Determination of heme in microorganisms using HPLC-MS/MS and cobalt(III) protoporphyrin IX inhibition of heme acquisition in Escherichia coli

Abstract

One of the main threats to the achievements in modern medicine is antimicrobial resistance. Molecular targeting of bacterial acquisition mechanisms of heme has been suggested to be an alternative to antibiotics. In the present study, HPLC-MS/MS combined with a simple clean-up based on liquid-liquid extraction has been developed and evaluated for simultaneous determination of heme and porphyrin heme precursors in microorganisms. Experimental design was used to optimize the extraction parameters, to obtain a method with high recovery, low matrix effects, and high precision. The effects of additives in the culture medium on the biosynthesis of heme were studied using Escherichia coli as a model microorganism. 5-Aminolaevulinic acid and hemin increased the heme concentration in E. coli by a factor of 1.5 and 4.5, respectively. Addition of 5-aminolaevulinic acid bypassed the E. coli negative feedback control of heme biosynthesis, which led to high amounts of intracellular porphyrins. The high heme concentration obtained when hemin was used as a culture additive shows that E. coli has an uptake of heme from its surroundings. In contrast, addition of cobalt protoporphyrin IX to the growth medium reduced the amount of heme in E. coli, demonstrating this compound's ability to mimic real heme and inhibit the heme acquisition mechanisms.

Keywords: Antimicrobial resistance; Escherichia coli; HPLC-MS/MS; Heme acquisition; Heme analysis; Porphyrins.

Fig. 1

Chemical structure of heme

Fig. 2

Scheme of the extraction and clean-up method. “Standard” points out were in the clean-up process the standard of heme for quantification purposes were added

Fig. 3

Response surface plot of heme extraction at 20 min, with concentration of acetonitrile versus concentration of HCl. Red color marks the region of highest response

Fig. 4

Relative yield (bars) of heme from S. cerevisiae using different extraction solvents and their protein precipitation efficiency (lines). Error bars represent the standard deviation for triplicate

Fig. 5

Hemin stability during 7 days when dissolved in six different solvents. A_x is the area ratio to a volumetric standard at the investigated day and A₀ is the area ratio to a volumetric standard at day 0. Organic top layer is the solvent used in the liquid-liquid extraction

Fig. 6

Total reconstructed ion chromatogram from the HPLC-MS/MS analysis of a standard mixture of porphyrins and hemin. UP, uroporphyrin; 7P, 7-carboxylporphyrin; 6P, 6-carboxylporphyrin; 5P, 5-carboxylporphyrin; CP, coproporphyrin; Hemin; MPIX, mesoporphyrin IX; PPIX, protoporphyrin IX. Flow rate = 1 mL/min. Injection volume = 5 μL. Detection: MRM using three compound-specific transitions for each analyte

Fig. 7

Heme concentrations in E. coli with different culturing conditions

Fig. 8

Porphyrin concentrations (A) and HPLC-MS/MS chromatogram (B) of a sample after addition of 5-ALA to E. coli growth medium. Due to the high response of protoporphyrin IX, the chromatogram has been enlarged in the range 0.00–1.50 min to make the other peaks visible. UP, uroporphyrin; 7P, 7-carboxylporphyrin; 5P, 5-carboxylporphyrin; CPI, coproporphyrin I; CPIII, coproporphyrin III; PPIX, protoporphyrin IX