMP and Western blot analysis confirmed high levels of MIMP expression. Attachment assays showed that the ability of enteropathogenic Escherichia coli (EPEC) to attach to NCM460/MIMP cells decreased significantly compared to adhesion observed to NCM460 cells. Western blot and quantitative RT-PCR showed that the expression levels of tight junction (TJ) proteins, including claudin-1, occludin, JAM-1 and ZO-1 in NCM460/MIMP cells infected with EPEC were similar to levels observed in uninfected NCM460 cells. Fluorescence further showed that NCM460/MIMP cells had significantly higher TJ protein staining intensity ocompared to NCM460 cells and transmission electron microscopy indicated that TJ structure was unchanged in NCM460/MIMP cells infected with EPEC compared to NCM460 cells after EPEC infection. Expression levels of PKC-η and phosphorylated occludin were also higher after EPEC infection in NCM460/MIMP cells compared to expression levels in EPEC-infected NCM460 cells. These data demonstrated that NCM460/MIMP cells possessed EPEC anti-infective properties related to the activation of protein kinase C-η and occludin phosphorylation.
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