Expression of the <i>Lactobacillus Plantarum</i> Surface Layer MIMP Protein Protected NCM460 Epithelial Cells from Enteroinvasive <i>Escherichia Coli</i> Infection

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.

Claudins in intestines

“…Occludin and claudin-1 are displaced from TJ membrane microdomains to cytoplasm after EPEC infection using sucrose density gradient centrifugation and western blot analysis 114 . It has been reported that the micro integral membrane protein (MIMP) can protect the intestinal barrier from injury by EPEC infection since MIMP-expressing NCM460 epithelial cells (NCM460/MIMP) show significantly higher immunostaining signals of occludin, claudin-1, JAM-1 and ZO-1 compared with parental NCM460 cells 115 . The associations between ZO-1, occludin and claudin-1 are progressively decreased after EPEC infection in human intestinal epithelial T84 cells, resulting in a loss of barrier function 116 …”

Differential profiles of gastrointestinal proteins interacting with peptidoglycans from Lactobacillus plantarum and Staphylococcus aureus

“…In addition, several lactobacilli are involved in the positive modulation of intestinal immune responses (Grangette et al, 2005;Liu et al, 2011;Macho Fernandez et al, 2011). On the other hand, Staphylococcus aureus is a versatile Grampositive pathogen causing a range of illnesses from minor skin infections to life-threatening infections such as sepsis, pneumonia, and endocarditis (Lowy, 1998).…”

Knockout of MIMP protein in lactobacillus plantarumlost its regulation of intestinal permeability on NCM460 epithelial cells through the zonulin pathway

“…Furthermore, soluble factors, including p75 and p40, extracted from Lactobacillus rhamnosus GG culture broth supernatant, showed protective effects on IECs, which was mediated by Akt pathway [ 13 , 14 ]. Only a few studies have, however, investigated the structure and functions of SLP [ 8 , 10 , 15 – 18 ], due to the specific hydrophilic and hydrophobic properties and technical difficulties associated with SLP purification, thus limiting the investigation of SLP binding domains.…”