Iron and citrate export by a major facilitator superfamily pump regulates metabolism and stress resistance in Salmonella Typhimurium

Abstract

The efficacy of antibiotics and host defenses has been linked to the metabolic and redox states of bacteria. In this study we report that a stress-induced export pump belonging to the major facilitator superfamily effluxes citrate and iron from the enteric pathogen Salmonella Typhimurium to arrest growth and ameliorate the effects of antibiotics, hydrogen peroxide, and nitric oxide. The transporter, formerly known as MdtD, is now designated IceT (iron citrate efflux transporter). Iron efflux via an iron-chelating tricarboxylic acid cycle intermediate provides a direct link between aerobic metabolism and bacterial stress responses, representing a unique mechanism of resistance to host defenses and antimicrobial agents of diverse classes.

Keywords: antibiotic resistance; oxidative stress.

Fig. 1.

MudJ transposon insertions in mdtABCD baeSR confer streptonigrin sensitivity. A screen for S. Typhimurium mutants with enhanced SN sensitivity identified three independent insertions in mdtB. The mdtABCD baeSR operon encodes an RND-family efflux pump (MdtABC) that forms a complex with TolC to efflux substrates, an uncharacterized MFS pump (MdtD/IceT), and a two-component regulatory system (BaeSR).

Fig. 2.

Expression of MdtD(IceT) is directly related to streptonigrin resistance. (A) In the absence of SN, EF221 [ΔmdtD(iceT); solid green line] grows as well as isogenic WT strain EF3 (solid blue line). In 6 μg mL⁻¹ SN, the mutant strain exhibits delayed exit from lag phase (dashed green line). Mean lag-time for EF221 to reach 50% max OD₆₀₀ (horizontal dashed line) was 5.73 h, P = 0.026. (B) An insertion in the araBAD locus does not affect growth of EF390 (blue), EF391 (green), and EF393 (orange) in LB (solid lines). In 6 μg mL⁻¹ SN, expression of mdtD(iceT) under control of its native P_mdtA promoter in strain EF393 (dashed orange line) complements the growth defect of EF391 [ΔmdtD(iceT), dashed green line], with similar growth to EF390, which contains an intact mdtD(iceT) gene (dashed blue line). (C) EF395 [Δfur ΔmdtD(iceT)] is more susceptible to killing by 10 μg mL⁻¹ SN than EF394 (Δfur) at 30 min P = 0.032, 60 min P = 0.008, 90 min P = 0.08, and 120 min P = 0.006. (D) EF35 (dark green columns) expressing mdtD(iceT) is more resistant to killing by 10 μg mL⁻¹ SN than EF39 (dark blue columns) containing empty vector at 30 min P = 0.0008, 60 min P = 0.0088, and 90 min P = 0.0046. Addition of 0.1 mM o-phenanthroline, an iron chelator, abrogated SN-killing in both strains (light blue and light green columns). Growth curves (A and B) are an average of seven experiments. Killing data are the mean of eight (C) and four (D) replicates ±1 SD. Statistical significance (*) was determined by two-tailed t test.

Fig. 3.

The mdtABCD baeSR operon is induced by NO· stress and disrupted iron homeostasis. (A) EF3 (WT) and EF214 (ΔbaeSR) were treated with 1 mM DEA mock treatment or 1 mM NO· DEA/NO and expression of mdtA, mdtB, and mdtD(iceT) assayed by qPCR. In the WT background, genes were significantly up-regulated upon DEA/NO treatment (P < 0.006). In the ΔbaeSR background, only a modest residual level of induction was observed (P < 0.05). (B) EF3 and EF221 were grown in 1 mM DEA/NO and 2 mM Sper/NO. The ΔmdtD(ΔiceT) mutant (green line) exhibited delayed exit from lag phase. Mean lag time for EF221 to reach 50% max OD₆₀₀ (dashed line) was 2.9 h (P = 0.013). (C) Expression of mdtA, mdtB, and mdtD(iceT) was compared in EF394 (Δfur) and EF3 (WT) by qPCR and shown to be significantly up-regulated (P < 0.03). Expression of bfd, sitA, and bfr was measured as a control for Fur-dependent gene expression. Expression data are the mean of three replicates ±1 SD with significance (*) determined by two-tailed t test. Growth curves are an average of five experiments with significance determined by paired two-tailed t test.

Fig. 4.

Cells expressing MdtD(IceT) are resistant to iron-mediated cell death and have reduced total iron content. (A) Strains HR111 and HR112 expressing the FeoAB iron import system were grown in LB containing 2 mM FeSO₄ for 30 min. HR112 expressing MdtD(IceT) (purple) better survived iron challenge by almost 2 log₁₀ compared with HR111 containing empty vector (blue). (B) HR111 and HR112 were grown in 1 mM FeSO₄ for 30 min before total iron content was determined by ICP-MS. HR111 (blue) contained more than twice as much iron as HR112 (purple). (C) HR111 and HR112 were induced for FeoAB expression, then grown in 1 mM FeSO₄ for 30 min before MdtD(IceT) expression was induced. Iron content was determined by ICP-MS at 30-min intervals post-iron addition. All data are the mean of three replicates ±1 SD, and significance (*) was determined by two-tailed t test (P < 0.02).

Fig. 5.

Cells expressing MdtD(IceT) secrete the CAS-reactive iron chelator citrate. (A) EF341 (ΔentB) is unable to produce siderophores and is CAS-negative (well 1). Expression of MdtD(IceT) (EF342) results in secretion of a CAS-positive iron chelator (well 2), whereas expression of MdtABC (EF336) does not (well 3). (B) The CAS-positive fraction was purified from growth medium and analyzed by ¹H NMR. Peaks indicated by an asterisk correspond to hydrogen atoms bound to the 1 and 3 carbons of citrate (indicated by an asterisk in Inset). (C) Identification of citrate was confirmed by determining the mass of the compound by mass spectrometry. MdtD was therefore renamed IceT. (D) Supernatant from EF345 and EF346 lacking citrate synthase (ΔentB ΔgltA, wells 3 and 4) are not CAS-positive even though EF346 expresses IceT (compare wells 2 and 4). When citrate synthase is expressed from a plasmid (EF347 and EF348, wells 5 and 6), IceT-dependent secretion of the chelator is restored (well 6).

Fig. 6.

IceT expression protects S. Typhimurium against the antibiotics ampicillin and ciprofloxacin. (A) EF356 expressing IceT (red) is less susceptible to ampicillin than EF355 containing empty vector (blue) at 30 min P = 0.0065, 60 min P = 0.0039, 90 min P = 0.0046, and 120 min P = 0.014. (B) EF35 expressing IceT (gold) is less susceptible to ciprofloxacin than EF39 containing empty vector (blue) at 30 min P = 0.0086, 60 min P = 0.0028, and 90 min P = 0.0055. Data are the mean of three replicates ±1 SD and significance (*) was determined by two-tailed t test.