Identification and characterization of zinc importers in Corynebacterium diphtheriae.

Corynebacterium diphtheriae is the causative agent of diphtheria, a severe respiratory disease in humans. C. diphtheriae colonizes the human upper respiratory tract, where it acquires zinc, an essential metal required for survival in the host. While the mechanisms for zinc transport by C. diphtheriae are not well characterized, four putative zinc ABC-type transporter loci were recently identified in strain 1737: iutABCD/E (iut), znuACB (znu), nikABCD1 (nik1), and nikABCD2 (nik2). A mutant deleted for all four loci (Δ4) exhibited similar growth to that of the wild-type strain in a zinc-limited medium, suggesting there are additional zinc transporters. Two additional gene loci predicted to be associated with metal import, mntABCD (mnt) and sidAB (sid), were deleted in the Δ4 mutant to construct a new mutant designated Δ6. The C. diphtheriae Δ6 mutant exhibited significantly reduced growth under zinc limitation relative to the wild type, suggesting a deficiency in zinc acquisition. Strains retaining the iut, znu, mnt, or sid loci grew to near-wild-type levels in the absence of the other five loci, indicating that each of these transporters may be involved in zinc uptake. Plasmid complementation with cloned iut, znu, mnt, or nik1 loci also enhanced the growth of the Δ6 mutant. Quantification of intracellular zinc content by inductively coupled plasma mass spectrometry was consistent with reduced zinc uptake by Δ6 relative to the wild type and further supports a zinc uptake function for the transporters encoded by iut, znu, and mnt. This study demonstrates that C. diphtheriae zinc transport is complex and involves multiple zinc uptake systems.IMPORTANCEZinc is a critical nutrient for all forms of life, including human bacterial pathogens. Thus, the tools that bacteria use to acquire zinc from host sources are crucial for pathogenesis. While potential candidates for zinc importers have been identified in Corynebacterium diphtheriae from gene expression studies, to date, no study has clearly demonstrated this function for any of the putative transporters. We show that C. diphtheriae encodes at least six loci associated with zinc import, underscoring the extent of redundancy for zinc acquisition. Furthermore, we provide evidence that a previously studied manganese-regulated importer can also function in zinc import. This study builds upon our knowledge of bacterial zinc transport mechanisms and identifies potential targets for future diphtheria vaccine candidates.

Analysis of the HbpA Protein from Corynebacterium diphtheriae Clinical Isolates and Identification of a Putative Hemoglobin-Binding Site on HbpA.

The Corynebacterium diphtheriae hemoglobin-binding protein HbpA is critical for the acquisition of iron from the hemoglobin-haptoglobin complex (Hb-Hp). Previous studies using C. diphtheriae strain 1737 showed that large aggregates formed by HbpA are associated with iron transport activity and enhanced binding to Hb-Hp; however, specific regions within HbpA required for Hb-Hp binding or iron uptake have not been identified. In this study, we characterized two clinical isolates from Austria, designated 07-18 and 09-15, which express HbpA proteins that share only 53% and 44% sequence identity, respectively, to the strain 1737 HbpA protein. The HbpA proteins expressed by the Austrian strains had functional and structural properties similar to those of the HbpA protein in strain 1737 despite the limited sequence similarity. These shared characteristics between the HbpA proteins included similar cellular localization, aggregate formation, and Hb and Hb-Hp binding. Additionally, the Austrian strains were able to acquire iron from Hb and Hb-Hp, and deletion of the hbpA gene from these two clinical isolates reduced their ability to use Hb-Hp as an iron source. A sequence comparison between the HbpA proteins from 1737 and the Austrian strains assisted in the identification of a putative Hb-binding site that shared similar characteristics with the Hb-binding regions in Staphylococcus aureus NEAT domains. Amino acid substitutions within this conserved Hb-binding region significantly reduced Hb and Hb-Hp binding and diminished the hemin-iron uptake function of HbpA. These findings represent important advances in our understanding of the interaction of HbpA with human hemoproteins. IMPORTANCE Hemoglobin (Hb) is the primary source of iron in humans, and the acquisition of hemin-iron from Hb is critical for many bacterial pathogens to infect and survive in the human host. In this study, we have examined the C. diphtheriae Hb-binding protein HbpA in two clinical isolates and show that these proteins, despite limited sequence similarity, are functionally equivalent to the previously described HbpA protein in strain 1737. A sequence comparison between these three strains led to the identification of a conserved Hb-binding site, which will further our understanding of how this novel protein functions in hemin-iron transport and, more generally, will expand our knowledge on how Hb interacts with proteins.

Analysis of the Manganese and MntR Regulon in Corynebacterium diphtheriae.

Corynebacterium diphtheriae is the causative agent of a severe respiratory disease in humans. The bacterial systems required for infection are poorly understood, but the acquisition of metals such as manganese (Mn) is likely critical for host colonization. MntR is an Mn-dependent transcriptional regulator in C. diphtheriae that represses the expression of the mntABCD genes, which encode a putative ABC metal transporter. However, other targets of Mn and MntR regulation in C. diphtheriae have not been identified. In this study, we use comparisons between the gene expression profiles of wild-type C. diphtheriae strain 1737 grown without or with Mn supplementation and comparisons of gene expression between the wild type and an mntR deletion mutant to characterize the C. diphtheriae Mn and MntR regulon. MntR was observed to both repress and induce various target genes in an Mn-dependent manner. Genes induced by MntR include the Mn-superoxide dismutase, sodA, and the putative ABC transporter locus, iutABCD. DNA binding studies showed that MntR interacts with the promoter regions for several genes identified in the expression study, and a 17-bp consensus MntR DNA binding site was identified. We found that an mntR mutant displayed increased sensitivity to Mn and cadmium that could be alleviated by the additional deletion of the mntABCD transport locus, providing evidence that the MntABCD transporter functions as an Mn uptake system in C. diphtheriae. The findings in this study further our understanding of metal uptake systems and global metal regulatory networks in this important human pathogen. IMPORTANCE Mechanisms for metal scavenging are critical to the survival and success of bacterial pathogens, including Corynebacterium diphtheriae. Metal import systems in pathogenic bacteria have been studied as possible vaccine components due to high conservation, critical functionality, and surface localization. In this study, we expand our understanding of the genes controlled by the global manganese regulator, MntR. We determined a role for the MntABCD transporter in manganese import using evidence from manganese and cadmium toxicity assays. Understanding the nutritional requirements of C. diphtheriae and the tools used to acquire essential metals will aid in the development of future vaccines.

The Corynebacterium diphtheriae HbpA Hemoglobin-Binding Protein Contains a Domain That Is Critical for Hemoprotein Binding, Cellular Localization, and Function.

The acquisition of hemin iron from hemoglobin-haptoglobin (Hb-Hp) by Corynebacterium diphtheriae requires the iron-regulated surface proteins HtaA, ChtA, and ChtC and the recently identified Hb-Hp-binding protein, HbpA. We previously showed that a purified form of HbpA (HbpA-S), lacking the C-terminal region, was able to bind Hb-Hp. In this study, we show that the C-terminal region of HbpA significantly enhances binding to Hb-Hp. A purified form of HbpA that includes the C-terminal domain (HbpA-FL) exhibits much stronger binding to Hb-Hp than HbpA-S. Size exclusion chromatography (SEC) showed that HbpA-FL as well as HtaA-FL, ChtA-FL, and ChtC-FL exist as high-molecular-weight complexes, while HbpA-S is present as a monomer, indicating that the C-terminal region is required for formation of large aggregates. Growth studies showed that expression of HbpA-FL in the ΔhbpA mutant restored wild-type levels of growth in low-iron medium that contained Hb-Hp as the sole iron source, while HbpA-S failed to complement the ΔhbpA mutant. Protein localization studies in C. diphtheriae showed that HbpA-FL is present in both the supernatant and membrane fractions and that the C-terminal region is required for membrane anchoring. Purified HbpA-FL was able to enhance growth of the ΔhbpA mutant when added to culture medium that contained Hb-Hp as a sole iron source, suggesting that secreted HbpA is involved in the use of hemin iron from Hb-Hp. These studies extend our understanding of this novel Hb-Hp binding protein in this important human pathogen. IMPORTANCE Hemoproteins, such as Hb, are an abundant source of iron in humans and are proposed to be required by numerous pathogens to cause disease. In this report, we expand on our previous studies in further defining the role of HbpA in hemin iron acquisition in C. diphtheriae. HbpA is unique to C. diphtheriae and appears to function unlike any previously described bacterial iron-regulated Hb- or Hb-Hp-binding protein. HbpA is both secreted and present in the membrane and exists as a large aggregate that enhances its ability to bind Hb-Hp and promote hemin iron uptake. Current studies with HbpA will increase our understanding of iron transport systems in C. diphtheriae.

Iron and Zinc Regulate Expression of a Putative ABC Metal Transporter in Corynebacterium diphtheriae.

Corynebacterium diphtheriae, a Gram-positive, aerobic bacterium, is the causative agent of diphtheria and cutaneous infections. While mechanisms required for heme iron acquisition are well known in C. diphtheriae, systems involved in the acquisition of other metals such as zinc and manganese remain poorly characterized. In this study, we identified a genetic region that encodes an ABC-type transporter (iutBCD) and that is flanked by two genes (iutA and iutE) encoding putative substrate binding proteins of the cluster 9 family, a related group of transporters associated primarily with the import of Mn and Zn. We showed that IutA and IutE are both membrane proteins with comparable Mn and Zn binding abilities. We demonstrated that the iutABCD genes are cotranscribed and repressed in response to iron by the iron-responsive repressor DtxR. Transcription of iutE was positively regulated in response to iron availability in a DtxR-dependent manner and was repressed in response to Zn by the Zn-dependent repressor Zur. Electrophoretic mobility shift assays showed that DtxR does not bind to the iutE upstream region, which indicates that DtxR regulation of iutE is indirect and that other regulatory factors controlled by DtxR are likely responsible for the iron-responsive regulation. Analysis of the iutE promoter region identified a 50-bp sequence at the 3' end of the iutD gene that is required for the DtxR-dependent and iron-responsive activation of the iutE gene. These findings indicate that transcription of iutE is controlled by a complex mechanism that involves multiple regulatory factors whose activity is impacted by both Zn and Fe.IMPORTANCE Vaccination against diphtheria prevents toxin-related symptoms but does not inhibit bacterial colonization of the human host by the bacterium. Thus, Corynebacterium diphtheriae remains an important human pathogen that poses a significant health risk to unvaccinated individuals. The ability to acquire iron, zinc, and manganese is critical to the pathogenesis of many disease-causing organisms. Here, we describe a gene cluster in C. diphtheriae that encodes a metal importer that is homologous to broadly distributed metal transport systems, some with important roles in virulence in other bacterial pathogens. Two metal binding components of the gene cluster encode surface exposed proteins, and studies of such proteins may guide the development of second-generation vaccines for C. diphtheriae.

Corynebacterium diphtheriae Iron-Regulated Surface Protein HbpA Is Involved in the Utilization of the Hemoglobin-Haptoglobin Complex as an Iron Source.

Corynebacterium diphtheriae utilizes various heme-containing proteins, including hemoglobin (Hb) and the hemoglobin-haptoglobin complex (Hb-Hp), as iron sources during growth in iron-depleted environments. The ability to utilize Hb-Hp as an iron source requires the surface-anchored proteins HtaA and either ChtA or ChtC. The ability to bind hemin, Hb, and Hb-Hp by each of these C. diphtheriae proteins requires the previously characterized conserved region (CR) domain. In this study, we identified an Hb-Hp binding protein, HbpA (38.5 kDa), which is involved in the acquisition of hemin iron from Hb-Hp. HbpA was initially identified from total cell lysates as an iron-regulated protein that binds to both Hb and Hb-Hp in situ HbpA does not contain a CR domain and has sequence similarity only to homologous proteins present in a limited number of C. diphtheriae strains. Transcription of hbpA is regulated in an iron-dependent manner that is mediated by DtxR, a global iron-dependent regulator. Deletion of hbpA from C. diphtheriae results in a reduced ability to utilize Hb-Hp as an iron source but has little or no effect on the ability to use Hb or hemin as an iron source. Cell fractionation studies showed that HbpA is both secreted into the culture supernatant and associated with the membrane, where its exposure on the bacterial surface allows HbpA to bind Hb and Hb-Hp. The identification and analysis of HbpA enhance our understanding of iron uptake in C. diphtheriae and indicate that the acquisition of hemin iron from Hb-Hp may involve a complex mechanism that requires multiple surface proteins.IMPORTANCE The ability to utilize host iron sources, such as heme and heme-containing proteins, is essential for many bacterial pathogens to cause disease. In this study, we have identified a novel factor (HbpA) that is crucial for the use of hemin iron from the hemoglobin-haptoglobin complex (Hb-Hp). Hb-Hp is considered one of the primary sources of iron for certain bacterial pathogens. HbpA has no similarity to the previously identified Hb-Hp binding proteins, HtaA and ChtA/C, and is found only in a limited group of C. diphtheriae strains. Understanding the function of HbpA may significantly increase our knowledge of how this important human pathogen can acquire host iron that allows it to survive and cause disease in the human respiratory tract.