The requirement of pathogenic bacteria for iron, a micronutrient that has limited bioavailability but is essential for virulence, requires specialized mechanisms for uptake and storage. Furthermore, the evolutionary struggle for iron between the host and invading bacterial pathogen sets up a competition that has shaped both the hosts defense strategies, and the iron-acquisition mechanisms deployed by the invading pathogen. By understanding the mechanisms by which pathogenic bacteria acquire heme from the host intervention strategies can be employed to reduce virulence. We have several projects ongoing in the laboratory to further understand the role of heme in bacterial pathogenesis.
Structure-Function of the Bacterial Heme Uptake Proteins
Heme is transported across the outer-membrane (OM) by a TonB-dependent OM-receptor where it is sequestered by a soluble heme binding protein that then acts as the soluble receptor for an ATP-dependent permease (ABC-transporter) that translocates heme to the cytoplasm. We have previously characterized the S. dysenteriae ShuA receptor. Kinetic analysis of heme transfer from hemoglobin revealed that transfer from the methemoglobin dimer or tetramer is unidirectional and on the order of 104 faster than the rate of free heme association. Biochemical characterization of the wild type, His-86 Ala and His-420 Ala mutants revealed that both residues were required for heme acquisition from methemoglobin. We are further characterizing the biochemical and biophysical properties of the OM the HasR and PhuR receptors of P. aeruginosa that have distinct substrate specificities and coordinating ligands. In combination with bacterial genetics, transcriptional and metabolomic profiling the contributions of each receptor to heme acquisition and pathogenesis are being determined. We were the first to show in vitro heme translocation by the ShuUV ABC-transporter. Through a combination of site-directed mutagenesis and translocation studies critical residues involved in transfer from ShuT to ShuU were identified. Furthermore, we determined conserved His-252 and His-262 residues located within the translocation channel are essential for heme transport. Site-directed mutagenesis studies combined with spectroscopic methods will aid in the elucidation of the molecular mechanism of heme translocation through the cytoplasmic membrane.
Intracellular Heme Trafficking and Homeostasis
We have recently characterized the P. aeruginosa cytoplasmic heme binding binding protein PhuS as a specific heme chaperone to the iron-regulated heme oxygenase (HemO). Biophysical studies have revealed heme coordination drives a conformational “induced fit” required for interaction with HemO. A combination of site-directed mutagenesis and spectroscopic studies confirmed the conserved His-209 as the proximal ligand to the heme. However, spectroscopic studies of the holo-PhuS H209A mutant also identified an overlapping but mutually exclusive heme binding site provided by coordination to His-212. Furthermore, His-210 in the absence of the proximal His-209 was shown to stabilize ligand coordination through His-212. Both His-212 and His-210, in addition to the proximal His-209, are required for the heme dependent conformational change and interaction with HemO. Furthermore, we describe a novel mechanism of heme transfer for the cytoplasmic heme binding proteins, where a ligand induced conformational change facilitates protein-protein interaction, and the free energy derived from this interaction triggers heme transfer via a histidine relay. Isotopic labeling (13C-heme) and transcriptional analysis further confirmed that HemO drives the metabolic flux of heme into the cell with PhuS acting as a “control-valve” (2). In the ΔhemO knockout there is no active transport of extracellular heme (13C-heme) into the cell. However, in the ΔphuS knockout the presence of HemO drives heme uptake, but the inability chaperone heme efficiently to HemO results in the degradation of intracellular heme. Consistent with inefficient heme utilization, increased expression levels of the OM-receptor PhuR and HemO are observed. Prelimanary studies suggest a link between PhuS controlled heme flux and the sRNA regulatory network. Coupling the metabolic heme flux to the regulatory RNA network would provide a novel mechanism for bacteria to rapidly respond and adapt to sudden changes in heme levels.
Antimicrobial Inhibitor Development
Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe infections in individuals with underlying conditions, including cystic fibrosis (CF). P. aeruginosa requires iron for virulence, biofilm formation, and is resistant to many antibiotics. We hypothesize that heme is a significant source of iron during infection and that inhibition of heme utilization may lead to novel therapeutic strategies. We have developed a series small molecule inhibitors directed toward HemO and PhuS. We have determined binding modes of two lead inhibitors of HemO. Both binding modes independently inhibit enzyme activity and lead to the reduced virulence of P. aeruginosa clinical strains. Fragment-based NMR screening approaches are being employed in the development of inhibitors that simultaneously target both sites and increase selectivity and efficacy. Such novel approaches targeting mechanisms required for virulence but not survival may lead to the development of antivirulants that reduce the pressure to undergo mutations that lead to resistance.