Pseudomonas aeruginosa heme metabolites biliverdin IXß and IXδ are integral to lifestyle adaptations associated with chronic infection.

Pseudomonas aeruginosa is a versatile opportunistic pathogen requiring iron for its survival and virulence within the host. The ability to switch to heme as an iron source and away from siderophore uptake provides an advantage in chronic infection. We have recently shown the extracellular heme metabolites biliverdin IXß (BVIXß) and BVIXδ positively regulate the heme-dependent cell surface signaling cascade. We further investigated the role of BVIXß and BVIXδ in cell signaling utilizing allelic strains lacking a functional heme oxygenase (hemOin) or one reengineered to produce BVIXα (hemOα). Compared to PAO1, both strains show a heme-dependent growth defect, decreased swarming and twitching, and less robust biofilm formation. Interestingly, the motility and biofilm defects were partially rescued on addition of exogenous BVIXß and BVIXδ. Utilizing liquid chromatography-tandem mass spectrometry, we performed a comparative proteomics and metabolomics analysis of PAO1 versus the allelic strains in shaking and static conditions. In shaking conditions, the hemO allelic strains showed a significant increase in proteins involved in quorum sensing, phenazine production, and chemotaxis. Metabolite profiling further revealed increased levels of Pseudomonas quinolone signal and phenazine metabolites. In static conditions, we observed a significant repression of chemosensory pathways and type IV pili biogenesis proteins as well as several phosphodiesterases associated with biofilm dispersal. We propose BVIX metabolites function as signaling and chemotactic molecules integrating heme utilization as an iron source into the adaptation of P. aeruginosa from a planktonic to sessile lifestyle. IMPORTANCE: The opportunistic pathogen Pseudomonas aeruginosa causes long-term chronic infection in the airways of cystic fibrosis patients. The ability to scavenge iron and to establish chronic infection within this environment coincides with a switch to utilize heme as the primary iron source. Herein, we show the heme metabolites biliverdin beta and delta are themselves important signaling molecules integrating the switch in iron acquisition systems with cooperative behaviors such as motility and biofilm formation that are essential for long-term chronic infection. These significant findings will enhance the development of viable multi-targeted therapeutics effective against both heme utilization and cooperative behaviors essential for survival and persistence within the host.

The heme-responsive PrrH sRNA regulates Pseudomonas aeruginosa pyochelin gene expression.

Pseudomonas aeruginosa is an opportunistic pathogen that requires iron for growth and virulence, yet this nutrient is sequestered by the innate immune system during infection. When iron is limiting, P. aeruginosa expresses the PrrF1 and PrrF2 small RNAs (sRNAs), which post-transcriptionally repress expression of nonessential iron-containing proteins, thus sparing this nutrient for more critical processes. The genes for the PrrF1 and PrrF2 sRNAs are arranged in tandem on the chromosome, allowing for the transcription of a longer heme-responsive sRNA, termed PrrH. While the functions of PrrF1 and PrrF2 have been extensively studied, the role of PrrH in P. aeruginosa physiology and virulence is not well understood. In this study, we performed transcriptomic and proteomic studies to identify the PrrH regulon. In shaking cultures, the pyochelin synthesis proteins were increased in two distinct prrH mutants compared to the wild type, while the mRNAs for these proteins were not affected by the prrH mutation. We identified complementarity between the PrrH sRNA and the sequence upstream of the pchE mRNA, suggesting the potential for PrrH to directly regulate the expression of genes for pyochelin synthesis. We further showed that pchE mRNA levels were increased in the prrH mutants when grown in static but not shaking conditions. Moreover, we discovered that controlling for the presence of light was critical for examining the impact of PrrH on pchE expression. As such, our study reports on the first likely target of the PrrH sRNA and highlights key environmental variables that will allow for future characterization of PrrH function. IMPORTANCE In the human host, iron is predominantly in the form of heme, which Pseudomonas aeruginosa can acquire as an iron source during infection. We previously showed that the iron-responsive PrrF small RNAs (sRNAs) are critical for mediating iron homeostasis during P. aeruginosa infection; however, the function of the heme-responsive PrrH sRNA remains unclear. In this study, we identified genes for pyochelin siderophore biosynthesis, which mediates uptake of inorganic iron, as a novel target of PrrH regulation. This study therefore highlights a novel relationship between heme availability and siderophore biosynthesis in P. aeruginosa.

The heme-responsive PrrH sRNA regulates Pseudomonas aeruginosa pyochelin gene expression.

Pseudomonas aeruginosa is an opportunistic pathogen that requires iron for growth and virulence, yet this nutrient is sequestered by the innate immune system during infection. When iron is limiting, P. aeruginosa expresses the PrrF1 and PrrF2 small regulatory RNAs (sRNAs), which post-transcriptionally repress expression of non-essential iron-containing proteins thus sparing this nutrient for more critical processes. The genes for the PrrF1 and PrrF2 sRNAs are arranged in tandem on the chromosome, allowing for the transcription of a longer heme-responsive sRNA, termed PrrH. While the functions of PrrF1 and PrrF2 have been studied extensively, the role of PrrH in P. aeruginosa physiology and virulence is not well understood. In this study, we performed transcriptomic and proteomic studies to identify the PrrH regulon. In shaking cultures, the pyochelin synthesis proteins were increased in two distinct prrH mutants compared to wild type, while the mRNAs for these proteins were not affected by prrH mutation. We identified complementarity between the PrrH sRNA and sequence upstream of the pchE mRNA, suggesting potential for PrrH to directly regulate expression of genes for pyochelin synthesis. We further showed that pchE mRNA levels were increased in the prrH mutants when grown in static but not shaking conditions. Moreover, we discovered controlling for the presence of light was critical for examining the impact of PrrH on pchE expression. As such, our study reports on the first likely target of the PrrH sRNA and highlights key environmental variables that will allow for future characterization of PrrH function.

Recombinant Production of Biliverdin IXß and δ Isomers in the T7 Promoter Compatible Escherichia coli Nissle.

The ability to obtain purified biliverdin IX (BVIX) isomers other than the commercially available BVIXα is limited due to the low yields obtained by the chemical coupled oxidation of heme. Chemical oxidation requires toxic chemicals, has very poor BVIX yields (<0.05%), and is not conducive to scalable production. Alternative approaches utilizing recombinant E. coli BL21 expressing a cyanobacterial heme oxygenase have been employed for the production BVIXα, but yields are limited by the rate of endogenous heme biosynthesis. Furthermore, the emerging roles of BVIXß and BVIXδ in biology and their lack of commercial availability has led to a need for an efficient and scalable method with the flexibility to produce all three physiologically relevant BVIX isomers. Herein, we have taken advantage of an optimized non-pathogenic E. coli Nissle (EcN(T7)) strain that encodes an endogenous heme transporter and an integrated T7 polymerase gene. Protein production of the Pseudomonas aeruginosa BVIXß and BVIXδ selective heme oxygenase (HemO) or its BVIXα producing mutant (HemOα) in the EcN(T7) strain provides a scalable method to obtain all three isomers, that is not limited by the rate of endogenous heme biosynthesis, due to the natural ability of EcN(T7) to transport extracellular heme. Additionally, we have optimized our previous LC-MS/MS protocol for semi-preparative separation and validation of the BVIX isomers. Utilizing this new methodology for scalable production and separation we have increased the yields of the BVIXß and -δ isomers >300-fold when compared to the chemical oxidation of heme.

Extracellular haem utilization by the opportunistic pathogen Pseudomonas aeruginosa and its role in virulence and pathogenesis.

Iron is an essential micronutrient for all bacteria but presents a significant challenge given its limited bioavailability. Furthermore, iron's toxicity combined with the need to maintain iron levels within a narrow physiological range requires integrated systems to sense, regulate and transport a variety of iron complexes. Most bacteria encode systems to chelate and transport ferric iron (Fe3+) via siderophore receptor mediated uptake or via cytoplasmic energy dependent transport systems. Pathogenic bacteria have further lowered the barrier to iron acquisition by employing systems to utilize haem as a source of iron. Haem, a lipophilic and toxic molecule, presents a significant challenge for transport into the cell. As such pathogenic bacteria have evolved sophisticated cell surface signaling (CSS) and transport systems to sense and obtain haem from the host. Once internalized haem is cleaved by both oxidative and non-oxidative mechanisms to release iron. Herein we summarize our current understanding of the mechanism of haem sensing, uptake and utilization in Pseudomonas aeruginosa, its role in pathogenesis and virulence, and the potential of these systems as antimicrobial targets.

Modeling the native ensemble of PhuS using enhanced sampling MD and HDX-ensemble reweighting.

The cytoplasmic heme binding protein from Pseudomonas aeruginosa, PhuS, plays two essential roles in regulating heme uptake and iron homeostasis. First, PhuS shuttles exogenous heme to heme oxygenase (HemO) for degradation and iron release. Second, PhuS binds DNA and modulates the transcription of the prrF/H small RNAs (sRNAs) involved in the iron-sparing response. Heme binding to PhuS regulates this dual function, as the unliganded form binds DNA, whereas the heme-bound form binds HemO. Crystallographic studies revealed nearly identical structures for apo- and holo-PhuS, and yet numerous solution-based measurements indicate that heme binding is accompanied by large conformational rearrangements. In particular, hydrogen-deuterium exchange mass spectrometry (HDX-MS) of apo- versus holo-PhuS revealed large differences in deuterium uptake, notably in α-helices 6, 7, and 8 (α6,7,8), which contribute to the heme binding pocket. These helices were mostly labile in apo-PhuS but largely protected in holo-PhuS. In contrast, in silico-predicted deuterium uptake levels of α6,7,8 from molecular dynamics (MD) simulations of the apo- and holo-PhuS structures are highly similar, consistent only with the holo-PhuS HDX-MS data. To rationalize this discrepancy between crystal structures, simulations, and observed HDX-MS, we exploit a recently developed computational approach (HDXer) that fits the relative weights of conformational populations within an ensemble of structures to conform to a target set of HDX-MS data. Here, a combination of enhanced sampling MD, HDXer, and dimensionality reduction analysis reveals an apo-PhuS conformational landscape in which α6, 7, and 8 are significantly rearranged compared to the crystal structure, including a loss of secondary structure in α6 and the displacement of α7 toward the HemO binding interface. Circular dichroism analysis confirms the loss of secondary structure, and the extracted ensembles of apo-PhuS and of heme-transfer-impaired H212R mutant, are consistent with known heme binding and transfer properties. The proposed conformational landscape provides structural insights into the modulation by heme of the dual function of PhuS.

Axial Heme Coordination by the Tyr-His Motif in the Extracellular Hemophore HasAp Is Critical for the Release of Heme to the HasR Receptor of Pseudomonas aeruginosa.

Pseudomonas aeruginosa senses extracellular heme via an extra cytoplasmic function σ factor that is activated upon interaction of the hemophore holo-HasAp with the HasR receptor. Herein, we show Y75H holo-HasAp interacts with HasR but is unable to release heme for signaling and uptake. To understand this inhibition, we undertook a spectroscopic characterization of Y75H holo-HasAp by resonance Raman (RR), electron paramagnetic resonance (EPR), and X-ray crystallography. The RR spectra are consistent with a mixed six-coordinate high-spin (6cHS), six-coordinate low-spin (6cLS) heme configuration and an H218O exchangeable FeIII-O stretching frequency with 16O/18O and H/D isotope shifts that support a two-body Fe-OH2 oscillator with (iron-hydroxy)-like character as both hydrogen atoms are engaged in short hydrogen bond interactions with protein side chains. Further support comes from the EPR spectrum of Y75H holo-HasAp that shows a LS rhombic signal with ligand-field splitting values intermediate between those of His-hydroxy and bis-His ferric hemes. The crystal structure of Y75H holo-HasAp confirmed the coordinated solvent molecule hydrogen bonded through H75 and H83. The long-range conformational rearrangement of HasAp upon heme binding can still take place in Y75H holo-HasAp, because the intercalation of a hydroxy ligand between the heme iron and H75 allows the variant to reproduce the heme binding pocket observed in wild-type holo-HasAp. However, in the absence of a covalent linkage to the Y75 loop combined with the malleability provided by the bracketing H75 and H83 hydrogen bonds, either the hydroxy sixth ligand remains bound after complexation of Y75H holo-HasAp with HasR or rearrangement and coordination of H85 prevent heme transfer.

Repurposing Acitretin as an Antipseudomonal Agent Targeting the Pseudomonas aeruginosa Iron-Regulated Heme Oxygenase.

Iron is an essential micronutrient for the survival and virulence of the bacterial pathogen Pseudomonas aeruginosa. To overcome iron withholding and successfully colonize a host, P. aeruginosa uses a variety of mechanisms to acquire iron, including the secretion of high-affinity iron chelators (siderophores) or the uptake and utilization of heme. P. aeruginosa heme oxygenase (HemO) plays pivotal roles in heme sensing, uptake, and utilization and has emerged as a therapeutic target for the development of antipseudomonal agents. Using a high-throughput fluorescence quenching assay combined with minimum inhibitory concentration measurements, we screened the Selleck Bioactive collection of 2100 compounds and identified acitretin, a Food and Drug Administration-approved oral retinoid, as a potent and selective inhibitor of HemO. Acitretin binds to HemO with a KD value of 0.10 ± 0.02 µM and inhibits the growth of P. aeruginosa PAO1 with an IC50 of 70 ± 18 µg/mL. In addition, acitretin showed good selectivity for HemO, which uniquely generates BVIXß/δ, over human heme oxygenase (hHO1) and other BVIXα-producing homologues such as the heme oxygenases from Neisseria meningitidis (nmHO) and Acinetobacter baumannii (abHO). The binding of acitretin within the HemO active site was confirmed by 1H-15N heteronuclear single-quantum coherence nuclear magnetic resonance, and molecular modeling provided further insight into potential interactions of acitretin with residues specific for orienting heme in the ß/δ selective HemO. Moreover, at 20 µM, acitretin inhibited the enzymatic activity of HemO in P. aeruginosa cells by >60% and effectively blocked the ability of P. aeruginosa to sense and acquire heme as demonstrated in the ß-galactosidase transcriptional reporter assay.

Understanding RNA Binding by the Nonclassical Zinc Finger Protein CPSF30, a Key Factor in Polyadenylation during Pre-mRNA Processing.

Cleavage and polyadenylation specificity factor 30 (CPSF30) is a zinc finger protein that regulates pre-mRNA processing. CPSF30 contains five CCCH domains and one CCHC domain and recognizes two conserved 3' pre-mRNA sequences: an AU hexamer and a U-rich motif. AU hexamer motifs are common in pre-mRNAs and are typically defined as AAUAAA. Variations within the AAUAAA hexamer occur in certain pre-mRNAs and can affect polyadenylation efficiency or be linked to diseases. The effects of disease-related variations on CPSF30/pre-mRNA binding were determined using a construct of CPSF30 that contains just the five CCCH domains (CPSF30-5F). Bioinformatics was utilized to identify the variability within the AU hexamer sequence in pre-mRNAs. The effects of this sequence variability on CPSF30-5F/RNA binding affinities were measured. Bases at positions 1, 2, 4, and 5 within the AU hexamer were found to be important for RNA binding. Bioinformatics revealed that the three bases flanking the AU hexamer at the 5' and 3' ends are twice as likely to be adenine or uracil as guanine and cytosine. The presence of A and U residues in these flanking regions was determined to promote higher-affinity CPSF30-5F/RNA binding than G and C residues. The addition of the zinc knuckle domain to CPSF30-5F (CPSF30-FL) restored binding to AU hexamer variants. This restoration of binding is connected to the presence of a U-rich sequence within the pre-mRNA to which the zinc knuckle binds. A mechanism of differential RNA binding by CPSF30, modulated by accessibility of the two RNA binding sites, is proposed.

The heme-binding protein PhuS transcriptionally regulates the Pseudomonas aeruginosa tandem sRNA prrF1,F2 locus.

Pseudomonas aeruginosa is an opportunistic pathogen requiring iron for its survival and virulence. P. aeruginosa can acquire iron from heme via the nonredundant heme assimilation system and Pseudomonas heme uptake (Phu) systems. Heme transported by either the heme assimilation system or Phu system is sequestered by the cytoplasmic protein PhuS. Furthermore, PhuS has been shown to specifically transfer heme to the iron-regulated heme oxygenase HemO. As the PhuS homolog ShuS from Shigella dysenteriae was observed to bind DNA as a function of its heme status, we sought to further determine if PhuS, in addition to its role in regulating heme flux through HemO, functions as a DNA-binding protein. Herein, through a combination of chromatin immunoprecipitation-PCR, EMSA, and fluorescence anisotropy, we show that apo-PhuS but not holo-PhuS binds upstream of the tandem iron-responsive sRNAs prrF1,F2. Previous studies have shown the PrrF sRNAs are required for sparing iron for essential proteins during iron starvation. Furthermore, under certain conditions, a heme-dependent read through of the prrF1 terminator yields the longer PrrH transcript. Quantitative PCR analysis of P. aeruginosa WT and ΔphuS strains shows that loss of PhuS abrogates the heme-dependent regulation of PrrF and PrrH levels. Taken together, our data show that PhuS, in addition to its role in extracellular heme metabolism, also functions as a transcriptional regulator by modulating PrrF and PrrH levels in response to heme. This dual function of PhuS is central to integrating extracellular heme utilization into the PrrF/PrrH sRNA regulatory network that is critical for P. aeruginosa adaptation and virulence within the host.

Metallotherapeutics development in the age of iron-clad bacteria.

Drug-resistant infections pose a significant risk to global health as pathogenic bacteria become increasingly difficult to treat. The rapid selection of resistant strains through poor antibiotic stewardship has reduced the number of viable treatments and increased morbidity of infections, especially among the immunocompromised. To circumvent such challenges, new strategies are required to stay ahead of emerging resistance trends, yet research and funding for antibiotic development lags other classes of therapeutics. Though the use of metals in therapeutics has been around for centuries, recent strategies have devoted a great deal of effort into the pathways through which bacteria acquire and utilize iron, which is critical for the establishment of infection. To target iron uptake systems, siderophore-drug conjugates have been developed that hijack siderophore-based iron uptake for delivery of antibiotics. While this strategy has produced several potential leads, the use of siderophores in infection is diminished over time when bacteria adapt to utilize heme as an iron source, leading to a need for the development of porphyrin mimetics as therapeutics. The use of such strategies as well as the inclusion of gallium, a redox-inert iron mimic, are herein reviewed.

Gallium(III)-Salophen as a Dual Inhibitor of Pseudomonas aeruginosa Heme Sensing and Iron Acquisition.

Pseudomonas aeruginosa is an opportunistic bacterium that causes life-threatening infections in immunocompromised patients. In infection, it uses heme as a primary iron source and senses the availability of exogenous heme through the heme assimilation system (Has), an extra cytoplasmic function σ-factor system. A secreted hemophore HasAp scavenges heme and, upon interaction with the outer-membrane receptor HasR, activates a signaling cascade, which in turn creates a positive feedback loop critical for sensing and adaptation within the host. The ability to sense and respond to heme as an iron source contributes to virulence. Consequently, the inhibition of this system will lead to a disruption in iron homeostasis, decreasing virulence. We have identified a salophen scaffold that successfully inhibits the activation of the Has signaling system while simultaneously targeting iron uptake via xenosiderophore receptors. We propose this dual mechanism wherein free Ga3+-salophen reduces growth through uptake and iron mimicry. A dual mechanism targeting extracellular heme signaling and uptake together with Ga3+-induced toxicity following active Ga3+salophen uptake provides a significant therapeutic advantage while reducing the propensity to develop resistance.

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

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.

Heme uptake and utilization by hypervirulent Acinetobacter baumannii LAC-4 is dependent on a canonical heme oxygenase (abHemO).

Acinetobacter baumannii is an opportunistic pathogen that causes serious infections in critically ill and immune compromised patients. The ability to acquire iron from the hosts iron and heme containing proteins is critical to their survival and virulence. The majority of A. baumannii hypervirulent strains encode a heme uptake system that includes a putative heme oxygenase (hemO). Despite reports indicating A. baumannii can grow on heme direct evidence of extracellular heme uptake and metabolism has not been shown. Through isotopic labeling (13C-heme) we show the hypervirulent A. baumannii LAC-4 metabolizes heme to biliverdin IXα (BVIXα), whereas ATC 17978 that lacks the hemO gene cluster cannot efficiently utilize heme. Expression and purification of the protein encoded by the A. baumannii LAC-4 hemO gene confirmed catalytic conversion of heme to BVIX. We further show inhibition of abHemO with previously characterized P. aeruginosa HemO inhibitors in a fluorescence based assay that couples HemO catalytic activity to the BVIXα binding phytochrome IFP1.4. Furthermore, the hemO gene cluster encodes genes with homology to heme-dependent extra cytoplasmic function (ECF) σ factor systems. The hemophore-dependent ECF system in Pseudomonas aeruginosa has been shown to play a critical role in heme sensing and virulence within the host. The prevalence of a hemO gene cluster in A. baumannii LAC4 and other hypervirulent strains suggests it is required within the host to adapt and utilize heme and is a major contributor to virulence.

Proteomic Analysis of the Pseudomonas aeruginosa Iron Starvation Response Reveals PrrF Small Regulatory RNA-Dependent Iron Regulation of Twitching Motility, Amino Acid Metabolism, and Zinc Homeostasis Proteins.

Iron is a critical nutrient for most microbial pathogens, and the immune system exploits this requirement by sequestering iron. The opportunistic pathogen Pseudomonas aeruginosa exhibits a high requirement for iron yet an exquisite ability to overcome iron deprivation during infection. Upon iron starvation, P. aeruginosa induces the expression of several high-affinity iron acquisition systems, as well as the PrrF small regulatory RNAs (sRNAs) that mediate an iron-sparing response. Here, we used liquid chromatography-tandem mass spectrometry to conduct proteomics of the iron starvation response of P. aeruginosa Iron starvation increased levels of multiple proteins involved in amino acid catabolism, providing the capacity for iron-independent entry of carbons into the tricarboxylic acid (TCA) cycle. Proteins involved in sulfur assimilation and cysteine biosynthesis were reduced upon iron starvation, while proteins involved in iron-sulfur cluster biogenesis were increased, highlighting the central role of iron in P. aeruginosa metabolism. Iron starvation also resulted in changes in the expression of several zinc-responsive proteins and increased levels of twitching motility proteins. Subsequent analyses provided evidence for the regulation of many of these proteins via posttranscriptional regulatory events, some of which are dependent upon the PrrF sRNAs. Moreover, we showed that iron-regulated twitching motility is partially dependent upon the prrF locus, highlighting a novel link between the PrrF sRNAs and motility. These findings add to the known impacts of iron starvation in P. aeruginosa and outline potentially novel roles for the PrrF sRNAs in iron homeostasis and pathogenesis.IMPORTANCE Iron is central for growth and metabolism of almost all microbial pathogens, and as such, this element is sequestered by the host innate immune system to restrict microbial growth. Here, we used label-free proteomics to investigate the Pseudomonas aeruginosa iron starvation response, revealing a broad landscape of metabolic and metal homeostasis changes that have not previously been described. We further provide evidence that many of these processes, including twitching motility, are regulated through the iron-responsive PrrF small regulatory RNAs. As such, this study demonstrates the power of proteomics for defining stress responses of microbial pathogens.

Post-transcriptional regulation of the Pseudomonas aeruginosa heme assimilation system (Has) fine-tunes extracellular heme sensing.

Pseudomonas aeruginosa is an opportunistic pathogen that utilizes heme as a primary iron source within the host. Extracellular heme is sensed via a heme assimilation system (has) that encodes an extracytoplasmic function (ECF) σ factor system. Herein, using has deletion mutants, quantitative PCR analyses, and immunoblotting, we show that the activation of the σ factor HasI requires heme release from the hemophore HasAp to the outer-membrane receptor HasR. Using RT-PCR and 5'-RACE, we observed that following transcriptional activation of the co-transcribed hasRAp, it is further processed into specific mRNAs varying in stability. We noted that the processing and variation in stability of the hasAp and hasR mRNAs in response to heme provide a mechanism for differential expression from co-transcribed genes. The multiple layers of post-transcriptional regulation of the ECF signaling cascade, including the previously reported post-transcriptional regulation of HasAp by the heme metabolites biliverdin IXß and IXδ, allow fine-tuning of the cell-surface signaling system in response to extracellular heme levels. We hypothesize that the complex post-transcriptional regulation of the Has system provides P. aeruginosa an advantage in colonizing a variety of physiological niches in the host.

Correction to: The Asp99-Arg188 salt bridge of the Pseudomonas aeruginosa HemO is critical in allowing conformational flexibility during catalysis.

In the original publication, fifth author's name was incorrectly published as Pierre Moenne-Loccoz.

The Asp99-Arg188 salt bridge of the Pseudomonas aeruginosa HemO is critical in allowing conformational flexibility during catalysis.

The P. aeruginosa iron-regulated heme oxygenase (HemO) is required within the host for the utilization of heme as an iron source. As iron is essential for survival and virulence, HemO represents a novel antimicrobial target. We recently characterized small molecule inhibitors that bind to an allosteric site distant from the heme pocket, and further proposed binding at this site disrupts a nearby salt bridge between D99 and R188. Herein, through a combination of site-directed mutagenesis and hydrogen-deuterium exchange mass spectrometry (HDX-MS), we determined that the disruption of the D99-R188 salt bridge leads to significant decrease in conformational flexibility within the distal and proximal helices that form the heme-binding site. The RR spectra of the resting state Fe(III) and reduced Fe(II)-deoxy heme-HemO D99A, R188A and D99/R188A complexes are virtually identical to those of wild-type HemO, indicating no significant change in the heme environment. Furthermore, mutation of D99 or R188 leads to a modest decrease in the stability of the Fe(II)-O2 heme complex. Despite this slight difference in Fe(II)-O2 stability, we observe complete loss of enzymatic activity. We conclude the loss of activity is a result of decreased conformational flexibility in helices previously shown to be critical in accommodating variation in the distal ligand and the resulting chemical intermediates generated during catalysis. Furthermore, this newly identified allosteric binding site on HemO represents a novel alternative drug-design strategy to that of competitive inhibition at the active site or via direct coordination of ligands to the heme iron.