The Corynebacterium diphtheriae HbpA hemoglobin-binding protein contains a domain that is critical for hemoprotein-binding, cellular localization and function.

2021 ◽  
Author(s):  
Lindsey R. Lyman ◽  
Eric D. Peng ◽  
Michael P. Schmitt
Keyword(s):  
Cellular Localization ◽  
Binding Protein ◽  
Protein Localization ◽  
Iron Acquisition ◽  
Corynebacterium Diphtheriae ◽  
Terminal Region ◽  
Surface Proteins ◽  
Size Exclusion ◽  
Transport Systems ◽  
Iron Source

The acquisition of hemin-iron from hemoglobin-haptoglobin (Hb-Hp) by Corynebacterium diphtheriae requires the iron-regulated surface proteins HtaA, ChtA, 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 Δ hbpA 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 Δ hbpA mutant. Protein localization studies in C. diphtheriae showed that HbpA-FL is present in both in the supernatant and in the membrane fractions, and that the C-terminal region is required for membrane anchoring. Purified HbpA-FL was able to enhance growth of the Δ hbpA 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 .

The role of lactoferrin binding protein B in mediating protection against human lactoferricin1This article is part of a Special Issue entitled Lactoferrin and has undergone the Journal's usual peer review process.

2012 ◽  
Vol 90 (3) ◽  
pp. 417-423 ◽  
Author(s):  
Ari Morgenthau ◽  
Margaret Livingstone ◽  
Paul Adamiak ◽  
Anthony B. Schryvers
Keyword(s):  
Peer Review Process ◽  
Binding Protein ◽  
Iron Acquisition ◽  
Protein A ◽  
Human Lactoferrin ◽  
Cationic Antimicrobial Peptide ◽  
Iron Source ◽  
Iron Deficient ◽  
Protein B

Bacteria that inhabit the mucosal surfaces of the respiratory and genitourinary tracts of mammals encounter an iron-deficient environment because of iron sequestration by the host iron-binding proteins transferrin and lactoferrin. Lactoferrin is also present in high concentrations at sites of inflammation where the cationic, antimicrobial peptide lactoferricin is produced by proteolysis of lactoferrin. Several Gram-negative pathogens express a lactoferrin receptor that enables the bacteria to use lactoferrin as an iron source. The receptor is composed of an integral membrane protein, lactoferrin binding protein A (LbpA), and a membrane-bound lipoprotein, lactoferrin binding protein B (LbpB). LbpA is essential for growth with lactoferrin as the sole iron source, whereas the role of LbpB in iron acquisition is not yet known. In this study, we demonstrate that LbpB from 2 different species is capable of providing protection against the killing activity of a human lactoferrin-derived peptide. We investigated the prevalence of lactoferrin receptors in bacteria and examined their sequence diversity. We propose that the protection against the cationic antimicrobial human lactoferrin-derived peptide is associated with clusters of negatively charged amino acids in the C-terminal lobe of LbpB that is a common feature of this protein.

scholarly journals The structure of lactoferrin-binding protein B fromNeisseria meningitidissuggests roles in iron acquisition and neutralization of host defences

2014 ◽  
Vol 70 (10) ◽  
pp. 1312-1317 ◽  
Author(s):  
Cory L. Brooks ◽  
Elena Arutyunova ◽  
M. Joanne Lemieux
Keyword(s):  
Binding Protein ◽  
Iron Acquisition ◽  
Protein A ◽  
Transport Systems ◽  
Cationic Peptide ◽  
Moraxella Bovis ◽  
Component Systems ◽  
Two Component Systems ◽  
Membrane Bound ◽  
Protein B

Pathogens have evolved a range of mechanisms to acquire iron from the host during infection. Several Gram-negative pathogens including members of the generaNeisseriaandMoraxellahave evolved two-component systems that can extract iron from the host glycoproteins lactoferrin and transferrin. The homologous iron-transport systems consist of a membrane-bound transporter and an accessory lipoprotein. While the mechanism behind iron acquisition from transferrin is well understood, relatively little is known regarding how iron is extracted from lactoferrin. Here, the crystal structure of the N-terminal domain (N-lobe) of the accessory lipoprotein lactoferrin-binding protein B (LbpB) from the pathogenNeisseria meningitidisis reported. The structure is highly homologous to the previously determined structures of the accessory lipoprotein transferrin-binding protein B (TbpB) and LbpB from the bovine pathogenMoraxella bovis. Docking the LbpB structure with lactoferrin reveals extensive binding interactions with the N1 subdomain of lactoferrin. The nature of the interaction precludes apolactoferrin from binding LbpB, ensuring the specificity of iron-loaded lactoferrin. The specificity of LbpB safeguards proper delivery of iron-bound lactoferrin to the transporter lactoferrin-binding protein A (LbpA). The structure also reveals a possible secondary role for LbpB in protecting the bacteria from host defences. Following proteolytic digestion of lactoferrin, a cationic peptide derived from the N-terminus is released. This peptide, called lactoferricin, exhibits potent antimicrobial effects. The docked model of LbpB with lactoferrin reveals that LbpB interacts extensively with the N-terminal lactoferricin region. This may provide a venue for preventing the production of the peptide by proteolysis, or directly sequestering the peptide, protecting the bacteria from the toxic effects of lactoferricin.

scholarly journals An exposed outer membrane haemin-binding protein facilitates haemin transport by a TonB-dependent receptor in Riemerella anatipestifer

2021 ◽  
Author(s):  
Mafeng Liu ◽  
Siqi Liu ◽  
Mi Huang ◽  
Yaling Wang ◽  
Mengying Wang ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Binding Protein ◽  
Bacterial Pathogen ◽  
Essential Element ◽  
Acid Sites ◽  
Transport Systems ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Riemerella Anatipestifer ◽  
Iron Source

Iron is an essential element for the replication of most bacteria, including Riemerella anatipestifer (R. anatipestifer, RA), a gram-negative bacterial pathogen of ducks and other birds. R. anatipestifer utilizes haemoglobin-derived haemin as an iron source; however, the mechanism by which this bacterium acquires haemin from haemoglobin is largely unknown. Here, RhuA disruption was shown to impair iron utilization from duck haemoglobin in R. anatipestifer CH-1. Moreover, the putative lipoprotein RhuA was identified as a surface-exposed, outer membrane haemin-binding protein, but it could not extract haemin from duck haemoglobin. Mutagenesis studies showed that recombinant RhuAY144A, RhuAY177A and RhuAH149A lost haemin-binding ability, suggesting that amino acid sites tyrosine 144 (Y144), Y177 and histidine 149 (H149) are crucial for haemin binding. Furthermore, RhuR, the gene adjacent to RhuA, encodes a TonB2-dependent haemin transporter. The function of RhuA in duck haemoglobin utilization was abolished in the RhuR mutant strain, and recombinant RhuA was able to bind the cell surface of R. anatipestifer CH-1ΔRhuA rather than R. anatipestifer CH-1ΔRhuRΔRhuA, indicating that RhuA associates with RhuR to function. The sequence of the RhuR-RhuA haemin utilization locus exhibits no similarity with those of characterized haemin transport systems. Thus, this locus is a novel haemin uptake locus with homologues distributed mainly in the Bacteroidetes phylum. IMPORTANCE In vertebrates, haemin from haemoglobin is an important iron source for infectious bacteria. Many bacteria can obtain haemin from haemoglobin, but the mechanisms of haemin acquisition from haemoglobin differ among bacteria. Moreover, most studies have focused on the mechanism of haemin acquisition from mammalian haemoglobin. In this study, we found that the RhuR-RhuA locus of R. anatipestifer CH-1, a duck pathogen, is involved in haemin acquisition from duck haemoglobin via a unique pathway. RhuA was identified as an exposed outer membrane haemin-binding protein, and RhuR was identified as a TonB2-dependent haemin transporter. Moreover, the function of RhuA in haemoglobin utilization is RhuR dependent, not vice versa. The homologues of RhuR and RhuA are widely distributed in bacteria in marine environments, animals, and plants, representing a novel haemin transportation system of gram-negative bacteria. This study not only was important for understanding haemin uptake in R. anatipestifer but also enriched the knowledge about the haemin transportation pathway in gram-negative bacteria.

scholarly journals Structure/Function studies on Glucan Binding Protein C of Streptococcus mutans

2014 ◽  
Vol 70 (a1) ◽  
pp. C1639-C1639
Author(s):  
Sangeetha Purushotham ◽  
Matthew Larson ◽  
Jeffrey Banas ◽  
Champion Deivanayagam
Keyword(s):  
Crystal Structure ◽  
Streptococcus Mutans ◽  
Protein C ◽  
Binding Protein ◽  
Surface Protein ◽  
Surface Proteins ◽  
Size Exclusion ◽  
Diffusion Method ◽  
Data Sets ◽  
Functional Studies

Streptococcus mutans is a known etiological agent in dental caries. In the past several years, we have taken a concerted effort toward understanding the adhesion mechanisms adopted by the surface proteins S. mutans. The Glucan Binding Protein C (GBPC) is a LPXTG anchored surface protein on S. mutans that has been widely implicated to play a significant role in biofilm formation. GBPC displays limited homology to the V-region of Antigen I/II (AgI/II), another surface protein of S. mutans (1,2). We undertook to crystallize and resolve the structure of GBPC. Recombinant GbpC111-552 (residues encompassing 111-552) was cloned into a pET23d vector, and thereafter expressed in E.coli BL21(DE3). GbpC111-552 purified initially using affinity chromatography (his-tag), followed by anion exchange (MonoQ) and finally polished with size exclusion (Superdex 75). GbpC111-552 was crystallized using the vapor diffusion method by scanning various commercial crystallization kits on a 96 well plate format through the Art Robbins Gryphon robot. Large crystals were obtained from a refined droplet condition that contained 1 μl of protein (43.3 mg/ml) mixed with 1 μl of reservoir made of 100 mM Bis-Tris pH 6.5 and 25% (w/v) PEG3350. The crystal structure could not be resolved by molecular replacement. Therefore crystals were soaked in Sodium Iodide (NaI) and thereafter flash frozen with 9% glycerol as crytoprotectant. MAD data sets were collected and the crystal structure was resolved. We will present the crystal structure, and how based on its structural homology we discovered similar functionalities among the surface proteins of S. mutans. The structural and functional studies of these surface proteins we hope would allow the identification of the adherence motifs, which would aid in development of inhibitors. The development of such anti-adhesive inhibitors would aid in disease preventative therapies.

scholarly journals Bacterial iron acquisition mediated by outer membrane translocation and cleavage of a host protein

2018 ◽  
Vol 115 (26) ◽  
pp. 6840-6845 ◽  
Author(s):  
Khedidja Mosbahi ◽  
Marta Wojnowska ◽  
Amaya Albalat ◽  
Daniel Walker
Keyword(s):  
Cell Surface ◽  
Outer Membrane ◽  
Pathogenic Bacteria ◽  
Protein Import ◽  
Binding Protein ◽  
Iron Acquisition ◽  
Host Protein ◽  
Transport Systems ◽  
Growth Enhancement ◽  
Periplasmic Binding Protein

Iron is an essential micronutrient for most bacteria and is obtained from iron-chelating siderophores or directly from iron-containing host proteins. For Gram-negative bacteria, classical iron transport systems consist of an outer membrane receptor, a periplasmic binding protein, and an inner membrane ABC transporter, which work in concert to deliver iron from the cell surface to the cytoplasm. We recently showed thatPectobacteriumspp. are able to acquire iron from ferredoxin, a small and stable 2Fe-2S iron sulfur cluster containing protein and identified the ferredoxin receptor, FusA, a TonB-dependent receptor that binds ferredoxin on the cell surface. The genetic context offusAsuggests an atypical iron acquisition system, lacking a periplasmic binding protein, although the mechanism through which iron is extracted from the captured ferredoxin has remained unknown. Here we show that FusC, an M16 family protease, displays a highly targeted proteolytic activity against plant ferredoxin, and that growth enhancement ofPectobacteriumdue to iron acquisition from ferredoxin is FusC-dependent. The periplasmic location of FusC indicates a mechanism in which ferredoxin is imported into the periplasm via FusA before cleavage by FusC, as confirmed by the uptake and accumulation of ferredoxin in the periplasm in a strain lackingfusC. The existence of homologous uptake systems in a range of pathogenic bacteria suggests that protein uptake for nutrient acquisition may be widespread in bacteria and shows that, similar to their endosymbiotic descendants mitochondria and chloroplasts, bacteria produce dedicated protein import systems.

Complex regulation and nuclear localization of JRK protein

2004 ◽  
Vol 32 (6) ◽  
pp. 920-923 ◽  
Author(s):  
R. Waldron ◽  
T. Moore
Keyword(s):  
Cellular Localization ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Distinct Pattern ◽  
Nuclear Bodies ◽  
Null Mouse ◽  
Fluorescence Confocal Microscopy ◽  
Pml Nuclear Bodies ◽  
Generalized Epilepsy ◽  
Green Fluorescent

The mouse jerky gene and its human orthologue, JRK/JH8, encode a putative DNA-binding protein with homology to the CENP-B (centromere-binding protein B). Disruption of the mouse jerky gene by transgene insertion causes generalized recurrent seizures reminiscent of human idiopathic generalized epilepsy. In addition (and similar to a cenp-b null mouse) jerky null mice exhibit postnatal weight loss and reduced fertility. Using fluorescence confocal microscopy, the cellular localization of a JRK–GFP fusion (where GFP stands for green fluorescent protein) was investigated in HeLa cells. JRK–GFP has a dynamic expression pattern in the interphase nucleus, localizing in a small number of punctate nuclear foci and in the nucleolus. The JRK–GFP foci number changes during the cell cycle, but a distinct pattern of three JRK–GFP foci is observed at G2. The endogenous protein behaves in a similar manner to the GFP-fusion protein. JRK–GFP was found to co-localize with CREST antigens (which recognize the centromere-binding proteins, CENP-A, -B and -C) through S and G2 phases of interphase and co-localized completely with a subset of PML nuclear bodies at G2. We speculate that JRK protein associates with a specific chromosomal centromeric locus in G2, where it associates fully with PML bodies. Research is underway to identify this locus.

scholarly journals Ferric Citrate Uptake is a Virulence Factor in Uropathogenic Escherichia coli

2021 ◽  
Author(s):  
Arwen E Frick-Cheng ◽  
Anna Sintsova ◽  
Sara N Smith ◽  
Ali Pirani ◽  
Evan S Snitkin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Iron Acquisition ◽  
Uropathogenic Escherichia Coli ◽  
Ferric Citrate ◽  
Uptake System ◽  
Lipocalin 2 ◽  
Compensatory Mechanisms ◽  
E Coli ◽  
Iron Source

More than half of women will experience a urinary tract infection (UTI) with uropathogenic Escherichia coli (UPEC) causing ~80% of uncomplicated cases. Iron acquisition systems are essential for uropathogenesis, and UPEC encode functionally redundant iron acquisition systems, underlining their importance. However, a recent UPEC clinical isolate, HM7 lacks this functional redundancy and instead encodes a sole siderophore, enterobactin. To determine if E. coli HM7 possesses unidentified iron acquisition systems, we performed RNA-sequencing under iron-limiting conditions and demonstrated that the ferric citrate uptake system (fecABCDE and fecIR) was highly upregulated. Importantly, there are high levels of citrate within urine, some of which is bound to iron, and the fec system is highly enriched in UPEC isolates compared to environmental or fecal strains. Therefore, we hypothesized that HM7 and other similar strains use the fec system to acquire iron in the host. Deletion of both enterobactin biosynthesis and ferric citrate uptake (ΔentB/ΔfecA) abrogates use of ferric citrate as an iron source and fecA provides an advantage in human urine in absence of enterobactin. However, in a UTI mouse model, fecA is a fitness factor independent of enterobactin production, likely due to the action of host Lipocalin-2 chelating ferrienterobactin. These findings indicate that ferric citrate uptake is used as an iron source when siderophore efficacy is limited, such as in the host during UTI. Defining these novel compensatory mechanisms and understanding the nutritional hierarchy of preferred iron sources within the urinary tract are important in the search for new approaches to combat UTI.

scholarly journals Epstein-Barr virus LMP1 modulates the CD63 interactome

2021 ◽  
Author(s):  
Mujeeb Cheerathodi ◽  
Dingani Nkosi ◽  
Allaura S. Cone ◽  
Sara B. York ◽  
David G. Meckes Jr.
Keyword(s):  
Intracellular Signaling ◽  
Ribosomal Proteins ◽  
Protein Localization ◽  
Cell Communication ◽  
Enrichment Analysis ◽  
Surface Proteins ◽  
Rab Gtpases ◽  
Interacting Proteins ◽  
Sorting Nexins ◽  
Cargo Sorting

Abstract Tetraspanin CD63 is a cluster of cell surface proteins with four transmembrane domains which associates with tetraspanin-enriched microdomains and typically localizes to late endosomes and lysosomes. CD63 plays an important role in cellular trafficking of different proteins, EV cargo sorting and vesicles formation. We have preciously shown that CD63 is important in LMP1 trafficking to EVs and this also affects LMP1 mediated intracellular signaling including MAPK/ERK, NF-κB and mTOR activation. Using the BioID combined with mass spectrometry, we sought to define the broad CD63 interactome and how LMP1 modulates this network of interacting proteins. We identified a total of 1600 total proteins as proximal interacting newtwork of proteins to CD63. Biological process enrichment analysis revealed significant involvement in signal transduction, cell communication, protein metabolism and transportation. The CD63 only interactome was enriched in Rab GTPases, SNARE proteins and sorting nexins while adding LMP1 into the interactome increased presence of signaling and ribosomal proteins. Our results showed that LMP1 alters the CD63 interactome, shifting the network of proteins enrichment from protein localization and vesicle mediated transportation to metabolic processes and translation. We also show that LMP1 interacts with mTor, Nedd4L and PP2A indicating formation of a multiprotein complex with CD63 thereby potentially regulating LMP1 dependent mTor signaling. Collectively, the comprehensive analysis of CD63 proximal interacting proteins provides insights into network of partners required for endocytic trafficking, extracellular vesicle cargo sorting, formation and secretion.

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