Heme and Hemoglobin Transport Systems of Listeria monocytogenes

Abstract

Heme (Hn) is an important iron source for Listeria monocytogenes, an intracellular pathogen that causes listeriosis. I characterized listerial Hn acquisition by two Fur-regulated genetic systems: the hup region that produces an ABC transporter for Hn and hemoglobin (Hb) and the srtB region that produces an ABC transporter and other proteins with currently unknown functions. Intrinsic fluorescence titration assay showed HupD binds Hn with high affinity (Kd=40 nM). Western immunoblot analysis with listerial fractions identified its localization in the envelope of L. monocytogenes. Nutrition tests showed that the deletions in either hup region or the srtB region reduced the uptake of Hn and Hb. To quantitatively characterize Hn acquisition by L. monocytogenes, I synthesized [59Fe]-Hn and for the first time measured thermodynamic and kinetic parameters of its binding and transport. The [59Fe]-Hn binding and uptake assay showed that L. monocytogenes binds Hn with high affinity (Kd¡Ö2 nM) and imports it with a Vmax (23 pMol/109 cells/min) comparable to those of ferric siderophore import systems. The Hup system was responsible for the majority of Hn uptake (Vmax=16 pMol/109 cells/min). The residual uptake system (in ¦¤hup) also had high affinity (Kd¡Ö2 nM) but lower rate (Vmax=7 pMol/109 cells/min). This quantitative assay also showed that the sortase B-anchored protein Lmo2185 binds Hn and deletions of lmo2185 or srtB severely impaired the uptake of [59Fe]-Hn by L. monocytogenes at low concentrations (¡Ü20 nM). However, at higher concentrations (¡Ý50 nM), Hn directly adsorbs to its high affinity binding protein of cytoplasmic membrane transporter. Deletion of sortase A, on the other hand, had no effect on Hn/Hb acquisition, in nutrition tests or [59Fe]-Hn binding and uptake assay. These data showed the participation of at least two cytoplasmic membrane permeases in Hn/Hb acquisition and the involvement of the SrtB-anchored cell envelope protein Lmo2185 in binding and uptake of Hn at low external concentrations. SrtA-anchored proteins, on the other hand, apparently do not function in Hn transport by L. monocytogenes.

Intracellular Hn concentration is tightly regulated. To prevent Hn toxicity, bacteria export excess Hn. Deletion of lmo0641 rendered the mutant intolerant to a moderate concentration of Hn/Hb (2 uM), implying its defect in Hn detoxification. [59Fe]-Hn uptake assay supported the hypothesis that Lmo0641 functions as a heme exporter: ∆lmo0641 accumulated intracellularly 1.6 fold higher amount of Hn than the wild type EGD-e did. [59Fe]-citrate uptake assay, on the other hand, displayed no difference in the ability of the two strains to uptake ferric citrate: the Vmax of [59Fe]-citrate uptake for EGD-e was 51.4 pMol/109 cells/min and 55 pMol/109 cells/min for ¦¤0641; the KM was 44.2 nM for EGD-e and 48.4 nM for ¦¤0641.

TonB has been proposed to be an energy transducer for the active transport of metal complexes across the OM of Gram-negative bacteria. Sequence analysis revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. My experiments confirmed that TonB physically binds to the peptidoglycan: the purified peptidoglycan precipitated MalE-TonB69C from solution, but not MalE nor FepB.