Contributions of the heme coordinating ligands of the Pseudomonas aeruginosa outer membrane receptor HasR to extracellular heme sensing and transport

Alecia T Dent,Angela Wilks

doi:10.1074/jbc.ra120.014081

Abstract

Pseudomonas aeruginosa exhibits a high requirement for iron, which it can acquire via several mechanisms, including the acquisition and utilization of heme. The P. aeruginosa genome encodes two heme uptake systems, the heme assimilation system (Has) and the Pseudomonas heme utilization (Phu) system. Extracellular heme is sensed via the Has system, which encodes an extracytoplasmic function (ECF) σ factor system. Previous studies have shown that the transfer of heme from the extracellular hemophore HasAp to the outer membrane receptor HasR is required for activation of the σ factor HasI and upregulation of has operon expression. Here, employing site-directed mutagenesis, allelic exchange, quantitative PCR analyses, immunoblotting, and 13C-heme uptake experiments, we delineated the differential contributions of the extracellular FRAP/PNPNL loop residue His-624 in HasR and of His-221 in its N-terminal plug domain required for heme capture to heme transport and signaling, respectively. Specifically, we show that substitution of the N-terminal plug His-221 disrupts both signaling and transport, leading to dysregulation of both the Has and Phu uptake systems. Our results are consistent with a model wherein heme release from HasAp to the N-terminal plug of HasR is required to initiate signaling, whereas His-624 is required for simultaneously closing off the heme transport channel from the extracellular medium and triggering heme transport. Our results provide critical insight into heme release, signaling, and transport in P. aeruginosa and suggest a functional link between the ECF σ factor and Phu heme uptake system.

Highlights

Iron is an essential micronutrient that is required for the survival and virulence of almost all bacterial pathogens
When heme was supplied in the form of holoHasAp, a significant inhibition in growth was observed for all of the variant strains compared with that of Pseudomonas aeruginosa O1 (PAO1) WT (Fig. 2B). These results suggest that heme, but not heme complexed to Pseudomonas aeruginosa HasA (HasAp), is accessible to the hasR variant strains via the Pseudomonas heme uptake (Phu) heme uptake system. quantitative PCR (qPCR) analysis of phuR on heme supplementation showed that the relative mRNA levels of the hasR loop variants are similar to that of PAO1 WT at all time points (Fig. S2)
Based on the crystal structure and previous NMR studies of Serratia marcescens HasA (HasAs), heme transfer was proposed to occur through a transient intermediate where His-32 of HasAs is displaced on protein–protein interaction with HasR, whereas the stronger Tyr-75 ligand remains coordinated because of deprotonation of Tyr-75 by the nearby His-83 [22, 23]

Summary

Introduction

Iron is an essential micronutrient that is required for the survival and virulence of almost all bacterial pathogens. The hasAp mRNA and protein levels for the hasR loop variants were very similar to those in the PAO1 WT strain (Fig. 3, C and D, and Fig. S1B).

Results

Conclusion