Purification and initial characterization of a novel Porphyromonas gingivalis HmuY protein expressed in Escherichia coli and insect cells

Porphyromonas gingivalis acquires iron and heme from the host environment using gingipains, lipoproteins, and outer-membrane receptors. Recently, we identified and characterized a heme receptor HmuR. The hmuR gene is localized in an operon together with a hmuY gene encoding a putative heme-binding protein. The aim of this study was to overexpress and perform a preliminary analysis of the recombinant HmuY protein. We constructed and examined several recombinant HmuY variants which were overexpressed and purified from Escherichia coli and insect cells. Recombinant HmuY protein was expressed in insect cells at levels similar to those in E. coli cells. This protein is predominantly present in a monomeric form but also dimerizes and several other oligomerization forms were found. Hemin and ATP binding to the purified HmuY showed that this protein may play a regulatory function in hemin utilization in P. gingivalis.     

Gallium(III), cobalt(III) and copper(II) protoporphyrin IX exhibit antimicrobial activity against Porphyromonas gingivalis by reducing planktonic and biofilm growth and invasion of host epithelial cells

Porphyromonas gingivalis acquires heme for growth, and initiation and progression of periodontal diseases. One of its heme acquisition systems consists of the HmuR and HmuY proteins. This study analyzed the antimicrobial activity of non-iron metalloporphyrins against P. gingivalis during planktonic growth, biofilm formation, epithelial cell adhesion and invasion, and employed hmuY, hmuR and hmuY-hmuR mutants to assess the involvement of HmuY and HmuR proteins in the acquisition of metalloporphyrins. Iron(III) mesoporphyrin IX (mesoheme) and iron(III) deuteroporphyrin IX (deuteroheme) supported planktonic growth of P. gingivalis cells, biofilm accumulation, as well as survival, adhesion and invasion of HeLa cells in a way analogous to protoheme. In contrast, cobalt(III), gallium(III) and copper(II) protoporphyrin IX exhibited antimicrobial activity against P. gingivalis, and thus represent potentially useful antibacterial compounds with which to target P. gingivalis. P. gingivalis hmuY, hmuR and hmuY-hmuR mutants showed decreased growth and infection of epithelial cells in the presence of all metalloporphyrins examined. In conclusion, the HmuY protein may not be directly involved in transport of free metalloporphyrins into the bacterial cell, but it may also play a protective role against metalloporphyrin toxicity by binding an excess of these compounds.     

Lysine-specific gingipain K and heme/hemoglobin receptor HmuR are involved in heme utilization in Porphyromonas gingivalis

We have previously reported on the identification and characterization of the Porphyromonas gingivalis A7436 strain outer membrane receptor HmuR, which is involved in the acquisition of hemin and hemoglobin. We demonstrated that HmuR interacts with the lysine- (Kgp) and arginine- (HRgpA) specific proteases (gingipains) and that Kgp and HRgpA can bind and degrade hemoglobin. Here, we report on the physiological significance of the HmuR-Kgp complex in heme utilization in P. gingivalis through the construction and characterization of a defined kgp mutant and a hmuR kgp double mutant in P. gingivalis A7436. The P. gingivalis kgp mutant exhibited a decreased ability to bind both hemin and hemoglobin. Growth of this strain with hemoglobin was delayed and its ability to utilize hemin as a sole iron source was diminished as compared to the wild type strain. Inactivation of both the hmuR and kgp genes resulted in further decreased ability of P. gingivalis to bind hemoglobin and hemin, as well as diminished ability to utilize either hemin or hemoglobin as a sole iron source. Collectively, these in vivo results further confirmed that both HmuR and Kgp are involved in the utilization of hemin and hemoglobin in P. gingivalis A7436.     

Characterization of a hemophore-like protein from Porphyromonas gingivalis

The porphyrin auxotrophic pathogen Porphyromonas gingivalis obtains the majority of essential iron and porphyrin from host hemoproteins. To achieve this, the organism expresses outer membrane gingipains containing cysteine proteinase domains linked to hemagglutinin domains. Heme mobilized in this way is taken up by P. gingivalis through a variety of potential portals where HmuY/HmuR of the hmu locus are best described. These receptors have relatively low binding affinities for heme. In this report, we describe a novel P. gingivalis protein, HusA, the product of PG2227, which rapidly bound heme with a high binding constant at equilibrium of 7 × 10(-10) M. HusA is both expressed on the outer membrane and released from the organism. Spectral analysis indicated an unusual pattern of binding where heme was ligated preferentially as a dimer. Further, the presence of dimeric heme induced protein dimer formation. Deletional inactivation of husA showed that expression of this moiety was essential for growth of P. gingivalis under conditions of heme limitation. This finding was in accord with the pronounced increase in gene expression levels for husA with progressive reduction of heme supplementation. Antibodies reactive against HusA were detected in patients with chronic periodontitis, suggesting that the protein is expressed during the course of infection by P. gingivalis. It is predicted that HusA efficiently sequesters heme from gingipains and fulfills the function of a high affinity hemophore-like protein to meet the heme requirement for growth of P. gingivalis during establishment of infection.     

A 35-kDa co-aggregation factor is a hemin binding protein in Porphyromonas gingivalis

It has been known that Porphyromonas gingivalis has an obligate requirement for hemin or selected heme- or Fe-containing compounds for its growth. In addition, the influence of hemin on the expression of several putative virulence factors produced by this bacterium has also been recently documented; however, the mechanisms involved in hemin uptake are poorly defined. We succeeded in cloning the gene coding for the 35-kDa protein, which was specifically expressed in P. gingivalis and seemed to confer colonizing activities. Recently, we have constructed the P. gingivalis 381 mutant defective in the 35-kDa protein by insertion mutagenesis. The beige mutant exhibited little co-aggregation and the virulence was also decreased. Based on these results and homology search analysis, we focused on assessing the hemin bindings and found the heme regulatory motif (HRM) as a hemin direct binding site. The 35-kDa protein did possess the binding ability of selected protoporphyrins involving the hemin. These results demonstrated that 35-kDa protein is one of the hemin binding proteins in P. gingivalis and suggested that hemin binding ability of 35-kDa protein is important for the expression of virulence in P. gingivalis.     

Burkholderia pseudomallei known siderophores and hemin uptake are dispensable for lethal murine melioidosis

Burkholderia pseudomallei is a mostly saprophytic bacterium, but can infect humans where it causes the difficult-to-manage disease melioidosis. Even with proper diagnosis and prompt therapeutic interventions mortality rates still range from >20% in Northern Australia to over 40% in Thailand. Surprisingly little is yet known about how B. pseudomallei infects, invades and survives within its hosts, and virtually nothing is known about the contribution of critical nutrients such as iron to the bacterium's pathogenesis. It was previously assumed that B. pseudomallei used iron-acquisition systems commonly found in other bacteria, for example siderophores. However, our previous discovery of a clinical isolate carrying a large chromosomal deletion missing the entire malleobactin gene cluster encoding the bacterium's major high-affinity siderophore while still being fully virulent in a murine melioidosis model suggested that other iron-acquisition systems might make contributions to virulence. Here, we deleted the major siderophore malleobactin (mba) and pyochelin (pch) gene clusters in strain 1710b and revealed a residual siderophore activity which was unrelated to other known Burkholderia siderophores such as cepabactin and cepaciachelin, and not due to increased secretion of chelators such as citrate. Deletion of the two hemin uptake loci, hmu and hem, showed that Hmu is required for utilization of hemin and hemoglobin and that Hem cannot complement a Hmu deficiency. Prolonged incubation of a hmu hem mutant in hemoglobin-containing minimal medium yielded variants able to utilize hemoglobin and hemin suggesting alternate pathways for utilization of these two host iron sources. Lactoferrin utilization was dependent on malleobactin, but not pyochelin synthesis and/or uptake. A mba pch hmu hem quadruple mutant could use ferritin as an iron source and upon intranasal infection was lethal in an acute murine melioidosis model. These data suggest that B. pseudomallei may employ a novel ferritin-iron acquisition pathway as a means to sustain in vivo growth.     

Novel hemin binding domains in the Corynebacterium diphtheriae HtaA protein interact with hemoglobin and are critical for heme iron utilization by HtaA

The human pathogen Corynebacterium diphtheriae utilizes hemin and hemoglobin as iron sources for growth in iron-depleted environments. The use of hemin iron in C. diphtheriae involves the dtxR- and iron-regulated hmu hemin uptake locus, which encodes an ABC hemin transporter, and the surface-anchored hemin binding proteins HtaA and HtaB. Sequence analysis of HtaA and HtaB identified a conserved region (CR) of approximately 150 amino acids that is duplicated in HtaA and present in a single copy in HtaB. The two conserved regions in HtaA, designated CR1 and CR2, were used to construct glutathione S-transferase (GST) fusion proteins (GST-CR1 and GST-CR2) to assess hemin binding by UV-visual spectroscopy. These studies showed that both domains were able to bind hemin, suggesting that the conserved sequences are responsible for the hemin binding property previously ascribed to HtaA. HtaA and the CR2 domain were also shown to be able to bind hemoglobin (Hb) by the use of an enzyme-linked immunosorbent assay (ELISA) method in which Hb was immobilized on a microtiter plate. The CR1 domain exhibited a weak interaction with Hb in the ELISA system, while HtaB showed no significant binding to Hb. Competitive binding studies demonstrated that soluble hemin and Hb were able to inhibit the binding of HtaA and the CR domains to immobilized Hb. Moreover, HtaA was unable to bind to Hb from which the hemin had been chemically removed. Alignment of the amino acid sequences of CR domains from various Corynebacterium species revealed several conserved residues, including two highly conserved tyrosine (Y) residues and one histidine (H) residue. Site-directed mutagenesis studies showed that Y361 and H412 were critical for the binding to hemin and Hb by the CR2 domain. Biological assays showed that Y361 was essential for the hemin iron utilization function of HtaA. Hemin transfer experiments demonstrated that HtaA was able to acquire hemin from Hb and that hemin bound to HtaA could be transferred to HtaB. These findings are consistent with a proposed mechanism of hemin uptake in C. diphtheriae in which hemin is initially obtained from Hb by HtaA and then transferred between surface-anchored proteins, with hemin ultimately transported into the cytosol by an ABC transporter.     

Roles of porphyrins and host iron transport proteins in regulation of growth of Porphyromonas gingivalis W50.

Porphyromonas gingivalis (Bacteroides gingivalis) requires iron in the form of hemin for growth and virulence in vitro, but the contributions of the porphyrin ring structure, porphyrin-associated iron, host hemin-sequestering molecules, and host iron-withholding proteins to its survival are unknown. Therefore, the effects of various porphyrins, host iron transport proteins, and inorganic iron sources on the growth of P. gingivalis W50 were examined to delineate the various types of iron molecules used for cellular metabolism. Cell envelope-associated hemin and iron stores contributed to the growth of P. gingivalis in hemin-free culture, and depletion of these endogenous reserves required eight serial transfers into hemin-free medium for total suppression of growth. Comparable growth of P. gingivalis was observed with 7.7 microM equivalents of hemin as hemoglobin (HGB), methemoglobin, myoglobin, hemin-saturated serum albumin, lactoperoxidase, cytochrome c, and catalase. Unrestricted growth was recorded in the presence of haptoglobin-HGB and hemopexin-hemin complexes, indicating that these host defense proteins do not sequester HGB and hemin from P. gingivalis. The iron chelator 2,2'-bipyridyl functionally chelated hemin-associated iron, resulting in dose-dependent inhibition of growth in hemin-restricted cultures at 1 to 25 microM 2,2'-bipyridyl concentrations. In the absence of an exogenous iron source, protoporphyrin IX did not support P. gingivalis growth. These findings suggest that the iron atom in the hemin molecule is the critical constituent for growth and that the tetrapyrrole porphyrin ring structure may represent an important vehicle for delivery of iron into the P. gingivalis cell. P. gingivalis does not have a strict requirement for porphyrins, since growth occurred with nonhemin iron sources, including high concentrations (200 muM) of ferric, ferrous, and nitrogenous inorganic iron, and P. gingivalis exhibited unrestricted growth in the presence of host transferrin, lactoferrin, and serum albumin. The diversity of iron substrates utilized by P. gingivalis and the observation that growth was not affected by the bacteriostatic effects of host iron-withholding proteins, which it may encounter in the periodontal pocket, may explain why P. gingivalis is such a formidable pathogen in the periodontal disease process.     

Effect of concentration of compounds containing iron on the growth of Porphyromonas gingivalis

Abstract We examined the effect of the concentration of various types of iron molecules on the regulation of growth of Porphyromonas gingivalis . Bacterial growth was monitored spectrophotometrically. The hemin-depleted cells of P. gingivalis 381 were incubated in the basal medium plus test substrates such as hemoglobin, hemin, transferrin and various inorganic iron compounds. The relationship between the specific growth rate of organisms and the concentration of iron-containing compounds was determined. The value of K _s, a parameter analogous to the Michaelis-Menten constant, was estimated. P . gingivalis 381 showed a K _ss value of 3.85, 4.91 and 0.0017 μM for hemin, transferrin and hemoglobin, respectively. However, the inorganic iron compounds tested did not support growth of P. gingivalis . These findings suggest that P. gingivalis utilizes hemoglobin as an iron source much more effectively than other iron-containing compounds under an iron-limited environment.     

Iron-regulated outer membrane proteins in the periodontopathic bacterium, Bacteroides gingivalis

Hemin has been implicated in the pathogenesis of the oral pathogen, Bacteroides gingivalis. In order to elucidate the role of hemin (iron) in the growth and expression of outer membrane proteins, B. gingivalis strain W50 was grown with and without hemin to induce iron-limitation. Cells grew slower under iron stress and growth was completely inhibited in the absence of added hemin. The outer membrane protein profiles of B. gingivalis grown under iron-replete and iron-restricted conditions were studied by extrinsic radiolabelling with [125I] and polyacrylamide gel-electrophoresis. The induction of 10 surface proteins, with apparent molecular weights of 26, 29, 50, 56, 58, 60, 62, 71, 77, and 80 Kd, was observed in B. gingivalis grown under iron-restricted conditions. These proteins were repressed under iron-replete conditions. We postulate the involvement of the iron-regulated proteins in hemin uptake and virulence in B. gingivalis.     

Characterization of MbrC involved in bacitracin resistance in Streptococcus mutans

Deferoxamine (DFO), an FDA-approved iron chelator used for treatment of iron poisoning, affects bacteria as iron availability is intimately connected with growth and several virulence determinants. However, little is known about the effect on oral pathogens. In this study, the effect of DFO on Porphyromonas gingivalis, a major periodontopathogen which has an essential growth requirement for hemin (Fe(3+)-protoporphyrin IX), was evaluated. The viability of P. gingivalis W83 was not affected by 0.06-0.24 mM DFO, whereas the doubling time of the bacterium was considerably prolonged by DFO. The inhibitory effect was evident at earlier stages of growth and reduced by supplemental iron. UV-visible spectra using the pigments from P. gingivalis cells grown on blood agar showed that DFO inhibited μ-oxo bisheme formation by the bacterium. DFO decreased accumulation and energy-driven uptake of hemin by P. gingivalis. Antibacterial effect of H(2)O(2) and metronidazole against P. gingivalis increased in the presence of DFO. Collectively, DFO is effective for hemin deprivation in P. gingivalis suppressing the growth and increasing the susceptibility of the bacterium to other antimicrobial agents such as H(2)O(2) and metronidazole. Further experiments are necessary to show that DFO may be used as a therapeutic agent for periodontal disease.     

Effects of non-iron metalloporphyrins on growth and gene expression of Porphyromonas gingivalis

The oral anaerobic bacterium Porphyromonas gingivalis, which is implicated as an important pathogen for chronic periodontitis, requires heme for its growth. Non-iron metalloporphyrins, In-PPIX and Ga-PPIX, were examined for antibacterial effects on P. gingivalis. Both In-PPIX and Ga-PPIX caused retardation of P. gingivalis growth in a dose-dependent fashion. Microarray and qPCR analyses revealed that In-PPIX treatment upregulated the expression of several genes encoding proteins including ClpB and ClpC, which are members of the Clp (caseinolytic protease, Hsp100) family, and aRNR, aRNR-activating protein and thioredoxin reductase, whereas In-PPIX treatment had no effect on the expression of genes encoding proteins involved in heme uptake pathways, Hmu-mediated, Iht-mediated and Tlr-mediated pathways. P. gingivalis ihtA and ihtB mutants were more resistant to In-PPIX than was the wild-type parent, whereas hmuR and tlr mutants did not show such resistance to In-PPIX. The results suggest that In-PPIX is incorporated by the Iht-mediated heme uptake pathway and that it influences protein quality control and nucleotide metabolism and retards growth of P. gingivalis.     

The RprY response regulator of Porphyromonas gingivalis

Porphyromonas gingivalis is a Gram-negative oral anaerobe associated with chronic adult periodontitis. Its ecological niche is the gingival crevice, where the organism adapts to the challenges of the infectious process such as host defence and bacterial products. Bacterial responses to environmental changes are partly regulated by two-component signal transduction systems. Several intact systems were annotated in the genome of P. gingivalis, as well as an orphan regulator encoding a homologue of RprY, a response regulator from Bacteroides fragilis. With the goal of defining the environmental cues that activate RprY in P. gingivalis, we used several strategies to identify its regulon. Results from gene expression and DNA-protein binding assays identified target genes that were either involved in transport functions or associated with oxidative stress, and indicated that RprY can act as an activator and a repressor. RprY positively activated the primary sodium pump, NADH : ubiquinone oxidoreductase (NQR), and RprY protein also interacted with the promoter regions of nqrA genes from B. fragilis and Vibrio cholerae. Given that gingival bleeding and infiltration of host defence cells are symptoms of periodontal infection, iron products released from blood and reactive oxygen species from polymorphonuclear leucocytes may be potential inducers of the RprY regulon.