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For many pathogenic microorganisms, iron acquisition represents a significant stress during the colonization of a mammalian host. Heme is the single most abundant source of soluble iron in this environment. While the importance of iron assimilation for nearly all organisms is clear, the mechanisms by which heme is acquired and utilized by many bacterial pathogens, even those most commonly found at sites of infection, remain poorly understood.
An alternative protocol for the production and purification of the outer membrane hemoglobin receptor HmbR from the pathogen Neisseria meningitidis has facilitated a biophysical characterization of this outer membrane transporter by electronic absorption, circular dichroism, electron paramagnetic resonance, and resonance Raman techniques. HmbR co-purifies with 5-coordinate high spin ferric heme bound. The heme binding site accommodates exogenous imidazole as a sixth ligand, which results in a 6-coordinate, low-spin ferric species.
Both the 5- and 6-coordinate complexes are reduced by sodium hydrosulfite. These findings are consistent with an emerging paradigm wherein the ferric iron center of bound heme is coordinated by a tyrosine ligand.
In summary, this study provides the first spectroscopic characterization for any heme or iron transporter in Neisseria meningitidisand suggests a coordination environment heretofore unobserved in a TonB-dependent hemin transporter. A detailed understanding of the nutrient acquisition pathways in common pathogens such as N.
Iron is essential for many types of biochemical reactions throughout biology. One obstacle faced by any organism that infects a host is the acquisition of iron from their environment, which is go here highly regulated by the host and tightly sequestered in heme and other iron binding proteins. Of interest to this work are gram-negative bacteria that have evolved rather sophisticated mechanisms to meet their iron requirements.
These are classified into two broad categories, referred to as siderophore and receptor mediated transport [ 12 ]. In the former, a low molecular weight compound with a high affinity for iron, referred to as a siderophore, is synthesized and secreted by the bacteria into the extracellular milieu.
Upon binding to ferric iron, the siderophore is recognized and transported into the cell by a TonB-dependent receptor. After subsequent Athens Georgia Dating Free Article Spinner Tool into the cytoplasm via an ABC ATP-binding cassette transport system, source iron is reduced and released [ 3 — 5 ].
The second mechanism involves direct binding of a heme-containing host protein to a specific TonB-dependent receptor on the surface of a Gram-negative cell.
The binding interaction excises the heme molecule from the native protein and transports it into the periplasm [ 6 Athens Georgia Dating Free Article Spinner Tool 8 ]. Both of these types of receptors require the proton motive force of the TonB accessory protein for proper transport, and thus are referred to as TonB dependent transporters TBDTs [ 9 ]. Once inside the periplasm, heme is further shuttled by a periplasmic binding protein to an ABC system on the inner membrane that transports it into the cytoplasm [ 10 ].
The bacterial pathogen Neisseria meningitidis is see more of the leading causes of bacterial meningitis and meningococcal septicemia [ 12 ]. The increasing number of strains resistant to traditional antibiotics has driven research into developing novel drugs for treating meningococcal infections [ 13 ].
Since virulence of this pathogen is tightly coupled to the availability of iron in the local environment of the host, one antimicrobial strategy is to disrupt its iron uptake system. Although this approach has been well recognized for several years, to date, no reliable drug treatment, based on targeting this essential pathway, exists. This deficiency is due, in part, to a Athens Georgia Dating Free Article Spinner Tool of fundamental understanding of how iron transport occurs in this, and other bacterial pathogens.
Targeting the iron uptake system of Neisseria meningitidis could be of particular interest, since synthesis of siderophores has not been definitively established in this organism, strongly suggesting that specific receptors for iron- and heme-containing host proteins are the sole means by which iron requirements are fulfilled [ 14 ].
Indeed, several such transport proteins have been identified in N. Disruption of any one of these transport systems should, in theory, decrease the iron available to the organism and cause a decrease in virulence. The hemoglobin receptor HmbR is one of the more promising targets due to its high prevalence in invasive strains, specificity for human hemoglobin, surface accessibility, and monomeric nature [ 1516 ]. Additionally, the expression of HmbR significantly increases when hemoglobin is the sole source of iron, suggesting that it is a major iron delivery protein that contributes to the aggressive nature of meningococcal septicemia [ 17 ].
Although HmbR is a promising drug target, like other heme transporters, very little is known about its structure, and how it extricates and transports heme from hemoglobin. In this study we have isolated HmbR and have obtained electronic absorption, circular dichroism CDelectron paramagnetic resonance EPRand resonance Raman rR spectra on the purified protein.
Hence, the design of drugs targeting heme transport proteins will likely need to take into account the differences between receptors of different organisms, even if they have identical substrates. In general, samples dialyzed using Slide-a-Lyzer cassettes as described above were diluted to 3. Google Scholar Download references.
This can be converted to a 6-coordinate low spin ferric state with the addition of imidazole, and both of these read more are susceptible to reduction upon treatment with sodium hydrosulfite. We also source residues involved in maintaining proper heme binding properties, and propose that a residue with an oxygen-centered partial negative charge, such as tyrosine, acts as an axial ligand in coordinating the heme, unlike a histidine residue observed in other TBDTs, such as HasA from Seratia marcesens [ 18 ] and ShuA from Shigella dysenteriae [ 719 ], and others [ 20 ].
As a whole, our investigation provides the first spectroscopic characterization of any heme or iron transporter in Neisseria meningitidis, and indicates that the cell-surface binding pocket of HmbR may provide a heme axial ligand environment distinct from the above TBDTs characterized to date.
The plasmids used in this study are listed in Table 1. Residues selected for mutational analysis were determined in two ways. Each mutation was engineered using Quickchange site-directed mutagenesis. In brief, complimentary mutagenic oligonucleotide primers were designed to contain the desired mutation flanked by a 15—20 base pair extension on each end homologous to the parent plasmid DPB Following the PCR, 1.
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Wild-type and single amino acid HmbR mutants were purified by identical methods. When the cells reached A 0. Owing to low yields, approximately 24 L of cell pellets were utilized for the purification of each HmbR variant. Cell pellets were collected and resuspended in mL of 10 mM potassium phosphate, pH 6. The cell suspension was then subsequently passed twice through a French Athens Georgia Dating Free Article Spinner Tool cell, and then spiked with 1 mM of MgSO 4and cleared of large cellular debris by spinning at g for 15 min.
The resulting supernatant was supplemented with hemin to a final concentration of 2. The isolated membranes were washed once in mL of 20 mM potassium phosphate, pH 6. The unsolubilized material was recovered atg for 1. Solubilized protein was recovered by centrifugationg for 1. The suspension was then transferred to a gravity-flow column, washed with 90 mL of 20 mM potassium phosphate, pH 7. Elution fractions containing the highest protein concentrations were pooled into a centrifugal filter unit with a 50 kDa nominal molecular weight cut-off NMWC This web pageand concentrated to an approximate volume of 1.
The imidazole was removed from these samples by two different methods. For all other applications, the imidazole was removed from the samples by iterative dilutions and concentrations in an Amicon concentrator NMWC using 20 mM potassium phosphate, pH 7. The final imidazole concentration was determined by calculating the dilution factor of the retentate after each spin.
Each method evaluated purified HmbR with or without the presence of mM imidazole, in both ferric and ferrous states. To record spectra under anaerobic conditions, samples and buffers were degassed using a vacuum manifold, and transferred to an anaerobic chamber. In both cases, the reduced sample was less stable and led to precipitation over time, however, reduction with sodium hydrosulfate occurred quickly and provided ample time to work with the sample. Electronic absorption spectra were obtained using samples that were anaerobically sealed in quartz cuvettes with link 1-cm path length.
All plots were generated using Excel graphing software from data electronically extracted from the nanodrop ND software program version 3. In order to determine the molar percent of heme present Athens Georgia Dating Free Article Spinner Tool purified HmbR samples, we performed a pyridine hemochromogen assay as previously described [ 23 ].
In general, samples dialyzed using Slide-a-Lyzer cassettes as described above were diluted to 3. Half the volume of these samples was removed and reduced with sodium hydrosulfite, and the absorbance difference between the reduced and unreduced samples was determined at nm. Heme concentrations were calculated using a millimolar extinction coefficient of These were compared to protein concentrations previously determined through a detergent compatible protein assay Bio-Rad DC.
Each HmbR variant was evaluated by this method a minimum of three independent times. A circular dichroism spectrum was recorded for purified HmbR obtained as described above in the ferric state. The sample was diluted to 3. A Jasco J spectropolarimeter obtained measurements from to nm using a 10 nm bandwidth.
The modulation amplitude was 6. The microwave frequency and power were 9. Resonance Raman scattering was excited with Samples were prepared in 0. Samples were contained in 5 mm NMR tubes and spun at 20 Hz at room temperature to avoid laser-induced degradation. Scattered light was collected, filtered to remove Rayleigh scattered light and focused onto the slit of a 0.
Spectra were recorded with a liquid nitrogen cooled CCD camera.
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Raman shift axes were calibrated against known Raman shifts for toluene and DMF. No spectral artifacts attributable to laser-induced sample damage were observed. The protocol described above Section 2. The removal of source from purified protein fractions resulted in a qualitative color change from bright red to rust brown.
Much like the rR spectra of heme catalases [ 45 ], this band is insensitive to changes in pH from 6. Additionally, the expression of HmbR significantly increases when hemoglobin is the sole source of iron, suggesting that it is a major iron delivery protein that contributes to the aggressive nature of meningococcal septicemia [ 17 ]. More importantly, in the absence of heme dissociation constants, it cannot be assumed that reduced heme loading is a result of reduced heme affinity. The plasmids used in this study are listed in Table 1.
Based on this observation, we hypothesized that the imidazole could interact with the heme that was bound to HmbR during purification, consistent with the behavior reported for other outer membrane heme transporters [ 71819 ]. In order to test this, we monitored the electronic absorption spectra of samples in the absence and presence of imidazole, in both the ferric and ferrous states Fig.
The ferric HmbR sample, in the presence of mM imidazole, exhibited a Soret maximum at nm that shifted to nm when Athens Georgia Dating Free Article Spinner Tool. These spectral features are characteristic of hexacoordinate, low spin heme hemophores [ 24 ]. HmbR samples that were free of imidazole conveyed slightly different behavior in their absorption spectra. Specifically, the ferric sample had a Soret maximum at nm that shifted to nm when reduced, consistent with high-spin pentacoordinate hemes Fig.
Reduction of HmbR under aerobic or anaerobic conditions was possible, however, the intensity of these bands was significantly click to see more relative to the more prominent peaks observed in the presence of imidazole. In contrast, and in order to confirm that the observed reduction in the intensity of absorption spectra peaks was not a result of spurious artifacts resulting from the re-oxidation of the aerobically-reduced HmbR during the course of the experiment, we also performed a titration experiment under anaerobic conditions using limiting quantities of sodium hydrosulfite, and acquired electronic absorption spectra from these samples.
No change in the overall spectra was seen when compared to the Athens Georgia Dating Free Article Spinner Tool obtained when measurements were recorded aerobically. In fact, attempts to reoxidize the sample were unsuccessful, leading to protein precipitation.
Hence, the scan of the reduced sample devoid of imidazole must be interpreted with caution, as some of the peaks could be attributed to a mix of non-native protein configurations in the conditions tested. Our purification strategy utilizes detergent micelles in order to solubilize HmbR, which complicates determining the absolute stoichiometry for heme binding to the receptor. However, the intensities of our absorption spectra were less than would be anticipated for a 1: In order to test continue reading heme binding mechanism, and to potentially obtain protein with an increased affinity for heme binding for facilitating spectroscopic studies, we created a number of point mutations either targeting tyrosines in highly conserved regions of HmbR compared to other proteins YC and YCor residues within regions previously determined to be functionally significant for heme binding within HmbR itself H87C, HA, YA, and YC [ 21 ].
These mutations were H87C The remaining two YC and YC had a diminished effect, but not pronounced enough to be considered statistically significant. These results are consistent with the previous deletion study of HmbR, which showed that partial heme transport capacity was retained, even when large stretches of amino acids were deleted from the transporter [ 21 ] see discussion.
We considered the possibility that the low molar binding ratio of heme to HmbR may be due to mild protein denaturation leading to a loss in affinity for heme. This would also explain why we were unable to identify mutants with enhanced heme binding.