Figure 1.
Growth of H. pylori in the presence of increasing concentrations of DHA.
A) DHA was added to the H. pylori culture strains 26695, SS1 and B128 after 12 h of growth. Each experiment, consisting of a control (non-treated H. pylori culture) and H. pylori incubated with DHA (concentrations of 50 µM, 100 µM, 250 µM and 500 µM) performed in triplicate. Ethanol (0.06% v/v) the solvent of DHA stock solutions was also tested in these experiments and it did not influence H. pylori growth. B) H. pylori was grown in liquid cultures with concentrations of DHA ranging from 50 µM to 1000 µM. Twenty-four hours later cells were pellet and put to grow into a fresh medium without DHA. H. pylori growth was irreversibly inhibited by DHA at the highest concentrations (≥500 µM of DHA). Data are expressed as the mean ± Standard Deviation and are representative of three independent experiments. * Refers to significant differences in H. pylori growth between controls and DHA-treated conditions (50 µM to 1000 µM of DHA).
Figure 2.
DHA impairs H. pylori metabolic activity.
Cultures of H. pylori 26695 grew for 24 h, and every six hours the relative ATP levels were measured as described in materials and methods. Luminescence units per 106 bacteria at each time point resulted from 3 independent experiments. T-test analysis confirmed a significant differences in ATP production between H. pylori untreated controls and H. pylori treated with 100 µM of DHA, (P<0.05) and with 250 µM of DHA (P<0.001).
Figure 3.
DHA treatment decreases H. pylori adhesion to epithelial gastric cells.
AGS cells are infected 24 h either with H. pylori strain 26695 pre-treated for 24 h with DHA 50 µM, 100 µM or 250 µM, or non-treated bacteria. A) Quantification of H. pylori attachment to AGS cells by ELISA using and anti-Helicobacter antibody. Pretreatment of H. pylori with DHA significantly decreased the bacterial adherence to AGS cells for treatment of 50 µM and above (*P<0.05). Data are expressed as the mean ± SE and are representative of three independent experiments. B) TEM observation of cells shows less attachment of H. pylori DHA – pretreated (100 µM) compared to cells infected with non-treated bacteria.
Figure 4.
DHA pre-treatment of H. pylori leads to a reduced inflammatory response.
A) AGS cells were cocultured with H. pylori strain 26695 for 24 h previously exposed to DHA, and then measured IL-8 production with an ELISA-like test. Pre-treatment of bacteria with DHA from 50 µM to 250 µM led to a significant decrease of 4-fold in IL-8 production as compared to non-DHA treated bacteria * P<0.05. Data are expressed as the mean ± SE and are representative of three independent experiments. B) Expression of COX2 and iNOS on protein extracts from AGS cells infected with H. pylori 26695 previously treated with DHA. α-tubulin corresponds to the loading control. The values are reported under the corresponding gel bands and showed a significant decrease of COX2, and iNOS levels in cells infected with DHA-pretreated H. pylori compared to untreated bacteria.
Figure 5.
Consequences of DHA exposure on H. pylori protein profile, LPS phenotype and MreC expression.
A) H. pylori 26695 proteins profile from outer membrane extracts in a 1D SDS-PAGE gel. Arrows are pointing out five bands that correspond to proteins with a decreased level after treatment of bacteria with 100 µM and 250 µM of DHA. Candidate proteins were identified and referred as FrpB4/HP1512 (arrow I), HorL/HP1395 (arrow II), HorB/HP0127 (arrow III), and HorJ/HP1469 (arrow IV). B) H. pylori 26695 proteins profile for outer membrane extracts in a 2D SDS-PAGE gel. Twelve proteins identified by MALDI TOF/TOF mass spectrometry presented a differential expression level when comparing untreated control and DHA-treated bacteria. These proteins were mostly related with induction of stress condition as listed in the Table 1. C) Western blot analysis of MreC protein level in DHA-treated bacteria. H. pylori 26695 grew as previously described until a maximum of 1 OD600. MreC expression was assessed using a rabbit anti-MreC antibody (1∶10000) [22]. D) H. pylori 26695 LPS profile. LPS was extracted from H. pylori strain 26695 cultured for 12 h in the presence of 100 µM of DHA and silver stained in a Trycine SDS-PAGE as described in material and methods. In absence of DHA, the H. pylori strain 26695 showed a low molecular weight rough (R)-form LPS phenotype. At concentrations of 100 µM of DHA, the LPS shifted from (R)-form to the high molecular weight smooth (S) form phenotype.
Table 1.
Identified H. pylori outer membrane proteins differentially expressed following DHA treatment.