Glutathione import in Haemophilus influenzae Rd is primed by the periplasmic heme-binding protein HbpA

Abstract

Glutathione (GSH) is a vital intracellular cysteine-containing tripeptide across all kingdoms of life and assumes a plethora of cellular roles. Such pleiotropic behavior relies on a finely tuned spatiotemporal distribution of glutathione and its conjugates, which is not only controlled by synthesis and breakdown, but also by transport. Here, we show that import of glutathione in the obligate human pathogen Haemophilus influenzae , a glutathione auxotrophe, is mediated by the ATP-binding cassette (ABC)-like dipeptide transporter DppBCDF, which is primed for glutathione transport by a dedicated periplasmic-binding protein (PBP). We have identified the periplasmic lipoprotein HbpA, a protein hitherto implicated in heme acquisition, as the cognate PBP that specifically binds reduced (GSH) and oxidized glutathione (GSSG) forms of glutathione with physiologically relevant affinity, while it exhibits marginal binding to hemin. Dissection of the ligand preferences of HbpA showed that HbpA does not recognize bulky glutathione S conjugates or glutathione derivatives with C-terminal modifications, consistent with the need for selective import of useful forms of glutathione and the concomitant exclusion of potentially toxic glutathione adducts. Structural studies of the highly homologous HbpA from Haemophilus parasuis in complex with GSSG have revealed the structural basis of the proposed novel function for HbpA-like proteins, thus allowing a delineation of highly conserved structure-sequence fingerprints for the entire family of HbpA proteins. Taken together, our studies unmask the main physiological role of HbpA and establish a paradigm for glutathione import in bacteria. Accordingly, we propose a name change for HbpA to glutathione-binding protein A.

Heme Utilization by Nontypeable Haemophilus influenzae Is Essential and Dependent on Sap Transporter Function

“…These periplasmic binding proteins share homology and structural characteristics with SapA. Preliminary titration experiments of SapA-heme complexes resolved by native PAGE revealed that the K D of heme association with SapA was greater than 50 M, similar to that described for the DppA-heme and HbpA-heme complexes (19,47). Further, SapA-heme complexes were detected using isothermal titration calorimetry.…”

“…The biochemical properties of heme and the low affinity for complex formation make the use of standard methodologies ineffective in the elucidation of precise dissociation constants (47). Despite these limitations, we attempted to estimate the relative affinity of heme for SapA by various methods previously used to determine the heme-binding affinities of the dipeptide and heme-binding proteins DppA and HbpA (19,47).…”

“…Despite these limitations, we attempted to estimate the relative affinity of heme for SapA by various methods previously used to determine the heme-binding affinities of the dipeptide and heme-binding proteins DppA and HbpA (19,47). These periplasmic binding proteins share homology and structural characteristics with SapA.…”

“…However, as observed with other low-affinity complexes, we were unable to determine a relative K D by this method due to increased signalto-noise ratio. Recently, Vergauwen et al determined that the relative K D of the well-described heme-binding lipoprotein (HbpA)-heme complex was weak (655 M) and that HbpA preferentially bound the glutathione substrate (47). Importantly, the authors determined the affinity of heme association by densitometric quantitation of apo-HbpA (unbound HbpA), which was observed to decrease with heme titration coincidently with the increase in the heme-bound HbpA complex.…”

Haemophilus influenzae stores and distributes hemin by using Protein E

“…We used this property of hemin to demonstrate the characteristics of hemin binding to PE using a UV-visible spectrophotometer (Vergauwen et al, 2010). Purified PE (15 μM) was incubated with 30 μM hemin in TS buffer for 1 h at room temperature and samples were scanned between 300 nm to 600 nm by using a UV-visible spectrophotometer.…”