Catecholate siderophores protect bacteria from pyochelin toxicity

Abstract

Background: Bacteria produce small molecule iron chelators, known as siderophores, to facilitate the acquisition of iron from the environment. The synthesis of more than one siderophore and the production of multiple siderophore uptake systems by a single bacterial species are common place. The selective advantages conferred by the multiplicity of siderophore synthesis remains poorly understood. However, there is growing evidence suggesting that siderophores may have other physiological roles besides their involvement in iron acquisition.

Methods and principal findings: Here we provide the first report that pyochelin displays antibiotic activity against some bacterial strains. Observation of differential sensitivity to pyochelin against a panel of bacteria provided the first indications that catecholate siderophores, produced by some bacteria, may have roles other than iron acquisition. A pattern emerged where only those strains able to make catecholate-type siderophores were resistant to pyochelin. We were able to associate pyochelin resistance to catecholate production by showing that pyochelin-resistant Escherichia coli became sensitive when biosynthesis of its catecholate siderophore enterobactin was impaired. As expected, supplementation with enterobactin conferred pyochelin resistance to the entE mutant. We observed that pyochelin-induced growth inhibition was independent of iron availability and was prevented by addition of the reducing agent ascorbic acid or by anaerobic incubation. Addition of pyochelin to E. coli increased the levels of reactive oxygen species (ROS) while addition of ascorbic acid or enterobactin reduced them. In contrast, addition of the carboxylate-type siderophore, citrate, did not prevent pyochelin-induced ROS increases and their associated toxicity.

Conclusions: We have shown that the catecholate siderophore enterobactin protects E. coli against the toxic effects of pyochelin by reducing ROS. Thus, it appears that catecholate siderophores can behave as protectors of oxidative stress. These results support the idea that siderophores can have physiological roles aside from those in iron acquisition.

Figure 1. Chemical structures of the siderophores pyochelin, watasemycin A, yersiniabactin, enterobactin and citrate.

Figure 2. Levels of reactive oxygen species in E. coli wild type and entE mutant.

Quantitation of ROS levels using the DCFA-DA probe. Fluorescence intensities are relative to that of the control. Control: wt grown in 2 mL M9 medium; PCH indicates cells grown in the presence of 15 µM pyochelin; ASC indicates supplementation with 1 mM ascorbic acid; ENT designates addition of 1 µM of pure enterobactin; CIT indicates supplementation with 50 µM citrate. Error bars = SEM, n = 3.