GroEL of Lactobacillus johnsonii La1 (NCC 533) is cell surface associated: potential role in interactions with the host and the gastric pathogen Helicobacter pylori

Abstract

Heat shock proteins of the GroEL or Hsp60 class are highly conserved proteins essential to all living organisms. Even though GroEL proteins are classically considered intracellular proteins, they have been found at the surface of several mucosal pathogens and have been implicated in cell attachment and immune modulation. The purpose of the present study was to investigate the GroEL protein of a gram-positive probiotic bacterium, Lactobacillus johnsonii La1 (NCC 533). Its presence at the bacterial surface was demonstrated using a whole-cell enzyme-linked immunosorbent assay and could be detected in bacterial spent culture medium by immunoblotting. To assess binding of La1 GroEL to mucins and intestinal epithelial cells, the La1 GroEL protein was expressed in Escherichia coli. We report here that La1 recombinant GroEL (rGroEL) binds to mucins and epithelial cells and that this binding is pH dependent. Immunomodulation studies showed that La1 rGroEL stimulates interleukin-8 secretion in macrophages and HT29 cells in a CD14-dependent mechanism. This property is common to rGroEL from other gram-positive bacteria but not to the rGroEL of the gastric pathogen Helicobacter pylori. In addition, La1 rGroEL mediates the aggregation of H. pylori but not that of other intestinal pathogens. Our in vitro results suggest that GroEL proteins from La1 and other lactic acid bacteria might play a role in gastrointestinal homeostasis due to their ability to bind to components of the gastrointestinal mucosa and to aggregate H. pylori.

FIG. 1.

(A) Localization of GroEL at the surface of La1. The presence of GroEL at the surface of La1 was determined by ELISA as described in Materials and Methods using a polyclonal anti-GroEL antibody as the primary antibody (•). An anti-β-lactoglobulin antibody (○) was used as a negative control. (B) GroEL release during logarithmic phase growth was monitored by Western blotting in La1 supernatants after concentration with 60% ammonium sulfate. The rGroEL protein was used as a positive control.

FIG. 2.

Binding properties of La1 GroEL. The capacity of La1 rGroEL (•) to bind mucins (A and B) and to bind to HT29 cells (C and D) was tested at pH 5.0 (A and C) and 7.2 (B and D). Two other La1 recombinant proteins were tested in parallel: rLJ1080 (⧫) and rLJ0752 (▴). Bound recombinant molecules were revealed using an anti-His tag antibody. Results are representative of three independent experiments and are expressed as the mean ± the range of variation (n = 2).

FIG. 3.

La1 binding assays to HT29 cells in the presence of La1 rGroEL. Nondifferentiated HT29 cells were incubated with increasing concentrations of La1 rGroEL followed by a second incubation with 2.5 × 10^{6 3}H-adenine-labeled La1 bacteria/well. Bound La1 cells were determined by radioactivity counting. Results are expressed as percentage of binding in the absence of La1 rGroEL (mean ± standard deviation; n = 6).

FIG. 4.

Stimulation of IL-8 secretion from HT29 cells by La1 rGroEL. IL-8 release from HT29 cells was measured by ELISA after stimulation for 24 h with increasing concentrations of La1 GroEL or LJ1080 recombinant proteins or E. coli LPS in the absence (open bars) or presence (solid bars) of 2% HM as a source of sCD14. DMEM with or without HM was used as a negative control. Where indicated (dotted bars), cells were simultaneously treated with anti-CD14 (MY4) or IgG2b antibodies or heat treated (℘). IL-8 secretion was measured in triplicates, and the results are expressed as the mean ± standard deviation. The figure shows one representative experiment of two independent experiments.

FIG. 5.

Stimulation of IL-8 secretion from macrophages by rGroEL proteins from different bacteria. Macrophages were isolated from healthy donors as described in Materials and Methods. IL-8 release was measured by ELISA after stimulation for 24 h with 1 μg/ml rGroEL proteins from different bacteria or 1 ng/ml E. coli LPS. Open and solid bars represent two different donors. Where indicated, cells were simultaneously treated with anti-CD14 (MY4) or IgG2b antibodies or heat treated (℘). The figure shows one representative experiment of four independent experiments using macrophages from two different donors. La1, L. johnsonii NCC 533; Lhe, L. helveticus ATCC 15009; Bsu, B. subtilis NCC 199; Lla, Lactococcus lactis strain MG 1363; Hp, H. pylori strain P1. RPMI, medium alone used as a negative control.

FIG. 6.

La1 rGroEL aggregating capacity. H. pylori and La1 (A) as well as S. enterica serovar Typhimurium SL1344 and E. coli E 2348/69 (B) in DMEM, pH 5.4, were incubated with no rGroEL as a control, with 0.1 μg of La1 rGroEL/ml, or with 1 μg of H. pylori rGroEL for 1 h at 37°C as indicated to the left of the panels. After elimination of bacterial clumps by manual shaking and vortexing, 10 μl of the preparation was loaded on a microscope slide and GroEL-mediated aggregation was assessed under microscope at a ×100 magnification and then photographed.