Strong decrease in invasive ability and outer membrane vesicle release in Crohn's disease-associated...

Area of Science:

• Microbiology
• Molecular Biology
• Cell Biology

Background:

• Adherent-invasive Escherichia coli (AIEC) strain LF82, isolated from Crohn's disease patients, invades intestinal epithelial cells.
• Outer membrane vesicles (OMVs) are implicated in bacterial pathogenesis and host-pathogen interactions.

Purpose of the Study:

• To investigate the role of the yfgL gene and its encoded lipoprotein (YfgL) in AIEC LF82's ability to invade intestinal epithelial cells.
• To determine the impact of yfgL deletion on the production and release of outer membrane proteins and vesicles.

Main Methods:

• Genetic manipulation to create yfgL-negative isogenic mutants of AIEC LF82.
• Invasion assays using Intestine-407 cell cultures.
• Analysis of outer membrane proteins in culture supernatants using SDS-PAGE and MALDI-TOF MS.
• Western blot analysis and transmission electron microscopy (TEM) to characterize outer membrane vesicles.

Main Results:

• Deletion of the yfgL gene significantly reduced the invasion ability of AIEC LF82.
• The yfgL mutant showed decreased release of outer membrane proteins (OmpA, OmpC) into the supernatant.
• Characterization confirmed that released proteins were part of outer membrane vesicles, and yfgL deletion decreased OMV release.
• Complementation with the yfgL gene restored both invasion ability and OMV release.

Conclusions:

• The YfgL lipoprotein plays a critical role in the biogenesis or release of outer membrane vesicles in AIEC LF82.
• Reduced OMV release due to yfgL deletion directly correlates with impaired intestinal epithelial cell invasion.
• Targeting YfgL or OMV production may represent a novel therapeutic strategy against AIEC infections in Crohn's disease.