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Iron, an essential nutrient with limited bioavailability, requires specialized cellular mechanisms for uptake. Although iron uptake into the cytoplasm in the form of heme has been well characterized in many bacteria, the subsequent trafficking is poorly understood. The cytoplasmic heme-binding proteins belong to a structurally related family though...
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... In all three systems, transport of the heme through the impermeable outer membrane of P. aeruginosa occurs through specialized receptors, called the TonB-dependent receptor (TBDR) family, in an energy-dependent process (11). The Phu system plays a major role in the heme acquisition through the TBDR PhuR that uptakes free heme directly (10,12). The Has system involves the secretion of a hemebinding protein, hemeophore HasAp, which captures free or hemoglobin-bound heme and shuttles it to the TBDR HasR (13), which further extracts heme from the hemeophore and transports it into the periplasm (8,10). ...
Bloodstream infections (BSIs) caused by Pseudomonas aeruginosa are associated with a high mortality rate in the clinic. However, the fitness mechanisms responsible for the evolution of virulence factors that facilitate the dissemination of P. aeruginosa to the bloodstream are poorly understood. In this study, a transcriptomic analysis of the BSI-associated P. aeruginosa clinical isolates showed a high-level expression of cell-surface signaling (CSS) system Hxu. Whole-genome sequencing and comparative genomics of these isolates showed that a mutation in rnfE gene was responsible for the elevated expression of the Hxu-CSS pathway. Most importantly, deletion of the hxuIRA gene cluster in a laboratory strain PAO1 reduced its BSI capability while overexpression of the HxuIRA pathway promoted BSI in a murine sepsis model. We further demonstrated that multiple components in the blood plasma, including heme, hemoglobin, the heme-scavenging proteins haptoglobin, and hemopexin, as well as the iron-delivery protein transferrin, could activate the Hxu system. Together, these studies suggested that the Hxu-CSS system was an important signal transduction pathway contributing to the adaptive pathogenesis of P. aeruginosa in BSI.
... In the opportunistic pathogen P. aeruginosa the iron regulated HemO oxidatively cleaves heme to release BVIXβ and BVIXδ (Ratliff et al., 2001). Heme uptake studies combined with LC-MS/MS analysis of the P. aeruginosa hemO deletion strain showed HemO is absolutely required to drive heme uptake into the cell (O'Neill et al., 2012). The significance of HemO in P. aeruginosa iron acquisition and its role in virulence has led to extensive study of the protein itself (Ratliff et al., 2001;Friedman et al., 2004). ...
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 BVIXβ 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 BVIXβ 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 BVIXβ and -δ isomers >300-fold when compared to the chemical oxidation of heme.
... Moreover, in contrast to the high degree of similarity between the apo-and holo-PhuS crystal structures, biophysical evidence suggests significant differences in the conformational landscapes of apo-and holo-PhuS in solution and points toward a more dynamic heme binding pocket in apo-PhuS. In the first of such studies, analytical ultracentrifugation, limited trypsin digests, and PhuS-HemO binding studies suggested that heme binding induces large conformational changes in PhuS that drive the interaction of PhuS with HemO (23). Subsequently, hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies (22) provided corroborating evidence for a conformational change upon heme binding, as large decreases in deuterium uptake were observed in various localized regions in holo-PhuS when compared with apo-PhuS ( Fig. 1, B, C, and D). ...
... Site-directed mutagenesis to create the PhuS H212R variant was performed as previously described (18,22). Expression and purification of the wild-type (WT) PhuS and H212R proteins were carried out as previously described (15,18,22,23) with slight modifications. Briefly, the PhuS or PhuS H212R mutant lysate was applied to a Sepharose-G column (Cytiva, Marlborough, MA) equilibrated in 20 mM Tris-HCl (pH 8.0) and washed with 5 column volumes of the same buffer. ...
... His212, located in a7, has been shown to serve as an alternate proximal coordinating residue for heme (5,23). It has also been proposed to play a role in heme transfer, in which the holo-PhuS/HemO interaction would trigger a ligand switch from His209 to His212 (23). ...
The cytoplasmic heme binding protein from P.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 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 vs holo-PhuS revealed large differences in deuterium uptake, notably in α-helices 6, 7 and 8 (α-6,7,8), 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 α-6,7,8 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) which 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 where α-6,7,8 are significantly rearranged compared to the crystal structure, including a loss of secondary structure in α6 and the displacement of α7 towards 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/transfer properties. The proposed conformational landscape provides structural insights into the modulation by heme of the dual function of PhuS.
... The rR studies of ferrous-CO complexes of WT and H111A HupZ, including their isotopically substituted analogs, places the ν(Fe-C)/ν(C-O) points on the inverse correlation line characteristic for histidine ligated proteins ( Figure 4); e.g., other proximal ligand candidates, such as Tyr residue or OH -/H2O, would result in a different location of the ν(Fe-C)/ν(C-O) point on the inverse correlation plots. There have been several other instances where non-enzymatic degradation of heme has led to the misannotation of heme-binding proteins [39][40][41]. Thus, it was crucial for us to interrogate the current form of HupZ and elucidate the nature of its heme degradation. We noted that the H111A variant had similar activity comparable to that of wild-type HupZ (Figure 8), suggesting that His111 does not contribute to the observed activity. ...
... The rR studies of ferrous-CO complexes of WT and H111A HupZ, including their isotopically substituted analogs, places the ν(Fe-C)/ν(C-O) points on the inverse correlation line characteristic for histidine ligated proteins ( Figure 4); e.g., other proximal ligand candidates, such as Tyr residue or OH -/H 2 O, would result in a different location of the ν(Fe-C)/ν(C-O) point on the inverse correlation plots. There have been several other instances where non-enzymatic degradation of heme has led to the mis-annotation of heme-binding proteins [39][40][41]. Thus, it was crucial for us to interrogate the current form of HupZ and elucidate the nature of its heme degradation. We noted that the H111A variant had similar activity comparable to that of wild-type HupZ (Figure 8), suggesting that His111 does not contribute to the observed activity. ...
... These results also emphasize the importance of considering exogenous protein tag(s) when interpreting experimental observations, as previously noted in the studying of a heme-utilization protein in Mycobacterium tuberculosis [45]. In the heme-degrading enzyme MhuD from mycobacteria, the C-terminal His 6 -tag interferes with heme-binding even though no interactions between heme and the tag were observed in the X-ray crystal structures of the enzyme in complex with heme [39][40][41]46]. ...
HupZ is an expected heme degrading enzyme in the heme acquisition and utilization pathway in Group A Streptococcus. The isolated HupZ protein containing a C-terminal V5-His6 tag exhibits a weak heme degradation activity. Here, we revisited and characterized the HupZ-V5-His6 protein via biochemical, mutagenesis, protein quaternary structure, UV–vis, EPR, and resonance Raman spectroscopies. The results show that the ferric heme-protein complex did not display an expected ferric EPR signal and that heme binding to HupZ triggered the formation of higher oligomeric states. We found that heme binding to HupZ was an O2-dependent process. The single histidine residue in the HupZ sequence, His111, did not bind to the ferric heme, nor was it involved with the weak heme-degradation activity. Our results do not favor the heme oxygenase assignment because of the slow binding of heme and the newly discovered association of the weak heme degradation activity with the His6-tag. Altogether, the data suggest that the protein binds heme by its His6-tag, resulting in a heme-induced higher-order oligomeric structure and heme stacking. This work emphasizes the importance of considering exogenous tags when interpreting experimental observations during the study of heme utilization proteins.
... Expression and Purification of apo-PhuS, and PhuSH212R. Protein expression was performed as previously reported with slight modification (10,43). The PhuS or PhuS H212R mutant lysate was applied to a Sepharose-G column (GE Life Sciences) equilibrated with 20 mM Tris-HCl (pH 8.0) and washed with 5 column volumes of the same buffer. ...
Pseudomonas aeruginosa is an opportunistic pathogen requiring iron for its survival and virulence. P. aeruginosa can acquire iron from heme via the non-redundant heme assimilation system (Has) and Pseudomonas heme uptake (Phu) systems. Heme transported by either the Has 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 CHIP-PCR, EMSA and fluorescence anisotropy we show 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. qPCR analysis of P. aeruginosa WT and ΔphuS strains shows loss of PhuS abrogates the heme-dependent regulation of PrrF and PrrH levels. Taken together, our data shows 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.
... This interaction was specific for the holo-PhuS whereas apo-PhuS did not interact with HemO. Moreover, biochemical and biophysical characterization of apo-and holo-PhuS showed that haem binding drives a conformational rearrangement of PhuS that promotes the interaction with HemO (O'Neill, Bhakta, Fleming, & Wilks, 2012). The conformational rearrangement was further investigated by site-directed mutagenesis, hydrogen deuterium exchange mass spectrometry (HDX-MS) and MD simulations, revealing long-range allostery between the N-terminal and C-terminal domains critical for the conformational rearrangement on haem binding (Deredge et al., 2017). ...
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.
... Protein expression was performed as previously reported with slight modification (10,43). The PhuS or PhuSH212R mutant lysate was applied to a Sepharose-G column (GE Life Sciences) equilibrated with 20 mM Tris-HCl (pH 8.0) and washed with five column volumes of the same buffer. ...
Pseudomonas aeruginosa is an opportunistic pathogen requiring iron for its survival and virulence. P. aeruginosa can acquire iron from heme via the heme assimilation system (Has) and Pseudomonas heme uptake (Phu) systems. The Has and Phu systems have non-redundant roles in heme sensing and transport, respectively. However, despite their respective roles heme taken up by either the Has or Phu system is regulated at the metabolic level by the cytoplasmic heme binding protein PhuS, which controls heme flux through the iron-regulated heme oxygenase HemO. Herein, through a combination of CHIP-PCR, EMSA and fluorescence anisotropy we show PhuS binds upstream of the tandem iron-responsive sRNAs prrF1,F2. Furthermore, qPCR analysis of the PAO1 WT and Δ phuS allelic strain shows loss of PhuS abrogates the heme dependent regulation of PrrH. Taken together our data shows PhuS, in addition to its role in regulating extracellular heme metabolism also functions as a transcriptional regulator of the heme-dependent sRNA, PrrH. This dual function of PhuS is central to integrating extracellular heme utilization into the PrrF/PrrH sRNA regulatory network critical for P. aeruginosa adaptation and virulence within the host.
... Notably,H emS proteins, often found within heme utilization operonsi nb acterial genomes,h ave been characterizeda nd exhibit functional variability despite high sequence similarity. [48][49][50][51][52][53][54][55][56] Although the E. anophelis Ag1 genome does not contain ac anonical HO, it does contain aH emS homologue with 33-45 %a mino acid identityt oc haracterized proteins from Pseudomonas aeruginosa, Escherichia coli O157:H7,a nd Yersinia pseudotuberculosis,t hus suggesting that this gene product might be responsible for biliverdin production in E. anophelis Ag1. Within the Pseudomonas sp. ...
Anopheles mosquito microbiomes are intriguing ecological niches. Within the gut, microbes adapt to oxidative stress due to heme and iron after blood meals. Although metagenomic sequencing has illuminated spatial and temporal fluxes of microbiome populations, limited data exist on microbial growth dynamics. Here, we analyze growth interactions between a dominant microbiome species, Elizabethkingia anophelis, and other Anopheles‐associated bacteria. We find E. anophelis inhibits a Pseudomonas sp. via an antimicrobial‐independent mechanism and observe biliverdins, heme degradation products, upregulated in cocultures. Purification and characterization of E. anophelis HemS demonstrates heme degradation, and we observe hemS expression is upregulated when cocultured with Pseudomonas sp. This study reveals a competitive microbial interaction between mosquito‐associated bacteria and characterizes the stimulation of heme degradation in E. anophelis when grown with Pseudomonas sp.
... The Phu system mediates the direct acquisition of free haem through the PhuR receptor, whereas the Has system involves the secretion of a haem-binding protein, the haemophore HasAp, and the HasR receptor that extracts the haem from the haemophore and transports it into the periplasm (Fig. 1C). Haem is internalized into the cytoplasm through the PhuSTUV system and degraded by the haem oxygenase HemO, which results in the release of iron (O'Neill et al., 2012;Fig. 1C). ...
... In the periplasm, the haem binding protein PhuT delivers the haem to the PhuUV ABC transporter that translocates it into the cytoplasm where haem is sequestered by the haem-trafficking protein PhuS. PhuS forms a complex with the haem oxygenase HemO that degrades haem to biliverdin IX and CO releasing the ferric ion (O'Neill et al., 2012). B, C. The Has system is arranged in three potential operons, with the first encoding a σ ECF /anti-σ factor pair (hasI-hasS). ...
Pathogens have developed several strategies to obtain iron during infection, including the use of iron‐containing molecules from the host. Heme accounts for the vast majority of the iron pool in vertebrates and thus represents an important source of iron for pathogens. Using a proteomic approach, we have identified in this work a previously uncharacterized system, which we name Hxu, that together with the known Has and Phu systems, is used by the human pathogen Pseudomonas aeruginosa to respond to heme. We show that the Has and Hxu systems are functional signal transduction pathways of the cell‐surface signaling class and report the mechanism triggering the activation of these signaling systems. Both signaling cascades involve an outer membrane receptor (HasR and HxuA, respectively) that upon sensing heme in the extracellular medium produces the activation of an σECF factor in the cytosol. HxuA has a major role in signaling and a minor role in heme acquisition in conditions in which the HasR and PhuR receptors or other sources of iron are present. Remarkably, P. aeruginosa compensates the lack of the HasR receptor by increasing the production of HxuA, which underscores the importance of heme signaling for this pathogen. This article is protected by copyright. All rights reserved.
... For example, PhuS is a heme-trafficking protein in Pseudomonas aeruginosa that delivers heme to the heme oxygenase, HemO (55). Mutation of both PhuS His ligands to the heme iron did not eliminate heme-binding even though both His ligands are required for protein-protein interaction with HemO and subsequent heme transfer, highlighting the flexibility of the heme environment (56). Similarly, S. aureus ChdC sitedirected mutants of eight distinct residues in the substrate binding site all bound coproheme with >80% occupancy and had measured KD values within an order of magnitude of wild-type (57). ...
Staphylococcus aureus infection relies on iron acquisition from its host. S. aureus takes up iron through heme uptake by the iron-responsive surface determinant (Isd) system and by the production of iron-scavenging siderophores. Staphyloferrin B (SB) is a siderophore produced by the 9-gene sbn gene cluster for SB biosynthesis and efflux. Recently, the ninth gene product, SbnI, was determined to be a free L-serine kinase that produces O-phospho-L-serine (OPS), a substrate for SB biosynthesis. Previous studies have also characterized SbnI as a DNA-binding regulatory protein that senses heme to control sbn gene expression for SB synthesis. Here, we present crystal structures at 1.9-2.1 Å resolution of a SbnI homolog from Staphylococcuspseudintermedius (SpSbnI) in both apo form and in complex with ADP, a product of the kinase reaction; the latter confirmed the active-site location. The structures revealed that SpSbnI forms a dimer through C-terminal domain swapping and a dimer of dimers through intermolecular disulfide formation. Heme binding had only a modest effect on SbnI enzymatic activity, suggesting that its two functions are independent and structurally distinct. We identified a heme-binding site and observed catalytic heme transfer between a heme-degrading protein of the Isd system, IsdI, and SbnI. These findings support the notion that SbnI has a bifunctional role contributing precursor OPS to SB synthesis and directly sensing heme to control expression of the sbn locus. We propose that heme transfer from IsdI to SbnI enables S. aureus to control iron source preference according to the sources available in the environment.
