The action of bismuth against Helicobacter pylori mimics but is not caused by intracellular iron deprivation

Abstract

Helicobacter pylori is highly susceptible to bismuth, a heavy metal with antimicrobial activity linked to its effect on bacterial iron uptake. Three strains of H. pylori were analyzed for indicators of iron limitation following exposure to the MIC of colloidal bismuth subcitrate (MIC(CBS)). Similar morphologic and outer membrane changes were observed following growth in iron-limiting medium and at the MIC(CBS) that inhibited the growth of all three strains. These changes, which were also observed for iron-limited bacteria, were alleviated by the addition of iron to the cultures. H. pylori ATP levels, reduced in iron-limiting medium, were below the limits of detection in two of the three strains following exposure to bismuth. The addition of iron partially restored bacterial ATP levels in these two strains, although not to normal concentrations. In contrast, exposure of the same strains to the MIC(CBS) failed to deplete intracellular levels of iron, which were significantly reduced by culturing in iron-limiting medium. Thus, the antimicrobial effect of bismuth and of iron limitation on H. pylori may be similar. However, the respective mechanisms of intracellular action would appear to be mediated by different pathways within the cell.

FIG. 1.

Effects of bismuth exposure and iron limitation on H. pylori morphology. TEM images (magnification, ×28,900) show strain 60190 following growth in 50 μM deferoxamine (A), MIC_CBS (B), MIC_CBS and protective iron (C), and normal culture conditions (D). The arrow in panel A indicates coccoid morphology; the arrowhead in panel B indicates distorted morphology. Similar results were obtained for strains Tx-30a and SS1 (data not shown).

FIG. 2.

Effects of bismuth exposure and iron limitation on H. pylori OMV protein composition. Shown is silver staining of SS1 (A) and Tx-30a (B) OMV-associated proteins following growth in 50 μM deferoxamine (lanes 1), MIC_CBS (lanes 2), MIC_CBS and protective iron (lanes 3), and normal culture conditions (lanes 4). The arrows indicate a 12-kDa protein band. Results obtained for SS1 were also representative of the 60190 OMV protein composition (data not shown).

FIG. 3.

Effects of bismuth exposure and iron limitation on H. pylori OMV LPS composition. Shown is silver staining of SS1 (A) and Tx-30a (B) OMV-associated LPS following growth in 50 μM deferoxamine (lanes 1), MIC_CBS (lanes 2), MIC_CBS and protective iron (lanes 3), and normal culture conditions (lanes 4). Similar results were obtained for 60190 (data not shown).

FIG. 4.

Effects of bismuth exposure and iron limitation on H. pylori OMV protease activity. Zymography was performed on OMV harvested from SS1 following growth in 50 μM deferoxamine (lane 1), MIC_CBS (lane 2), MIC_CBS and protective iron (lane 3), and normal culture conditions (lane 4). Protease activity is indicated (arrowheads). Similar results were obtained for 60190 and Tx-30a (data not shown). Lane M, markers.

FIG. 5.

Effects of bismuth exposure and iron limitation on H. pylori intracellular iron levels. Iron levels per microgram of bacterial dry weight were determined for H. pylori 60190, SS1, and Tx-30a cultured in the presence of normal growth conditions (control), iron-limiting conditions (De), MIC_CBS (Bi), MIC_CBS plus protective iron (Bi + Fe), and protective iron alone (Fe). Data are the means and standard errors of two independent experiments performed in duplicate. An asterisk indicates that the result is statistically significantly different (P < 0.05) from that for control (untreated) bacteria.

FIG. 6.

Effects of bismuth exposure and iron limitation on H. pylori intracellular ATP levels. ATP levels per microgram of bacterial dry weight were determined for H. pylori 60190, SS1, and Tx-30a following growth in the presence of normal growth conditions (control), iron-limiting conditions (De), MIC_CBS (Bi), MIC_CBS plus protective iron (Bi + Fe), and protective iron alone (Fe). Data are the means and standard errors of two independent experiments performed in duplicate. An asterisk indicates that the result is statistically significantly different (P < 0.05) from that for control (untreated) bacteria.