• Characterization of Heme Transport in Pseudomonas aeruginosa and the Preferential Pathway for Heme Uptake

      Smith, Aaron Dennison; Wilks, Angela (2015)
      Bacterial pathogens require iron for their survival and virulence and have evolved multiple mechanisms to acquire this scarce micro-nutrient. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa acquires heme as an iron source through the Phu (Pseudomonas heme utilization) and Has (Heme assimilation system) systems. The studies herein detail the initial purification and characterization of the outer membrane (OM) HasR and PhuR receptors. A series of site-directed mutagenesis and spectroscopic studies confirmed HasR, in keeping with previously characterized OM receptors, coordinates heme through the conserved N-terminal plug His-221 and His-624 of the surface exposed FRAP-loop. In contrast PhuR coordinates heme through His-124 and Tyr-519 ligands not previously reported in OM receptors but associated with high affinity heme binding proteins. In vivo studies utilizing a combination of bacterial genetics, isotopic labeling (13C-heme), and qRT-PCR further revealed that both receptors are required for optimal heme uptake. However, whereas deletion of hasR leads to an inability to regulate heme uptake, loss of PhuR results in decreased efficiency in heme uptake, despite a significant up regulation in HasR protein levels. The results are consistent with PhuR being the major heme uptake receptor, while HasR senses and regulates extracellular heme uptake. Thus PhuR and HasR represent non-redundant receptors required for accessing and regulating heme uptake across a wide range of physiological conditions found upon infection. The research presented herein also involved optimization of the ABC-transporter ShuUV along with the soluble periplasmic heme binding ShuT proteins from Shigella dysenteriae, which are involved in the transport of heme across the cytoplasmic membrane and into the cell. By generating and screening a series of expression constructs we were able to obtain a construct that resulted in increased expression levels of ShuUV homodimer. Reconstitution of ShuUV in lipososmes with heme loaded ShuT trapped in the interior of the liposome gave a functional system that could transport heme on activation with ATP. Taken together, the current research lays the foundation for future spectroscopic and structural studies aimed at understanding the molecular mechanisms of membrane bound heme transport proteins.
    • The Cytoplasmic Heme Binding Protein PhuS of P. aeruginosa: A Heme Oxygenase (HemO) Titratable Regulator of Extracellular Heme Uptake

      O'Neill, Maura Jean; Wilks, Angela (2013)
      Iron acquisition is critical for pathogenic bacteria and as such they have evolved sophisticated mechanisms to utilize the hosts heme containing proteins as an iron source. The Pseudomonas aeruginosa cytoplasmic heme binding protein (PhuS) has been shown to interact specifically with and deliver heme to the iron regulated heme oxygenase (HemO). HemO then oxidatively cleaves heme to release iron with biliverdin (BV) IX delta and IX beta and CO as by-products of the reaction. A combination of site directed mutagenesis and spectroscopic studies of holo-PhuS reveal a dynamic heme with overlapping but distinct binding sites through alternate heme ligands, His-209 or His-212. We have further investigated the role of the histidine triad (His-209, His-210 and His-212) in complex formation and heme transfer. A series of biophysical studies has shown that a heme induced conformational change drives interaction of holo-PhuS with HemO. We further show that in addition to the proximal ligand His-209 both His-210 and His-212 are required for complex formation and heme transfer. Based on these studies we propose a mechanism that couples the heme-dependent conformational switch in PhuS to protein-protein interaction, the subsequent free energy of which drives heme transfer via a His-ligand switch from His-209 to His-212, and subsequent release of heme to HemO. The in vitro characterization of PhuS as a heme trafficking protein was further confirmed in vivo utilizing a combination of isotopic labeling (13C-heme) and qRT-PCR. Under conditions of active heme uptake wild type P. aeruginosa produced exclusively 13C- BVIX delta and IX beta. In contrast the -phuS knockout strain led to loss of the heme-dependent regulation of the heme uptake proteins and an uncoupling of heme trafficking to HemO. The resulting elevated expression of the heme uptake proteins leads to increased heme uptake and degradation of heme via both HemO (13C-BVIX delta and IX beta) and the alternate non-iron-regulated BphO (13C-BVIX alpha). We propose a testable model whereby PhuS acts as a HemO titratable regulator of extracellular heme uptake that couples the metabolic flux of heme through PhuS-HemO to the regulatory RNA network.
    • Design and Discovery of Novel Small Molecule Inhibitors targeting Heme Oxygenase (HemO) Dependent Iron Acquisition and Heme Signaling in Pseudomonas aeruginosa.

      Robinson, Elizabeth; Wilks, Angela; Xue, Fengtian, Ph.D.; 0000-0002-7770-4060 (2021)
      The recent rise in antibiotic resistance particularly those pertaining to hospital acquired infections has highlighted the need for alternative therapeutic approaches. Alternative approaches include anti-virulence strategies that differ from current treatments targeting essential pathways in pathogens and instead target systems and factors required for virulence and infection. Gram-negative opportunistic multi-drug resistant (MDR) pathogens like Pseudomonas aeruginosa are ranked at a serious threat level by the CDC and are infamous for causing life threatening infections in immunocompromised populations such as patients with ventilator-assisted pneumonia, open surgical wounds, and cystic fibrosis. Pathogenic bacteria including P. aeruginosa, require the essential micronutrient iron for their survival and virulence. It has been reported that during acute and chronic infections P. aeruginosa preferentially utilizes heme as its iron source over siderophore mechanisms. P. aeruginosa encodes two non-redundant heme uptake systems that both utilize the iron regulated heme oxygenase enzyme (HemO) to release iron and the biliverdin (BVIX) metabolites BVIXβ and -δ. It has been shown that HemO catalytic activity is required to drive heme uptake into the cell. Furthermore, the products of extracellular heme metabolism BVIXβ and -δ function as signaling and regulatory molecules in several virulence traits. Therefore, HemO represents an ideal therapeutic target due to its dual function in limiting both iron and the heme metabolites that regulate several virulence traits. We hypothesize such a dual function strategy will also increase the barrier to resistance. The work herein applies a structure-based design and high-throughput screening approach followed by in vitro and in cell characterization of lead compounds. Further computer-aided drug design (CADD) and guided chemical synthesis optimization were employed to design and discover novel small molecule scaffolds and inhibitors of HemO. The approaches resulted in lead compounds with nanomolar binding affinity and inhibition of HemO enzyme activity both in vitro and in vivo. Additionally, I developed a new method for the production and purification of BVIXβ and -δ, in > 500-fold increased yields, to further study their role in P. aeruginosa virulence and infection.
    • Determining the Mechanism and Regulation of the Heme Assimilation System (Has) in Pseudomonas aeruginosa Heme Signaling and Acquisition

      Dent, Alecia T.; Wilks, Angela; 0000-0002-6376-7482 (2020)
      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.
    • Inhibiting the Iron-regulated Heme Oxygenase (HemO) of Pseudomonas aeruginosa via Competitive and Non-competitive Mechanisms

      Heinzl, Geoffrey Addison; Wilks, Angela; Xue, Fengtian; 0000-0001-5291-5999 (2016)
      The discovery and development of new antimicrobials has become a top priority as resistance to known therapeutics continues to grow. While most antimicrobials target essential functions, some in the field question this historical approach and instead propose targeting virulence factors, rendering pathogens non-pathogenic. In most Gram-negative bacteria, virulence is globally regulated by iron via the ferric uptake regulator (Fur). Recent studies show that in the host, iron is preferentially acquired via heme uptake and utilization. Pseudomonas aeruginosa encodes two heme uptake systems, both of which terminate in the oxidative cleavage of heme by the iron-regulated heme oxygenase (HemO). HemO is required for the efficient utilization of heme as an iron source in P. aeruginosa. Thus, inhibiting HemO will globally reduce virulence via disrupting the utilization of heme as an iron source. Previous work identified small-molecule inhibitors of HemO via computer-aided drug design techniques, which were validated in vitro and in vivo. Several of those compounds were further explored for optimization using medicinal chemistry, biochemistry, and microbiology techniques. Compounds were synthesized, characterized, and assessed for binding, inhibitory activity in cellulo, and antimicrobial activity. Binding was analyzed by fluorescence quenching, saturation transfer difference (STD)-NMR, heteronuclear single quantum coherence (HSQC) NMR, molecular dynamics simulations, and hydrogen-deuterium exchange mass spectrometry (HXMS). Two lead compounds were shown to bind in the heme-binding site of HemO with low micromolar affinity. Another lead compound was shown to bind to a previously unidentified back site of HemO, which was identified in silico and verified with HXMS. To analyze the mechanism of back side inhibition of HemO, site-directed mutagenesis eliminated a salt bridge (D99-R188) adjacent to the back site. These mutations disrupted the essential hydrogen-bonding network in the distal pocket, as evidenced by poor stability of intermediates and altered structural dynamics. Together, these data show that inhibiting HemO with small-molecules can be achieved on two sites of the enzyme, both the heme-binding site and the newly discovered back site. Future work includes improvement of heme-binding site inhibitors, development of novel inhibitors, and confirming the antivirulent activity of HemO inhibitors in an infection model.