Escherichia Coli Outer Membrane Vesicles Induced DNA Double-Strand Breaks in Intestinal Epithelial Caco-2 Cells

Abstract

BACKGROUND Recent studies have shown that Escherichia coli induced digestive tract diseases may be related to outer membrane vesicles (OMVs) induced intestinal double-strand breaks (DSBs) in intestinal epithelial cells. This study aimed to compare the impact of OMVs forces on DSBs in intestinal epithelial Caco-2 cells, and provide a new treatment for digestive diseases caused by E. coli. MATERIAL AND METHODS E.coli OMVs were prepared and co-cultured with Caco-2 cells. The uptake of OMVs by Caco-2 cells was observed by confocal microscopy. The γ-H2AX protein was detected by western-blots. The DSBs caused by OMVs was detected by single cell gel electrophoresis. RESULTS The particle size analyzer showed that the average diameters of OMVs centrifuged at 20 000×g and 50 000×g were 217.5±7.29 nm and 186.3±6.59 nm (P<0.05), respectively. Transmission electron microscopy of the OMVs revealed a lipid bilayer structure with a variety of different sizes. Confocal fluorescence microscopy revealed that OMVs almost completely entered Caco-2 cells after 24 hours. The ratio of γ-H2AX protein band gray value normalized data in the OMVs centrifuged at 20 000×g and 50 000×g, and the control group (without OMVs) were 2.23±0.18, 1.58±0.20, 1±0.30 (P<0.05), respectively, while DNA levels of the comet tail (TailDNA%, TDNA%) were 72.21±14.61%, 23.11±4.98%, and 1.02±1.41% (P<0.05), respectively. The corresponding DNA damage was categorized as high (grade 3), moderate (grade 2), and no damage (grade 0). CONCLUSIONS Different sizes of OMVs induced different degrees of DNA damage in intestinal epithelial Caco-2 cells.

Figure 1

Escherichia coli outer membrane vesicles (OMV) size and morphology. (A) OMV obtained by 20 000×g and 50 000×g centrifugation (n=3). (B) Size comparison between 20 000×g and 50 000×g OMV groups (n=3, per group). (C) 20 000×g and 50 000×g OMV group morphology.

Figure 2

Confocal microscopy imaging of Dio-labeled outer membrane vesicles (OMVs) taken up by Caco-2 cells.

Figure 3

γ-H2AX protein levels. (A) Expression of γ-H2AX protein. (B) Expression of γ-H2AX protein in Escherichia coli outer membrane vesicles (OMVs) obtained with different centrifugal forces.

Figure 4

Impact of outer membrane vesicle (OMV) size on DNA damage in Caco-2 cells. (A) Single-cell gel electrophoresis; (B) TailDNA% in Caco-2 cells for Escherichia coli OMVs obtained with different centrifugal forces.