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. 2007 Jul;189(13):4860-71.
doi: 10.1128/JB.00233-07. Epub 2007 Apr 20.

The oxidoreductase DsbA plays a key role in the ability of the Crohn's disease-associated adherent-invasive Escherichia coli strain LF82 to resist macrophage killing

Marie-Agnès Bringer  1 , Nathalie Rolhion, Anne-Lise Glasser, Arlette Darfeuille-Michaud
Affiliations

The oxidoreductase DsbA plays a key role in the ability of the Crohn's disease-associated adherent-invasive Escherichia coli strain LF82 to resist macrophage killing

Marie-Agnès Bringer et al. J Bacteriol. 2007 Jul.

Abstract

Adherent-invasive Escherichia coli (AIEC) isolated from Crohn's disease patients is able to adhere to and invade intestinal epithelial cells and to replicate in mature phagolysosomes within macrophages VSports手机版. Here, we show that the dsbA gene, encoding a periplasmic oxidoreductase, was required for AIEC strain LF82 to adhere to intestinal epithelial cells and to survive within macrophages. The LF82-DeltadsbA mutant did not express flagella and, probably as a consequence of this, did not express type 1 pili. The role of DsbA in adhesion is restricted to the loss of flagella and type 1 pili, as forced contact between bacteria and cells and induced expression of type 1 pili restored the wild-type phenotype. In contrast, the dsbA gene is essential for AIEC LF82 bacteria to survive within macrophages, irrespective of the loss of flagella and type 1 pilus expression, and the survival ability of LF82-DeltadsbA was as low as that of the nonpathogenic E. coli K-12, which was efficiently killed by macrophages. We also provide evidence that the dsbA gene is needed for LF82 bacteria to grow and survive in an acidic and nutrient-poor medium that partly mimics the harsh environment of the phagocytic vacuole. In addition, under such stress conditions dsbA transcription is highly up-regulated. Finally, the CpxRA signaling pathway does not play a role in regulation of dsbA expression in AIEC LF82 bacteria under conditions similar to those of mature phagolysosomes. .

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V体育平台登录 - Figures

FIG. 1.
FIG. 1.
Deletion of the dsbA gene affects neither the growth of bacteria nor yihE expression in the LF82-ΔdsbA mutant. (A) Growth of the wild-type strain LF82 and of LF82-ΔdsbA in RPMI medium supplemented with 10% heat-inactivated FCS. (B) Analysis of yihE mRNAs of LF82 and LF82-ΔdsbA by two-step RT-PCR using primers annealing the 5′and 3′ ends of the yihE cDNA. (C) yihE mRNA level of LF82-ΔdsbA relative to that of the wild-type strain LF82 determined by real-time RT-PCR experiments after growth of the bacteria in LB medium. Only experiments showing the same levels of 16S rRNA for each sample were taken into account.
FIG. 2.
FIG. 2.
The dsbA gene is required for survival of strain LF82 within J774 macrophages. (A) Uptake of the LF82, LF82-ΔdsbA, LF82-ΔdsbA transformed with the cloned dsbA gene (pPBI11), and LF82-ΔdsbA harboring the vector alone (pBAD33). Results are expressed as the number of intracellular bacteria after 1 h of gentamicin treatment relative to that obtained for strain LF82, taken as 100%. (B) Bacterial survival and replication after 24 h of gentamicin treatment. Results are expressed as the number of intracellular bacteria at 24 h relative to that obtained at 1 h after gentamicin exposure, taken as 100%. The nonpathogenic E. coli K-12 strain C600, which is efficiently killed by J774, was used as a negative control. Data are means ± standard errors of the means for four separate experiments. *, P < 0.05. (C to F) Confocal microscopic examinations of J774 macrophages infected with wild-type strain LF82 and LF82-ΔdsbA. Bacteria were transformed with plasmid pFPV25.1 for constitutive expression of GFP. The actin cytoskeleton of J774 cells was stained with TRITC-labeled phalloidin. Shown are J774-A1 macrophages infected with the LF82 wild-type strain (C and D) and with LF82-ΔdsbA (E and F) at 1 h postinfection (C and E) and at 24 h postinfection (D and F).
FIG. 3.
FIG. 3.
Phenotype of the LF82-ΔdsbA mutant in Intestine-407 epithelial cells. (A) Adhesion of LF82, LF82-ΔdsbA, LF82-ΔdsbA transformed with the cloned dsbA gene (pPBI11), and LF82-ΔdsbA harboring the vector alone (pBAD33) to Intestine-407 cells. Cell-associated bacteria were quantified after a 3-h infection period. The results are expressed as levels of cell-associated bacteria (adherent plus intracellular) relative to those obtained for wild-type strain LF82, taken as 100%. (B) Bacterial invasion of Intestine-407 cells was determined after gentamicin treatment for an additional 1 h. The results are expressed as levels of intracellular bacteria relative to those obtained for wild-type strain LF82, taken as 100%. (C) Percentage of intracellular bacteria relative to adherent bacteria. Data are means ± standard errors of the means for four separate experiments. *, P < 0.05.
FIG. 4.
FIG. 4.
Lack of expression of type 1 pili and flagella in LF82-ΔdsbA. Experiments were performed with strain LF82, LF82-ΔdsbA, LF82-ΔdsbA transformed with the cloned dsbA gene (pPBI11), and LF82-ΔdsbA harboring the vector alone (pBAD33) (A) Motility was visualized on a 0.3% agar plate as a halo of radial diffusion of bacteria around the primary inoculum after 16 h at 37°C. (B) Colony immunoblotting using polyclonal antibodies raised against purified type 1 pili. (C) Determination by PCR analysis of the invertible element orientation of the fim operon. A 450-bp product revealed the phase-ON orientation, and a 750-bp product revealed the phase-OFF orientation of the invertible element (58).
FIG. 5.
FIG. 5.
TEM of gold immunolabeling of LF82 bateria (A), LF82-ΔdsbA (B), and LF82-ΔdsbA transformed with plasmid pPBI11 harboring cloned dsbA (C), with plasmid pPBI01 harboring the cloned E. coli K-12 fim operon (D), or with cosmid JE7 harboring the cloned LF82 fim operon (E) using polyclonal antibodies raised against purified type 1 pili. Arrows indicate a flagellar structure. Bars, 0.5 μm.
FIG. 6.
FIG. 6.
Effects of induced type 1 pilus expression and forced contact between bacteria and host cells on LF82-ΔdsbA interactions with intestinal epithelial cells and macrophages. (A) Adhesion and invasion abilities of LF82-ΔdsbA transformed with pPBI01, harboring the entire fim operon, with Intestine-407 epithelial cells. Experiments were performed with an initial centrifugation step to establish close contact between bacteria and epithelial cells to bypass the absence of bacterial motility. (B) Intramacrophagic survival ability of LF82 and LF82-ΔdsbA with or without induced type 1 pilus expression obtained by transformation with pPBI01, harboring the entire fim operon, and LF82-ΔfliC. See the legends to Fig. 2 and 3. Data are means ± standard errors of the means for four separate experiments. *, P < 0.05.
FIG. 7.
FIG. 7.
Involvement of DsbA in bacterial growth and dsbA gene expression in AIEC strain LF82 under in vitro phagocytic vacuole stress conditions. (A) Growth of the wild-type strain LF82, LF82-ΔdsbA, and LF82-ΔdsbA transcomplemented with plasmid pPBI11, harboring the dsbA gene, in acidic and nutrient-poor medium. (B) dsbA and htrA mRNA levels of the wild-type strain LF82 and the LF82-ΔcpxR mutant determined by RT-PCR experiments after growth of the bacteria in acidic and nutrient-poor medium relative to that of bacteria grown in LB broth. Only experiments showing the same levels of 16S rRNA for each sample were taken into account. Data are means ± standard errors of the means for three separate experiments. *, P < 0.05. (C) Intracellular survival ability of LF82, LF82-ΔdsbA, and LF82-ΔhtrA within macrophages. See the legend to Fig. 2 for experiment setup details. Data are means ± standard errors of the means for three separate experiments. *, P < 0.05.
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