Source Of Iron For Growth Research Articles

The involvement of PacIRA system of Stenotrophomonas maltophilia in the uptake of Pseudomonas aeruginosa pyochelin and intraspecies competition for iron acquisition.

Stenotrophomonas maltophilia, a species of highly genetic diversity, has emerged as an important nosocomial pathogen. S. maltophilia and Pseudomonas aeruginosa are often co-isolated from pneumonia patients. In our previous study, we have demonstrated that the pacIRA cluster present in some but not all clinical S. maltophilia isolates. Proteins encoded by pacIRA operon are an extracytoplasmic function (ECF) sigma factor, a transmembrane anti-sigma regulator, and a TonB-dependent receptor. This study aimed to elucidate PacIRA system function and its significance to S. maltophilia. The pacI, pacR, and pacA genes were individually or totally deleted from the chromosome of KJΔEnt, a pacIRA-positive and siderophore-null strain. Growth promotion assay was performed to examine the implication of pacIRA system in iron utilization. Gene expression was quantified by quantitative real time PCR (qRT-PCR). Growth competition assay was executed to investigate the significance of pacIRA operon to S. maltophilia. PacIRA system contributed to utilize ferri-pyochelin of P. aeruginosa as iron sources for growth in an iron-depleted condition, but hardly utilized ferric citrate, hemin, ferri-stenobactin, and ferri-pyoverdine. PacIRA was founded to belong to Fur regulon and upregulated in response to iron-depleted stress. Growth competition assay demonstrated that pacIRA-positive S. maltophilia had a superiority over pacIRA-negative S. maltophilia in iron acquisition when they were co-cultured in P. aeruginosa ferri-pyochelin-supplemented medium. PacIRA system of S. maltophilia is a xenosiderophore uptake implement, involving in the acquisition of pyochelin of P. aeruginosa.

Transferrin Binding Protein B and Transferrin Binding Protein A2 Expand the Transferrin Recognition Range of Histophilus somni.

The bacterial bipartite transferrin receptor is an iron acquisition system that several important human and animal pathogens require for survival. It consists of the TonB-dependent transporter transferrin binding protein A (TbpA) and the surface lipoprotein transferrin binding protein B (TbpB). Curiously, the Tbps are only found in host-specific pathogens and are themselves host specific, meaning that they will bind to the transferrin of their host species but not to the transferrins of other animal species. While this phenomenon has long been established, neither the steps in the evolutionary process that led to this exquisite adaptation for the host nor the steps that could alter it are known. We sought to gain insight into these processes by studying Tbp specificity in Histophilus somni, an economically important pathogen of cattle. A past study showed that whole cells of H. somni specifically bind bovine transferrin but not transferrin from sheep and goats, two bovids whose transferrins share 93% amino acid sequence identity with bovine transferrin. To our surprise, we found that H. somni can use sheep and goat transferrins as iron sources for growth and that HsTbpB, but not HsTbpA, has detectable affinity for sheep and goat transferrins. Furthermore, a third transferrin binding protein found in H. somni, HsTbpA2, also showed affinity for sheep and goat transferrins. Our results suggest that H. somni TbpB and TbpA2 may contribute to broadening the host transferrin recognition range of H. somniIMPORTANCE Host-restricted pathogens infect a single host species or a narrow range of host species. Histophilus somni, a pathogen that incurs severe economic losses for the cattle industry, infects cattle, sheep, and goats but not other mammals. The transferrin binding proteins, TbpA and TbpB, are thought to be a key iron acquisition system in H. somni; however, despite their importance, H. somni TbpA and TbpB were previously shown to be cattle transferrin specific. In our study, we find that H. somni TbpB and another little-studied Tbp, TbpA2, bind sheep and goat transferrins, as well as bovine transferrin. Our results suggest that TbpB and TbpA2 may allow for host range expansion and provide a mechanism for how host specificity in Tbp-encoding pathogens can be altered.

Are lactoferrin receptors in Gram-negative bacteria viable vaccine targets?

A number of important Gram-negative pathogens that reside exclusively in the upper respiratory or genitourinary tract of their mammalian host rely on surface receptors that specifically bind host transferrin and lactoferrin as a source of iron for growth. The transferrin receptors have been targeted for vaccine development due to their critical role in acquiring iron during invasive infection and for survival on the mucosal surface. In this study, we focus on the lactoferrin receptors, determining their prevalence in pathogenic bacteria and comparing their prevalence in commensal Neisseria to other surface antigens targeted for vaccines; addressing the issue of a reservoir for vaccine escape and impact of vaccination on the microbiome. Since the selective release of the surface lipoprotein lactoferrin binding protein B by the NalP protease in Neisseria meningitidis argues against its utility as a vaccine target, we evaluated the release of outer membrane vesicles, and transferrin and lactoferrin binding in N. meningitidis and Moraxella catarrhalis. The results indicate that the presence of NalP reduces the binding of transferrin and lactoferrin by cells and native outer membrane vesicles, suggesting that NalP may impact all lipoprotein targets, thus this should not exclude lactoferrin binding protein B as a target.

TonB-Dependent Utilization of Dihydroxamate Xenosiderophores in Synechocystis sp. PCC 6803.

In Gram-negative bacteria, transport of ferric siderophores through outer membrane is a complex process that requires specific outer membrane transporters and energy-transducing TonB-ExbB-ExbD system in the cytoplasmic membrane. The genome of the non-siderophore-producing cyanobacterium Synechocystis sp. PCC 6803 encodes all putative components of the siderophore-mediated iron uptake system. So far, there has been no experimental evidence for the existence of such a pathway in this organism. On the contrary, its reductive iron uptake pathway has been studied in detail. We demonstrate that Synechocystis sp. PCC 6803 is capable of using dihydroxamate xenosiderophores, either ferric schizokinen (FeSK) or a siderophore of the filamentous cyanobacterium Anabaena variabilis ATCC 29413 (SAV), as the sole source of iron. Inactivation of the tonB gene or the exbB1-exbD1 gene cluster resulted in an inability to utilize these siderophores. At the same time, the inactivation of the feoB gene encoding FeoB plasma membrane ferrous iron transporter, or one of the futB or futC genes encoding permease and ATPase subunit of FutABC ferric iron transporter, did not impair the ability of cells to utilize FeSK or SAV as the sole source of iron for growth. Our data suggest that cyanobacterium Synechocystis sp. PCC 6803 is capable of acquiring iron-siderophore complexes in a TonB-dependent manner without iron reduction in the periplasm.

The ChrSA and HrrSA Two-Component Systems Are Required for Transcriptional Regulation of the hemA Promoter in Corynebacterium diphtheriae.

Corynebacterium diphtheriae utilizes heme and hemoglobin (Hb) as iron sources for growth in low-iron environments. In C. diphtheriae, the two-component signal transduction systems (TCSs) ChrSA and HrrSA are responsive to Hb levels and regulate the transcription of promoters for hmuO, hrtAB, and hemA ChrSA and HrrSA activate transcription at the hmuO promoter and repress transcription at hemA in an Hb-dependent manner. In this study, we show that HrrSA is the predominant repressor at hemA and that its activity results in transcriptional repression in the presence and absence of Hb, whereas repression of hemA by ChrSA is primarily responsive to Hb. DNA binding studies showed that both ChrA and HrrA bind to the hemA promoter region at virtually identical sequences. ChrA binding was enhanced by phosphorylation, while binding to DNA by HrrA was independent of its phosphorylation state. ChrA and HrrA are phosphorylated in vitro by the sensor kinase ChrS, whereas no kinase activity was observed with HrrS in vitro Phosphorylated ChrA was not observed in vivo, even in the presence of Hb, which is likely due to the instability of the phosphate moiety on ChrA. However, phosphorylation of HrrA was observed in vivo regardless of the presence of the Hb inducer, and genetic analysis indicates that ChrS is responsible for most of the phosphorylation of HrrA in vivo Phosphorylation studies strongly suggest that HrrS functions primarily as a phosphatase and has only minimal kinase activity. These findings collectively show a complex mechanism of regulation at the hemA promoter, where both two-component systems act in concert to optimize expression of heme biosynthetic enzymes. Understanding the mechanism by which two-component signal transduction systems function to respond to environmental stimuli is critical to the study of bacterial pathogenesis. The current study expands on the previous analyses of the ChrSA and HrrSA TCSs in the human pathogen C. diphtheriae The findings here underscore the complex interactions between the ChrSA and HrrSA systems in the regulation of the hemA promoter and demonstrate how the two systems complement one another to refine and control transcription in the presence and absence of Hb.

Crystal structure of the N-lobe of lactoferrin binding protein B from Moraxella bovis1 1This paper is an invited article as a result of a presentation at the International Lactoferrin Conference held in Mazatlan, Mexico (May 2011), and has undergone the Journal’s usual peer review process.

Lactoferrin (Lf) is a bi-lobed, iron-binding protein found on mucosal surfaces and at sites of inflammation. Gram-negative pathogens from the Neisseriaceae and Moraxellaceae families are capable of using Lf as a source of iron for growth through a process mediated by a bacterial surface receptor that directly binds host Lf. This receptor consists of an integral outer membrane protein, lactoferrin binding protein A (LbpA), and a surface lipoprotein, lactoferrin binding protein B (LbpB). The N-lobe of the homologous transferrin binding protein B, TbpB, has been shown to facilitate transferrin binding in the process of iron acquisition. Currently there is little known about the role of LbpB in iron acquisition or how Lf interacts with the bacterial receptor proteins. No structural information on any LbpB or domain is available. In this study, we express and purify from Escherichia coli the full-length LbpB and the N-lobe of LbpB from the bovine pathogen Moraxella bovis for crystallization trials. We demonstrate that M. bovis LbpB binds to bovine but not human Lf. We also report the crystal structure of the N-terminal lobe of LbpB from M. bovis and compare it with the published structures of TbpB to speculate on the process of Lf mediated iron acquisition.

The ChrA Response Regulator in Corynebacterium diphtheriae Controls Hemin-Regulated Gene Expression through Binding to the hmuO and hrtAB Promoter Regions

Corynebacterium diphtheriae, the etiologic agent of diphtheria, utilizes heme and hemoglobin (Hb) as iron sources for growth. Heme-iron utilization involves HmuO, a heme oxygenase that degrades cytosolic heme, resulting in the release of heme-associated iron. Expression of the hmuO promoter is under dual regulation, in which transcription is repressed by DtxR and iron and activated by a heme source, such as hemin or Hb. Hemin-dependent activation is mediated primarily by the ChrAS two-component system, in which ChrS is a putative heme-responsive sensor kinase while ChrA is proposed to serve as a response regulator that activates transcription. It was recently shown that the ChrAS system similarly regulates the hrtAB genes, which encode an ABC transporter involved in the protection of C. diphtheriae from hemin toxicity. In this study, we characterized the phosphorelay mechanism in the ChrAS system and provide evidence for the direct regulation of the hmuO and hrtAB promoters by ChrA. A fluorescence staining method was used to show that ChrS undergoes autophosphorylation and that the phosphate moiety is subsequently transferred to ChrA. Promoter fusion studies identified regions upstream of the hmuO and hrtAB promoters that are critical for the heme-dependent regulation by ChrA. Electrophoretic mobility shift assays revealed that ChrA specifically binds at the hmuO and hrtAB promoter regions and that binding is phosphorylation dependent. A phosphorylation-defective mutant of ChrA [ChrA(D50A)] exhibited significantly diminished binding to the hmuO promoter region relative to that of wild-type ChrA. DNase I footprint analysis further defined the sequences in the hmuO and hrtAB promoters that are involved in ChrA binding, and this analysis revealed that the DtxR binding site at the hmuO promoter partially overlaps the binding site for ChrA. DNase I protection studies as well as promoter fusion analysis suggest that ChrA and DtxR compete for binding at the hmuO promoter. Collectively, these data demonstrate that the ChrA response regulator directly controls the expression of hmuO and the hrtAB genes and the binding activity of ChrA is dependent on phosphorylation by its cognate sensor kinase ChrS.

Iron-Saturated Lactoferrin and Pathogenic Protozoa: Could this Protein be an Iron Source for Their Parasitic Style of Life?

Iron is an essential nutrient for the survival of pathogens inside a host. As a general strategy against microbes, mammals have evolved complex iron-withholding systems for efficiently decreasing the iron accessible to invaders. Pathogens that inhabit the respiratory, intestinal and genitourinary tracts encounter an iron-deficient environment on the mucosal surface, where ferric iron is chelated by lactoferrin, an extracellular glycoprotein of the innate immune system. However, parasitic protozoa have developed several mechanisms to obtain iron from host holo-lactoferrin. Tritrichomonas fetus, Trichomonas vaginalis, Toxoplasma gondii and Entamoeba histolytica express lactoferrin-binding proteins and use holo-lactoferrin as an iron source for growth in vitro; in some species, these binding proteins are immunogenic and, therefore, may serve as potential vaccine targets. Another mechanism to acquire lactoferrin iron has been reported in Leishmania spp. promastigotes, which use a surface reductase to recognize and reduce ferric iron to the accessible ferrous form. Cysteine proteases that cleave lactoferrin have been reported in E. histolytica. This review summarizes the available information on how parasites uptake and use the iron from lactoferrin to survive in hostile host environments.

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.

The ABC Transporter HrtAB Confers Resistance to Hemin Toxicity and Is Regulated in a Hemin-Dependent Manner by the ChrAS Two-Component System inCorynebacterium diphtheriae

Corynebacterium diphtheriae, the causative agent of the severe respiratory disease diphtheria, utilizes hemin and hemoglobin as iron sources for growth in iron-depleted environments. Because of the toxicity of high levels of hemin and iron, these compounds are often tightly regulated in bacterial systems. In this report, we identify and characterize the C. diphtheriae hrtAB genes, which encode a putative ABC type transporter involved in conferring resistance to the toxic effects of hemin. Deletion of the hrtAB genes in C. diphtheriae produced increased sensitivity to hemin, which was complemented by a plasmid harboring the cloned hrtAB locus. The HrtAB system was not involved in the uptake and use of hemin as an iron source. The hrtAB genes are located on the C. diphtheriae genome upstream from the chrSA operon, which encodes a previously characterized two-component signal transduction system that regulates gene expression in a heme-dependent manner. The hrtB promoter is activated by the ChrAS system in the presence of hemin or hemoglobin, and mutations in the chrSA genes abolish heme-activated expression from the hrtB promoter. It was also observed that transcription from the hrtB promoter is reduced in a dtxR deletion mutant, suggesting that DtxR is required for optimal expression of hrtAB. Previous studies proposed that the ChrS sensor kinase may be responsive to an environmental signal, such as hemin. We show that specific point mutations in the ChrS N-terminal transmembrane domain result in a reduced ability to activate the hrtB promoter in the presence of a heme source, suggesting that this putative sensor region is essential for the detection of a signal produced in response to hemin exposure. This study shows that the HrtAB system is required for protection from hemin toxicity and that expression of the hrtAB genes is regulated by the ChrAS two-component system. This study demonstrates a direct correlation between the detection of heme or a heme-associated signal by the N-terminal sensor domain of ChrS and the transcriptional activation of the hrtAB genes.

The lactoferrin receptor complex in gram negative bacteria

Bacteria that inhabit the respiratory and genitourinary tracts of mammals encounter an iron-deficient environment on the mucosal surface where iron is complexed by the host iron-binding proteins transferrin and lactoferrin. Lactoferrin is also present in high concentrations at sites of inflammation where the cationic anti-microbial peptide lactoferricin is produced by proteolysis of lactoferrin. Several members of the Neisseriaceae and Moraxellaceae families express surface receptors, capable of specifically binding host lactoferrin and extracting the iron from lactoferrin as a source of iron for growth. The receptor is comprised of an integral outer membrane protein, lactoferrin binding protein A (LbpA), and a largely exposed surface lipoprotein, lactoferrin binding protein B (LbpB). LbpA is essential for mediating growth using lactoferrin as a sole iron source whereas LbpB only plays a facilitating role. LbpB, with the presence of a large tract of negatively charged residues, appears to protect the bacterial cell from the bactericidal effects of the lactoferricin. The lactoferrin receptors in these species appear to be essential for survival and thus may serve as potential vaccine targets.

Transcriptional Profile of Haemophilus influenzae : Effects of Iron and Heme

Haemophilus influenzae requires either heme or a porphyrin and iron source for growth. Microarray studies of H. influenzae strain Rd KW20 identified 162 iron/heme-regulated genes, representing approximately 10% of the genome, with > or =1.5-fold changes in transcription in response to iron/heme availability in vitro. Eighty genes were preferentially expressed under iron/heme restriction; 82 genes were preferentially expressed under iron/heme-replete conditions.

Isolation of a hemin and hemoglobin binding outer membrane protein of Vibrio vulnificus biotype 2 (serogroup E).

The eel pathogen Vibrio vulnificus biotype 2 (serogroup E) is able to use hemin (Hm) or hemoglobin (Hb) as the sole iron source for growth in vitro and in vivo. The mechanism of heme-iron acquisition in this bacterium requires a direct interaction through binding sites on the bacterial surface (constitutive outer membrane proteins). Using affinity chromatography techniques, a unique protein of around 36.5 kDa was isolated from cell envelopes of E86 strain regardless of the affinity ligand used, hemoglobin or hemin. This protein was purified from both iron-enriched and iron-restricted grown cells. These results support the hypothesis that in this pathogen Hm- and Hb-iron acquisition is mediated by a common protein receptor which recognizes the heme prosthetic group of Hb.

Acquisition of haemoglobin-bound iron by Histophilus somni

Histophilus somni is an important pathogen of cattle and sheep. H. somni requires iron and can use ruminant transferrins as iron sources for growth. Here, we investigated the abilities of bovine (strains 649 and 2336) and ovine (strains 9L and 3384Y) isolates of H. somni to acquire iron from haemoglobins. Using growth assays, the bovine isolates were shown to acquire iron from bovine haemoglobin, but not from ovine, porcine or human haemoglobins; the ovine isolates, however, failed to use any of these haemoglobins as iron sources for growth. In solid phase binding assays, the bovine isolates, grown under iron-restricted conditions in the presence of bovine haemoglobin, bound not only bovine but also ovine and human haemoglobins. Competition binding assays indicated that all three haemoglobins were bound by the same receptor(s) and SDS-PAGE of membrane fractions revealed that expression of haemoglobin-binding activity was associated with the production of an ∼120-kDa outer membrane protein. PCR approaches allowed the amplification and sequencing of hgbA, and also hugX and hugZ homologues from strains 649, 9L and 3384Y. While hgbA of strain 649 was predicted to encode an HgbA precursor that is processed to yield a mature, 123.9-kDa haemoglobin-binding protein, the hgbA genes of strains 9L and 3384Y were predicted to give rise to truncated products. RT-PCR experiments revealed that in strain 649, hugX, hugZ and hgbA are co-transcribed and iron-regulated and additional sequencing suggested that in strain 2336, expression of HgbA is subject to phase variation involving a poly C tract within hgbA.

Iron acquisition by Actinobacillus suis: Identification and characterization of a single-component haemoglobin receptor and encoding gene

Actinobacillus suis is an important swine pathogen. As with other pathogens, the ability of A. suis to acquire iron within the host is crucial for virulence. Here, we investigated the ability of seven strains of A. suis to acquire iron from haemoglobins. In growth assays, all strains could use porcine, bovine and human haemoglobins as iron sources for growth. Using solid phase binding assays, membranes derived from all strains, grown under iron-restricted conditions, were shown to bind all three haemoglobins. Competition binding assays indicated that these haemoglobins were bound by the same receptor and an affinity procedure allowed the isolation and identification of an iron-repressible, haemoglobin-binding polypeptide (∼105 kDa) from all strains. Nucleotide sequence analyses revealed that A. suis possesses a gene ( hgbA) that encodes a homologue of the Actinobacillus pleuropneumoniae haemoglobin-binding protein, HgbA. hgbA, encoding a mature protein of 105 kDa, was shown to be preceded by a hugZ homologue; putative promoter sequences and a putative Fur box were located upstream of hugZ and RT-PCR revealed that hugZ and hgbA are co-transcribed and iron-repressible. It is concluded that the acquisition of haemoglobin-bound iron by A. suis involves a single-component receptor that is up-regulated in response to iron restriction.

Identification of a conserved Moraxella catarrhalis haemoglobin-utilization protein, MhuA

Moraxella catarrhalis is a leading cause of acute otitis media in children and is a cause of respiratory disease in adults with underlying lung disease. This organism is a strict human pathogen that has an absolute requirement for iron in order to grow and cause disease. Previous studies identified transferrin and lactoferrin receptors used by M. catarrhalis to obtain iron from the human host, yet other iron-acquisition systems remain undefined. In this study, it is demonstrated that this strict mucosal pathogen can utilize haemoglobin (Hb) as a sole source of iron for growth. A novel 107 kDa outer-membrane protein involved in Hb utilization by this pathogen was also identified. An isogenic mutant defective in this Moraxella Hb-utilization protein (MhuA), 7169 : : mhuA, showed a significant lag during growth in the presence of Hb as the sole iron source. This protein appears to be expressed constitutively, regardless of growth conditions, and a mAb directed to MhuA demonstrated that this protein contains highly conserved, surface-exposed epitopes. Data demonstrating that expression of MhuA may be highly specific to isolates of M. catarrhalis are also presented, suggesting a potential role as a diagnostic marker. To our knowledge, this is the first report demonstrating that M. catarrhalis expresses an Hb-binding protein and that this bacterium can utilize Hb as a sole iron source for growth.

Production of Catechol-Siderophore and Utilization of Transferrin-Bound Iron in Bacillus cereus

In the present study we attempted to ascertain whether Bacillus cereus was able to produce catechol-siderophore(s), and whether it was able to utilize transferrin-bound iron. The growth of B. cereus was stimulated in proportion to the iron-saturation level of the transferrin, and catechol-siderophores were produced in inverse proportion to this level. B. cereus was proved to uptake iron from partially iron-saturated transferrin or holotransferrin, without destroying the transferrin by its proteases. The catechol-siderophores from B. cereus were able to sustain and augment its growth on the transferrin-bound iron. These results indicate that B. cereus has the ability to produce catechol-siderophores, and to utilize transferrin-bound iron as an iron source for growth, via the siderophore-mediated iron-uptake system.

Identification of a Hemin Utilization Protein of Moraxella catarrhalis (HumA)

Moraxella catarrhalis is a major cause of acute otitis media in young children and has also been implicated as an important cause of exacerbations in adults with underlying pulmonary disease. Due to the considerable level of antibiotic resistance and the high degree of carriage rates in young children, it is likely that the incidence of M. catarrhalis infections will continue to rise. M. catarrhalis is a strict human respiratory pathogen, and this bacterium uses both transferrin and lactoferrin receptors to fulfill the essential iron requirement for survival in vivo. However, these are the only described iron acquisition systems for this organism. In this report we have demonstrated that M. catarrhalis can also utilize hemin as a sole source of iron for growth. In addition, we have identified and characterized an outer membrane protein with homology (26 to 28% similarity) to other known hemin binding and uptake proteins in related gram-negative organisms (i.e., Bordetella and Yersinia spp.). This newly described M. catarrhalis protein, termed HumA, is capable of directly binding to hemin coupled to a solid-phase matrix. M. catarrhalis HumA expressed on the surface of an Escherichia coli hemA-deficient strain (K-12 EB53) is fully capable of complementing the defect and thus restoring the ability of this strain to grow in the presence of hemin. When M. catarrhalis is grown in the presence of hemin, HumA expression is clearly increased as shown by Western blotting with polyclonal antiserum developed against a HumA peptide. In addition, growth analyses revealed that a HumA-deficient mutant of M. catarrhalis (7169::humA) is restricted for growth in the presence of hemin as the sole iron source compared to the wild-type strain. We conclude that HumA is an essential component of a hemin uptake and utilization system previously undescribed for M. catarrhalis, thus providing another mechanism of iron acquisition that may facilitate persistent colonization of the mucosal surface.

Molecular cloning of haemoglobin-binding protein HgbA in the outer membrane of Actinobacillus pleuropneumoniae.

From the porcine pathogen Actinobacillus pleuropneumoniae cultivated in iron-deficient or haem-deficient media, haemoglobin (Hb)-agarose affinity purification was exploited to isolate an outer-membrane protein of approximately 105 kDa, designated HgbA. Internal peptide sequences of purified HgbA were used to design oligonucleotide primers for PCR amplification, yielding amplicons that showed partial sequences with homology to hgbA of Pasteurella multocida. Upon screening two genomic libraries of A. pleuropneumoniae serotype 1 strain 4074, positive clones were assembled into an ORF of 2838 bp. HgbA (946 aa) includes a signal peptide of 23 aa and the deduced HgbA sequence (104 890 Da) also demonstrated a possible Ton box. The promoter region of hgbA from A. pleuropneumoniae serotype 1 showed consensus for -35 and -10 sequences and a putative Fur-binding site. RT-PCR confirmed that hgbA of A. pleuropneumoniae is upregulated in response to diminished levels of iron in the culture medium. While an internally deleted hgbA mutant was unable to use pig Hb as sole source of iron for growth, flow cytometry confirmed its Hb binding; the internally deleted sequences may not be required for Hb binding, but appear necessary for the iron supply from Hb. HgbA is required for growth of A. pleuropneumoniae in the presence of Hb as sole iron source.

Iron acquisition by Actinobacillus suis: identification and characterization of transferrin receptor proteins and encoding genes

Actinobacillus suis is an important pathogen of swine, especially in high-health-status herds. A published report mentioning the binding of porcine transferrin (Tf) by at least one strain of A. suis suggested that A. suis, like other members of the Pasteurellaceae, can acquire Tf-bound iron by means of a siderophore-independent, receptor-mediated mechanism. The objective of the present study was to characterize the components involved in this process, if present. Growth assays, with seven strains, confirmed that A. suis can use porcine (but not human or bovine) Tf as an iron source for growth. In solid phase binding assays, total membranes derived from all strains exhibited strong binding of porcine Tf, but only if the membranes were from organisms grown under iron-restricted conditions. An affinity-isolation procedure allowed the isolation of putative Tf-binding polypeptides (∼100 and ∼63 kDa) from comparable membranes from all strains. PCR approaches allowed the amplification, cloning and sequencing of A. suis tonB, exbB, exbD, tbpB and tbpA homologues. Reverse transcription (RT)–PCR, using RNA from organisms grown under iron-replete and iron-restricted conditions, revealed that tonB, exbB, exbD, tbpB and tbpA are transcribed as a single unit with expression being up-regulated in response to iron restriction. The calculated molecular masses of the predicted, mature TbpA (104.3 kDa) and TbpB (63.4 kDa) proteins suggest strongly that the affinity-isolated, ∼100 and ∼63 kDa Tf-binding polypeptides represent TbpA and TbpB, respectively. It is concluded that the acquisition of Tf-bound iron by A. suis involves mechanisms analogous to those found in other members of the Pasteurellaceae.