Determining the Mechanism and Regulation of the Heme Assimilation System (Has) in Pseudomonas aeruginosa Heme Signaling and Acquisition

Abstract

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes infections in immunocompromised populations including patients with cystic fibrosis, surgical site wounds and pneumonia. Like most other bacterial pathogens, Pseudomonas requires iron for survival and virulence and has adapted several mechanisms including utilizing heme as an iron source. P. aeruginosa encodes two nonredundant heme uptake systems, the heme assimilation system (has) and Pseudomonas heme utilization (phu) pathways. Proteomic and RNA seq analysis of P. aeruginosa show the Has pathway is one of the most upregulated during infection and knockout strains of HasR reduce the pathogenicity of the bacteria in mice elevating it as a potential drug target. Despite previous studies of the S. marcescens Has pathway there has been no comprehensive study of the molecular mechanism by which heme is sensed and transported by the Has pathway. The work herein utilizes a combination of site-directed mutagenesis of the extracellular hemophore HasAp, allelic exchange, quantitative PCR analyses, immunoblotting and 13C-heme uptake studies to elucidate both the mechanism of heme release from HasAp to HasR and its requirement for initiation of the extracellularcytoplasmic function (ECF) HasIS sigma/anti-sigma factor system. Furthermore, I show in contrast to the S. marcescens system the hasIS operon is not subject to autoregulation by HasI, but rather post-transcriptional regulation through modulation of HasAp. Employing similar approaches with the outer membrane receptor HasR, I determined heme capture by H221 on the plug domain of HasR is required for signaling and transport, whereas mutations to the extracellular FRAP/PNPL loop H624 and L8 loop Ile694 are competent to signal but not transport heme. Based on my studies, I propose a model for heme signaling and transport by the P. aeruginosa Has system that provides a foundation for further studies of heme uptake and a starting point for the development of novel antimicrobial strategies.