DNA, cell wall and general oxidative damage underlie the tellurite/cefotaxime synergistic effect in Escherichia coli

Abstract

The constant emergence of antibiotic multi-resistant pathogens is a concern worldwide. An alternative for bacterial treatment using nM concentrations of tellurite was recently proposed to boost antibiotic-toxicity and a synergistic effect of tellurite/cefotaxime (CTX) was described. In this work, the molecular mechanism underlying this phenomenon is proposed. Global changes of the transcriptional profile of Escherichia coli exposed to tellurite/CTX were determined by DNA microarrays. Induction of a number of stress regulators (as SoxS), genes related to oxidative damage and membrane transporters was observed. Accordingly, increased tellurite adsorption/uptake and oxidative injuries to proteins and DNA were determined in cells exposed to the mixture of toxicants, suggesting that the tellurite-mediated CTX-potentiating effect is dependent, at least in part, on oxidative stress. Thus, the synergistic tellurite-mediated CTX-potentiating effect depends on increased tellurite uptake/adsorption which results in damage to proteins, DNA and probably other macromolecules. Our findings represent a contribution to the current knowledge of bacterial physiology under antibiotic stress and can be of great interest in the development of new antibiotic-potentiating strategies.

Figure 1. Extracellular tellurite concentration in culture supernatants.

Tellurite concentration was determined in supernatants of E. coli cultures that had been exposed to tellurite (20 µg ml⁻¹) in the absence (control) or presence of the indicated CTX concentrations. Values represent the average of 3 independent experiments. Statistical significance was determined using t-test. (**) p<0.01.

Figure 2. Hydroxyl radical generation in E. coli exposed to CTX or Te/CTX.

E. coli was exposed for 3 h to tellurite/CTX (A) and to different antibiotic concentrations (B). As positive control, cells were exposed for 3 h to ampicillin. Units are expressed in µg ml⁻¹. The data correspond to a representative result of 3 independent trials.

Figure 3. Tellurite/CTX-mediated damage to macromolecules.

A, protein oxidation was assessed by determining the content of carbonyl groups after 15 µg ml⁻¹ tellurite, 0.5 µg ml⁻¹ CTX or tellurite/CTX at the same concentrations. H₂O₂ (2.5 mM) was used as positive control of oxidation. B, DNA damage was determined by real time PCR using total DNA from cultures exposed during 3 h to 0.05 µg ml⁻¹ tellurite, 0.13 µg ml⁻¹ CTX or TeO₃ ^2−/CTX (at the same concentrations). FeSO₄ and H₂O₂ (50 µM and 100 mM, respectively) were used as positive control of DNA damage (Fenton reaction).

Figure 4. Tellurite/CTX damage in E. coli.

Peptidoglycan integrity (stability) is affected by CTX (1), favoring tellurite entry (2). Tellurite/CTX administration generates global transcriptional changes on different stress response pathways, transport, [Fe-S] clusters assembly, protein folding and different oxidative stress regulators (3). Finally, CTX and tellurite generate hydroxyl radical and superoxide respectively, damaging DNA, proteins and most probably other macromolecules.