• 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.
    • Iron-Regulated Production of Antimicrobial Metabolites by Pseudomonas aeruginosa

      Nguyen, Angela; Oglesby, Amanda G. (2016)
      Cystic fibrosis (CF) is a hereditary disease characterized by the accumulation of thick, viscous mucus in the lungs. CF disease results in decreased pulmonary function and makes patients prone to chronic bacterial infections. The CF lung is a polymicrobial environment and includes many bacterial species. Early infection with Staphylococcus aureus is common, while Pseudomonas aeruginosa becomes the dominant pathogenic resident as disease progresses. This shift in microbial populations is still not well understood, but the ability of pathogens to obtain limiting nutrients, such as iron, may play a role. The work in this dissertation employed metabolomics, genetics, and biochemical approaches to show that P. aeruginosa alters its iron uptake mechanisms over the course of CF lung infection to adapt to the changing environment of the CF lung and to maintain iron homeostasis. Furthermore, I show that depletion of iron, an essential nutrient for P. aeruginosa growth and survival, enhances antimicrobial activity of this pathogen against S. aureus. I show that iron not only regulates the production of antimicrobial metabolites by P. aeruginosa, but also impacts the susceptibility of S. aureus to the effects of these metabolites. This work has expanded on the knowledge of how iron availability impacts polymicrobial interactions and the progression of CF lung disease.