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.

The basal and major pilins in the Corynebacterium diphtheriae SpaA pilus adopt similar structures that competitively react with the pilin polymerase.

Many species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus from Corynebacterium diphtheriae is built by the Cd SrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that Cd SrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA (N SpaA) that is also crosslinked by Cd SrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed "latch" mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting N SpaA for access to a shared thioester enzyme-substrate reaction intermediate.

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.

Crystal structures of FadD32 and pks13-ACP domain from Corynebacterium diphtheriae.

Mycolic acids (MAs) are unique components of cell envelope of Mycobacterium or Corynebacterium and are key factors of their virulence to human. In order to develop new anti-Tuberculosis (TB) drugs, many efforts have paid on investigation of structures and functions of proteins involved in the biosynthesis pathway of MAs. FadD32 and polyketide synthase 13 (pks13) catalyze the last step of MAs synthesis. Here we present the crystal structures of FadD32 with substrates and holo-form of ACP-domain from Corynebacterium diphtheriae. The crystal structures and in vitro biochemical assays provide new insights into the assembly of FadD32 and pks13.

Reaction intermediate rotation during the decarboxylation of coproheme to heme b in C. diphtheriae.

Monoderm bacteria utilize coproheme decarboxylases (ChdCs) to generate heme b by a stepwise decarboxylation of two propionate groups of iron coproporphyrin III (coproheme), forming two vinyl groups. This work focuses on actinobacterial ChdC from Corynebacterium diphtheriae (CdChdC) to elucidate the hydrogen peroxide-mediated decarboxylation of coproheme via monovinyl monopropionyl deuteroheme (MMD) to heme b, with the principal aim being to understand the reorientation mechanism of MMD during turnover. Wild-type CdChdC and variants, namely H118A, H118F, and A207E, were studied by resonance Raman and ultraviolet-visible spectroscopy, mass spectrometry, and molecular dynamics simulations. As actinobacterial ChdCs use a histidine (H118) as a distal base, we studied the H118A and H118F variants to elucidate the effect of 1) the elimination of the proton acceptor and 2) steric constraints within the active site. The A207E variant mimics the proximal H-bonding network found in chlorite dismutases. This mutation potentially increases the rigidity of the proximal site and might impair the rotation of the reaction intermediate MMD. We found that both wild-type CdChdC and the variant H118A convert coproheme mainly to heme b upon titration with H2O2. Interestingly, the variant A207E mostly accumulates MMD along with small amounts of heme b, whereas H118F is unable to produce heme b and accumulates only MMD. Together with molecular dynamics simulations, the spectroscopic data provide insight into the reaction mechanism and the mode of reorientation of MMD, i.e., a rotation in the active site versus a release and rebinding.

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.

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.

Seroprevalence of diphtheria toxoid IgG antibodies in the Malaysian population.

BACKGROUND: Despite high childhood immunization coverage, sporadic cases of diphtheria have been reported in Malaysia in recent years. This study aims to evaluate the seroprevalence of diphtheria among the Malaysian population. METHODS: A total of 3317 respondents age 2 years old to 60 years old were recruited in this study from August to November 2017. Enzyme-linked immunosorbent assay (ELISA) was used to measure the level of IgG antibody against the toxoid of C. diphtheriae in the blood samples of respondents. We classified respondent antibody levels based on WHO definition, as protective (≥0.1 IU/mL) and susceptible (< 0.1 IU/mL) to C. diphtheriae infection. RESULTS: Among the 3317 respondents, 57% were susceptible (38.1% of children and 65.4% of adults) and 43% (61.9% of children and 34.6% of adults) had protective antibody levels against diphtheria. The mean antibody level peaked among individuals aged 1-2 years old (0.59 IU/mL) and 6-7 years old (0.64 IU/mL) but generally decreased with age, falling below 0.1 IU/mL at around 4-6 years old and after age 20 years old. There was a significant association between age [Children: χ2 = 43.22(df = 2),p < 0.001)], gender [Adults: χ2 = 5.58(df = 1),p = 0.018] and ethnicity [Adults: χ2 = 21.49(df = 5),p = 0.001] with diphtheria toxoid IgG antibody level. CONCLUSIONS: About 57% of the Malaysian population have inadequate immunity against diphtheria infection. This is apparently due to waning immunity following childhood vaccination without repeated booster vaccination in adults. Children at age 5-6 years old are particularly vulnerable to diphtheria infection. The booster vaccination dose normally given at 7 years should be given earlier, and an additional booster dose is recommended for high-risk adults.

Imported Toxin-Producing Cutaneous Diphtheria - Minnesota, Washington, and New Mexico, 2015-2018.

From September 2015 to March 2018, CDC confirmed four cases of cutaneous diphtheria caused by toxin-producing Corynebacterium diphtheriae in patients from Minnesota (two), Washington (one), and New Mexico (one). All patients had recently returned to the United States after travel to countries where diphtheria is endemic. C. diphtheriae infection was not clinically suspected in any of the patients; treating institutions detected the organism through matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF) testing of wound-derived coryneform isolates. MALDI-TOF is a rapid screening platform that uses mass spectrometry to identify bacterial pathogens. State public health laboratories confirmed C. diphtheriae through culture and sent isolates to CDC's Pertussis and Diphtheria Laboratory for biotyping, polymerase chain reaction (PCR) testing, and toxin production testing. All isolates were identified as toxin-producing C. diphtheriae. The recommended public health response for cutaneous diphtheria is similar to that for respiratory diphtheria and includes treating the index patient with antibiotics, identifying close contacts and observing them for development of diphtheria, providing chemoprophylaxis to close contacts, testing patients and close contacts for C. diphtheriae carriage in the nose and throat, and providing diphtheria toxoid-containing vaccine to incompletely immunized patients and close contacts. This report summarizes the patient clinical information and response efforts conducted by the Minnesota, Washington, and New Mexico state health departments and CDC and emphasizes that health care providers should consider cutaneous diphtheria as a diagnosis in travelers with wound infections who have returned from countries with endemic diphtheria.

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.

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.

Comparing Galactan Biosynthesis in Mycobacterium tuberculosis and Corynebacterium diphtheriae.

The suborder Corynebacterineae encompasses species like Corynebacterium glutamicum, which has been harnessed for industrial production of amino acids, as well as Corynebacterium diphtheriae and Mycobacterium tuberculosis, which cause devastating human diseases. A distinctive component of the Corynebacterineae cell envelope is the mycolyl-arabinogalactan (mAG) complex. The mAG is composed of lipid mycolic acids, and arabinofuranose (Araf) and galactofuranose (Galf) carbohydrate residues. Elucidating microbe-specific differences in mAG composition could advance biotechnological applications and lead to new antimicrobial targets. To this end, we compare and contrast galactan biosynthesis in C. diphtheriae and M. tuberculosis In each species, the galactan is constructed from uridine 5'-diphosphate-α-d-galactofuranose (UDP-Galf), which is generated by the enzyme UDP-galactopyranose mutase (UGM or Glf). UGM and the galactan are essential in M. tuberculosis, but their importance in Corynebacterium species was not known. We show that small molecule inhibitors of UGM impede C. glutamicum growth, suggesting that the galactan is critical in corynebacteria. Previous cell wall analysis data suggest the galactan polymer is longer in mycobacterial species than corynebacterial species. To explore the source of galactan length variation, a C. diphtheriae ortholog of the M. tuberculosis carbohydrate polymerase responsible for the bulk of galactan polymerization, GlfT2, was produced, and its catalytic activity was evaluated. The C. diphtheriae GlfT2 gave rise to shorter polysaccharides than those obtained with the M. tuberculosis GlfT2. These data suggest that GlfT2 alone can influence galactan length. Our results provide tools, both small molecule and genetic, for probing and perturbing the assembly of the Corynebacterineae cell envelope.

Protein Labeling via a Specific Lysine-Isopeptide Bond Using the Pilin Polymerizing Sortase from Corynebacterium diphtheriae.

Proteins that are site-specifically modified with peptides and chemicals can be used as novel therapeutics, imaging tools, diagnostic reagents and materials. However, there are few enzyme-catalyzed methods currently available to selectively conjugate peptides to internal sites within proteins. Here we show that a pilus-specific sortase enzyme from Corynebacterium diphtheriae (CdSrtA) can be used to attach a peptide to a protein via a specific lysine-isopeptide bond. Using rational mutagenesis we created CdSrtA3M, a highly activated cysteine transpeptidase that catalyzes in vitro isopeptide bond formation. CdSrtA3M mediates bioconjugation to a specific lysine residue within a fused domain derived from the corynebacterial SpaA protein. Peptide modification yields greater than >95% can be achieved. We demonstrate that CdSrtA3M can be used in concert with the Staphylococcus aureus SrtA enzyme, enabling dual, orthogonal protein labeling via lysine-isopeptide and backbone-peptide bonds.

Biochemical studies of inositol N-acetylglucosaminyltransferase involved in mycothiol biosynthesis in Corynebacterium diphtheria.

Mycothiol (MSH) is the predominant low molecular weight thiol produced by actinomycetes, and it plays a pivotal role in the bacterial detoxication process. 1L-myo-Inositol-1-phosphate (1L-Ins-1-P) α-N-acetylglucosaminyltransferase (GlcNAc-T), known as MshA, is the only glycosyltransferase involved in MSH biosynthesis. In this work, the MshA from Corynebacterium diphtheria, named as CdMshA, was expressed, purified, and studied in detail. Its enzymatic activity to transfer GlcNAc to 1L-Ins-1-P was confirmed by the isolation and rigorous characterization of its reaction product 3-phospho-1-d-myo-inositol-2-acetamido-2-deoxy-α-d-glucopyranoside. CdMshA was shown to accept only UDP-GlcNAc and 1L-Ins-1-P as its substrates among various tested glycosyl donors, such as UDP-GlcNAc, UDP-Gal, UDP-Glc, UDP-GalNAc and UDP-GlcA, and glycosyl acceptors, such as myo-inositol, 1L-Ins-1-P and 1D-Ins-1-P. The results have demonstrated the strict substrate selectivity of CdMshA. Furthermore, its reaction kinetics with UDP-GlcNAc and 1L-Ins-1-P as substrates were characterized, while site-directed mutagenesis of CdMshA disclosed that its amino acid residues N28, K81 and R157 were essential for its enzymatic activity.

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

UNLABELLED: 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. IMPORTANCE: 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.

Disulfide-Bond-Forming Pathways in Gram-Positive Bacteria.

Disulfide bonds are important for the stability and function of many secreted proteins. In Gram-negative bacteria, these linkages are catalyzed by thiol-disulfide oxidoreductases (Dsb) in the periplasm. Protein oxidation has been well studied in these organisms, but it has not fully been explored in Gram-positive bacteria, which lack traditional periplasmic compartments. Recent bioinformatics analyses have suggested that the high-GC-content bacteria (i.e., actinobacteria) rely on disulfide-bond-forming pathways. In support of this, Dsb-like proteins have been identified in Mycobacterium tuberculosis, but their functions are not known. Actinomyces oris and Corynebacterium diphtheriae have recently emerged as models to study disulfide bond formation in actinobacteria. In both organisms, disulfide bonds are catalyzed by the membrane-bound oxidoreductase MdbA. Remarkably, unlike known Dsb proteins, MdbA is important for pathogenesis and growth, which makes it a potential target for new antibacterial drugs. This review will discuss disulfide-bond-forming pathways in bacteria, with a special focus on Gram-positive bacteria.

Utilization of host iron sources by Corynebacterium diphtheriae: multiple hemoglobin-binding proteins are essential for the use of iron from the hemoglobin-haptoglobin complex.

The use of hemin iron by Corynebacterium diphtheriae requires the DtxR- and iron-regulated ABC hemin transporter HmuTUV and the secreted Hb-binding protein HtaA. We recently described two surface anchored proteins, ChtA and ChtC, which also bind hemin and Hb. ChtA and ChtC share structural similarities to HtaA; however, a function for ChtA and ChtC was not determined. In this study, we identified additional host iron sources that are utilized by C. diphtheriae. We show that several C. diphtheriae strains use the hemoglobin-haptoglobin (Hb-Hp) complex as an iron source. We report that an htaA deletion mutant of C. diphtheriae strain 1737 is unable to use the Hb-Hp complex as an iron source, and we further demonstrate that a chtA-chtC double mutant is also unable to use Hb-Hp iron. Single-deletion mutants of chtA or chtC use Hb-Hp iron in a manner similar to that of the wild type. These findings suggest that both HtaA and either ChtA or ChtC are essential for the use of Hb-Hp iron. Enzyme-linked immunosorbent assay (ELISA) studies show that HtaA binds the Hb-Hp complex, and the substitution of a conserved tyrosine (Y361) for alanine in HtaA results in significantly reduced binding. C. diphtheriae was also able to use human serum albumin (HSA) and myoglobin (Mb) but not hemopexin as iron sources. These studies identify a biological function for the ChtA and ChtC proteins and demonstrate that the use of the Hb-Hp complex as an iron source by C. diphtheriae requires multiple iron-regulated surface components.

Heme Binding by Corynebacterium diphtheriae HmuT: Function and Heme Environment.

The heme uptake pathway (hmu) of Corynebacterium diphtheriae utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. In this study, the axial ligation of the heme in ferric HmuT is probed by examination of wild-type (WT) HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, and M292A. Characterization by UV-visible, resonance Raman, and magnetic circular dichroism spectroscopies indicates that H136 and Y235 are the axial ligands in ferric HmuT. Consistent with this assignment of axial ligands, ferric WT and H136A HmuT are difficult to reduce while Y235A is reduced readily in the presence of dithionite. The FeCO Raman shifts in WT, H136A, and Y235A HmuT-CO complexes provide further evidence of the axial ligand assignments. Additionally, these frequencies provide insight into the nonbonding environment of the heme pocket. Ferrous Y235A and the Y235A-CO complex reveal that the imidazole of H136 exists in two forms, one neutral and one with imidazolate character, consistent with a hydrogen bond acceptor on the H136 side of the heme. The ferric fluoride complex of Y235A reveals the presence of at least one hydrogen bond donor on the Y235 side of the heme. Hemoglobin utilization assays showed that the axial Y235 ligand is required for heme uptake in HmuT.

Characterization of DIP0733, a multi-functional virulence factor of Corynebacterium diphtheriae.

Corynebacterium diphtheriae is typically recognized as an extracellular pathogen. However, a number of studies revealed its ability to invade epithelial cells, indicating a more complex pathogen-host interaction. The molecular mechanisms controlling and facilitating internalization of Cor. diphtheriae are poorly understood. In this study, we investigated the role of DIP0733 as virulence factor to elucidate how it contributes to the process of pathogen-host cell interaction. Based on in vitro experiments, it was suggested recently that the DIP0733 protein might be involved in adhesion, invasion of epithelial cells and induction of apoptosis. A corresponding Cor. diphtheriae mutant strain generated in this study was attenuated in its ability to colonize and kill the host in a Caenorhabditis elegans infection model system. Furthermore, the mutant showed an altered adhesion pattern and a drastically reduced ability to adhere and invade epithelial cells. Subsequent experiments showed an influence of DIP0733 on binding of Cor. diphtheriae to extracellular matrix proteins such as collagen and fibronectin. Furthermore, based on its fibrinogen-binding activity, DIP0733 may play a role in avoiding recognition of Cor. diphtheriae by the immune system. In summary, our findings support the idea that DIP0733 is a multi-functional virulence factor of Cor. diphtheriae.

[Features of biofilm morphology, formed by Corynebacterium diphtheriae gravis tox+ strains].

AIM: Study the structure of homogenous microbial communities of Corynebacterium diphtheriae gravis tox+ strains during formation of biofilms in vitro. MATERIALS AND METHODS: Object of study--typical and biofilm cultures of C. diphtheriae gravis tox+ museum and circulating strains. Intensity of biofilm formation was evaluated by OD on microplate reader at 540 nm wave length studying 120 and 720 hour cultures. S-450 (Hitachi, Japan) scanning electron microscope was used. RESULTS: The peak of exopolysaccharide matrix (EPS) formation, that is formed in the process of biofilm formation, by museum strain takes place at earlier terms of cultivation (120 hours) than circulating (720 hours). An inverse correlation was established during analysis of bacterial cells of museum and circulating strains of C. diphtheriae during biofilm formation between them and intensity of EPS formation. At maximum EPS content, that took place at various terms of cultivation of the 2 studied strains of diphtheria causative agent, a reduction of corynebacteria cells was observed. CONCLUSION: Bacterial biofilms of museum and circulating strains of C. diphtheriae and patterns of dynamics of EPS reflect, probably, adaptive abilities of the causative agent, that determine its competitiveness in the fight for adhesion sites, resistance to factors of natural immunity and as a result--prolonged persistence in the organism of bacterial carriers.