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. 2005 Apr;187(7):2286-96.
doi: 10.1128/JB.187.7.2286-2296.2005.

Strong decrease in invasive ability and outer membrane vesicle release in Crohn's disease-associated adherent-invasive Escherichia coli strain LF82 with the yfgL gene deleted (VSports手机版)

Nathalie Rolhion  1 , Nicolas Barnich, Laurent Claret, Arlette Darfeuille-Michaud
Affiliations

Strong decrease in invasive ability and outer membrane vesicle release in Crohn's disease-associated adherent-invasive Escherichia coli strain LF82 with the yfgL gene deleted

Nathalie Rolhion et al. J Bacteriol. 2005 Apr.

Abstract

Adherent-invasive Escherichia coli strain LF82 recovered from a chronic lesion of a patient with Crohn's disease is able to invade cultured intestinal epithelial cells VSports手机版. Three mutants with impaired ability to invade epithelial cells had the Tn5phoA transposon inserted in the yfgL gene encoding the YfgL lipoprotein. A yfgL- negative isogenic mutant showed a marked decrease both in its ability to invade Intestine-407 cells and in the amount of the outer membrane proteins OmpA and OmpC in the culture supernatant, as shown by analysis of the culture supernatant protein contents by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Transcomplementation of the LF82-DeltayfgL isogenic mutant with the cloned yfgL gene restored invasion ability and outer membrane protein release in the culture supernatant. The outer membrane proteins in the culture supernatant of strain LF82 resulted from the formation of vesicles. This was shown by Western blot analysis of periplasmic and outer membrane fraction markers typically found in outer membrane vesicles and by transmission electron microscopic analysis of ultracentrifuged cell-free LF82 supernatant pellets, indicating the presence of vesicles with a bilayered structure surrounding a central electron-dense core. Thus, deletion of the yfgL gene in strain LF82 resulted in a decreased ability to invade intestinal epithelial cells and a decreased release of outer membrane vesicles. .

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Figures

FIG. 1.
FIG. 1.
Invasion ability, motility, and type 1 pilus expression of E. coli mutants with the Tn5phoA transposon inserted in the yfgL gene. A. Intracellular bacteria in human intestinal epithelial cells (Intestine-407 cells) were quantified after a 3-h infection period, followed by gentamicin treatment for an additional hour. Results are expressed as the percentage of intracellular bacteria relative to that of E. coli strain LF82, which is set at 100%. The invasion level of the wild-type strain LF82 expressed as a mean percentage of the original inoculum was 0.95% ± 0.10%. Each value is the mean ± standard error of the mean (error bar) of at least four separate experiments. Values that were significantly different from the value for wild-type strain LF82 (P < 0.001) are indicated by an asterisk. B. Motility assay on 0.3% agar after 18 h at 37°C. Motility was visualized as a halo of radial diffusion of bacteria around the primary inoculum. C. Expression of type 1 pili, analyzed by colony immunoblotting using rabbit antiserum raised against purified type 1 pili. Mutant 52D11 with an insertion of Tn5phoA within the fimA gene was used as a negative control for pilus type 1 expression (7).
FIG. 2.
FIG. 2.
Phenotype of the E. coli LF82-ΔyfgL isogenic mutant. A. Time course of bacterial growth in LB broth of AIEC LF82 wild-type strain (open circles) and LF82-ΔyfgL isogenic mutant (black circles). The initial inoculum was 4 × 106 bacteria per ml. Results are means ± standard errors of the means (error bars) of six separate experiments. OD620nm, optical density at 620 nm. B. Motility assay on 0.3% agar after 18 h at 37°C. Motility was visualized as a halo of radial diffusion of bacteria around the primary inoculum.
FIG. 3.
FIG. 3.
Transmission electron micrograph of negatively stained E. coli LF82 bacteria (A) and LF82-ΔyfgL isogenic mutant (B). Magnifications: ×22,000 (A) and ×27,000 (B).
FIG. 4.
FIG. 4.
Restoration of the invasion defect of the E. coli LF82-ΔyfgL isogenic mutant after transcomplementation with the plasmid pPBI07 harboring the entire LF82 yfgL gene. Invasion of Intestine-407 cells was determined after a 3-h infection period, followed by gentamicin treatment for an additional hour. Results are expressed as the percentage of intracellular bacteria relative to that obtained for wild-type strain LF82, which was set at 100%. The invasion level of the wild-type strain LF82 expressed as the mean percentage of the original inoculum was 1.31% ± 0.13%. Each value is the mean ± standard error of the mean (error bar) of at least three separate experiments. Values that were significantly different from the value for wild-type strain LF82 (P < 0.001) are indicated by an asterisk.
FIG. 5.
FIG. 5.
Transmission electron micrographs of Intestine-407 cells infected with E. coli strain LF82 (A) and LF82-ΔyfgL isogenic mutant (B). Strain LF82 adhered to Intestine-407 cells and induced the elongation of membrane extensions upon contact with the eukaryotic cell membranes. Numerous bacteria were found inside Intestine-407 cells. In contrast, no intracellular bacteria were observed with the LF82-ΔyfgL isogenic mutant, even if adhering bacteria induced membrane elongations. Magnification,×6,000.
FIG. 6.
FIG. 6.
Release of proteins into the E. coli LF82 culture supernatant. Bacterial cells were grown without shaking at 37°C in LB broth to early stationary phase. A. Whole-cell lysates (WC) and secreted proteins in culture supernatants (S) of strain LF82 and LF82-ΔyfgL isogenic mutant were separated by SDS-PAGE and stained with Coomassie blue. The positions of secreted proteins in the culture supernatant of strain LF82 identified by MS are marked by a small black triangle. B. Western immunoblot analysis of OmpC/F (top) and OmpA (bottom) outer membrane proteins in whole-cell lysates (WC) and culture supernatants (S). The positions of OmpC/F and OmpA protein bands detected by antibodies correlate with the protein bands analyzed by MS. C. Outer membrane preparations (OM) of strain LF82 and LF82-ΔyfgL isogenic mutant were separated by SDS-PAGE and stained with Coomassie blue. The positions of molecular mass markers (in kilodaltons) are shown at the sides of the gels. Lanes: 1, strain LF82; 2, LF82-ΔyfgL isogenic mutant.
FIG. 7.
FIG. 7.
Analysis of protein content of OMVs released by E. coli strain LF82 and LF82-ΔyfgL isogenic mutant. Bacterial cells were grown overnight without shaking at 37°C in LB broth. A. Whole-cell lysates (WC) and OMV preparations were separated by SDS-PAGE and stained with Coomassie blue. The positions of molecular mass markers (in kilodaltons) are shown to the left of the gel. B. Western immunoblot analysis of the OmpC/F and OmpA outer membrane proteins, the MalE periplasmic protein, the Lep inner membrane protein, and the α subunit of RNA polymerase cytoplasmic protein in whole-cell lysates (WC) and OMV preparations. Lanes: 1, strain LF82; 2, LF82-ΔyfgL isogenic mutant; 3, LF82-ΔyfgL isogenic mutant transcomplemented with pPBI07 harboring the entire yfgL gene; 4, LF82-ΔyfgL isogenic mutant transformed with the pBAD33 vector alone.
FIG. 8.
FIG. 8.
Transmission electron micrographs of negatively stained OMVs isolated from the culture supernatant of E. coli strain LF82 (A and B) and immunogold labeling of OMV preparation (C) using antibodies raised against E. coli lipopolysaccharide O83. Bars, 100 nm.
FIG. 9.
FIG. 9.
Analysis of protein content of OMVs released by E. coli strain MC4100 and MC4100-ΔyfgL isogenic mutant. Bacterial cells were grown overnight without shaking at 37°C in LB broth. A. OMV preparations were separated by SDS-PAGE and stained with Coomassie blue. The positions of molecular mass markers (in kilodaltons) are shown to the left of the gel. B and C. Western immunoblot analysis of OmpA (B) and MalE (C) in OMV preparation. Lanes: 1, strain MC4100; 2, MC4100-ΔyfgL isogenic mutant. Compared to Fig. 7, the E. coli K-12 OMV preparations were concentrated threefold.
FIG. 10.
FIG. 10.
Effect of coinfection with wild-type E. coli strain LF82 or pretreatment of intestinal epithelial cells with LF82 OMVs on the invasive level of LF82-ΔyfgL isogenic mutant. For coinfection experiments, invasion of Intestine-407 cells by LF82-ΔyfgL isogenic mutant was determined after a 3-h infection period, followed by gentamicin treatment for an additional hour and determination of the number of bacteria on Mueller-Hinton agar plates containing kanamycin. For experiments in the presence of LF82 OMVs, Intestine-407 cells were pretreated with OMVs for 1 h and then infected by bacteria. Results are expressed as the percentage of intracellular bacteria relative to that obtained for LF82-ΔyfgL isogenic mutant, which was set at 100%. The invasion level of LF82-ΔyfgL isogenic mutant expressed as the mean percentage of the original inoculum was 0.15% ± 0.11%. Each value is the mean ± standard error of the mean (error bar) of at least three separate experiments. Values that were significantly different from the value for the LF82-ΔyfgL isogenic mutant alone (P < 0.001) are indicated by an asterisk.
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