Accumulation of heme biosynthetic intermediates contributes to the antibacterial action of the metalloid tellurite

The metalloid tellurite is highly toxic to microorganisms. Several mechanisms of action have been proposed, including thiol depletion and generation of hydrogen peroxide and superoxide, but none of them can fully explain its toxicity. Here we use a combination of directed evolution and chemical and biochemical approaches to demonstrate that tellurite inhibits heme biosynthesis, leading to the accumulation of intermediates of this pathway and hydroxyl radical. Unexpectedly, the development of tellurite resistance is accompanied by increased susceptibility to hydrogen peroxide. Furthermore, we show that the heme precursor 5-aminolevulinic acid, which is used as an antimicrobial agent in photodynamic therapy, potentiates tellurite toxicity. Our results define a mechanism of tellurite toxicity and warrant further research on the potential use of the combination of tellurite and 5-aminolevulinic acid in antimicrobial therapy.


Selection of mutants conferring increased tellurite resistance. a)
Representative growth curves of strains BW25113, EM40, EM41 and EM2 in LB medium in the presence of the indicated tellurite concentrations. b) Representative growth curves of strains EM42, EM43,44,64,70,and 71 in M9 medium in the presence of the indicated tellurite concentrations. Values represent the mean of 3 biological replicates. In all figures, error bars represent SD. c) Strains EM 44, EM64, EM70 and EM71 were grown in M9 medium and the heme content was measured in cell extracts. For comparison, the level of heme in strains BW25113 (magenta line), EM43 (blue line) and EM2 (black line) are shown. Values represent the mean of three biological replicates. Error bars represent SD. TeO 3 2-: tellurite.
Representative growth curves of strains BW25113 and EM2 with tellurite. Representative growth curves of strains BW25113 and EM2 in M9 medium in the presence of the indicated tellurite concentrations. Values represent the mean of 3 biological replicates. Error bars represent SD. TeO 3 2-: tellurite.
Increased thiols, H 2 O 2 detoxification or resistance are not required for tellurite resistance. a) Strains BW25113 and EM2 were grown in M9 medium with or without 25 µg/mL ALA to an OD 600nm of ˜0.4 and treated with 2 mM H 2 O 2 (left panel) or tellurite (middle and right panel, 25 µg/mL for strain BW25113 and 400 µg/mL for strain EM2) and catalase (left and middle panels) or superoxide dismutase (SOD, right panel) activity were determined at the indicated time points. SOD activity was normalized by mg of protein. Values represent the mean of three biological replicates. Error bars represent SD. b) Overnight cultures of strains BW25113 and EM2 were inoculated into M9 medium supplemented or not with 25 µg/mL ALA, challenged or not with 2 mM H 2 O 2 and CFU/mL were quantified at the indicated time points. Values represent the mean of three biological replicates. Error bars represent SD. c) Representative growth curves of strains BW25113, EM2, ΔhemN and ΔhemF in M9 medium with 0.3 mM H 2 O 2 . Values represent the mean of three biological replicates. Error bars represent SD. d) Strains BW25113 and EM2 were grown as in a), exposed to tellurite and tellurite reductase (TR) activity was determined with NADH or NADPH as cofactor. Values represent the mean of three biological replicates. Error bars represent SD. e) Strains BW25113, ΔhemN, hemN + and EM2 were grown as in a), exposed to 25 µg/mL tellurite and total intracellular thiols were determined and normalized by mg of protein. Strain EM2 was also treated with 400 µg/mL tellurite, and thiols were determined (blue dashed line). Values represent the mean of three biological replicates. Error bars represent SD.TeO 3 2-: tellurite; RSH: µM thiols. Conservation of HemA residue mutated in strain EM2. The amino acid sequences of HemA from strain EM2 was aligned against that of other organisms. Residue that was mutated in strain EM2 is indicated by a magenta box along all the sequences.

Representative growth curves of strains
1 H NMR spectra of ALA in M9 medium with or without tellurite. All the measurements were carried out in M9 medium with 5 mg ALA and with or without 5 mg tellurite. TeO 3 2-: tellurite.
Evaluation of the effect of ALA and tellurite on selected pathogens. Representative growth curves of selected pathogens in M9 medium supplemented with 25 µg/mL ALA in the presence of the indicated tellurite concentrations. TeO 3 2-: tellurite.
E. coli growth in the presence of high tellurite concentrations results in black deposits of elemental tellurium. Representative growth curves in LB medium containing or not 100 µg/mL tellurite of colonies isolated from each passage during directed evolution experiment. Number at the top of each well indicates the passage at which the corresponding colony was isolated. Strains EM40, EM41 and EM2 were isolated at passages 3, 13, and 26, respectively. Blackening of the growth culture is due to tellurite reduction to tellurium. TeO 3 2-: tellurite.