scholarly journals Porphyromonas gingivalis HmuY and Bacteroides vulgatus Bvu—A Novel Competitive Heme Acquisition Strategy

2021 ◽  
Vol 22 (5) ◽  
pp. 2237
Author(s):  
Klaudia Siemińska ◽  
Patryk Cierpisz ◽  
Michał Śmiga ◽  
Teresa Olczak
Keyword(s):  
Porphyromonas Gingivalis ◽  
Gut Microbiome ◽  
Inflammatory Diseases ◽  
Functional Characterization ◽  
Etiologic Agent ◽  
Heme Iron ◽  
Leading Role ◽  
Heme Acquisition ◽  
Reducing Conditions

Human oral and gut microbiomes are crucial for maintenance of homeostasis in the human body. Porphyromonas gingivalis, the key etiologic agent of chronic periodontitis, can cause dysbiosis in the mouth and gut, which results in local and systemic infectious inflammatory diseases. Our previous work resulted in extensive biochemical and functional characterization of one of the major P. gingivalis heme acquisition systems (Hmu), with the leading role played by the HmuY hemophore-like protein. We continued our studies on the homologous heme acquisition protein (Bvu) expressed by Bacteroides vulgatus, the dominant species of the gut microbiome. Results from spectrophotometric experiments showed that Bvu binds heme preferentially under reducing conditions using Met145 and Met172 as heme iron-coordinating ligands. Bvu captures heme bound to human serum albumin and only under reducing conditions. Importantly, HmuY is able to sequester heme complexed to Bvu. This is the first study demonstrating that B. vulgatus expresses a heme-binding hemophore-like protein, thus increasing the number of members of a novel HmuY-like family. Data gained in this study confirm the importance of HmuY in the context of P. gingivalis survival in regard to its ability to cause dysbiosis also in the gut microbiome.

scholarly journals Prevotella intermedia produces two proteins homologous to Porphyromonas gingivalis HmuY but with different heme coordination mode

2020 ◽  
Vol 477 (2) ◽  
pp. 381-405
Author(s):  
Marcin Bielecki ◽  
Svetlana Antonyuk ◽  
Richard W. Strange ◽  
Klaudia Siemińska ◽  
John W. Smalley ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Heme Iron ◽  
Prevotella Intermedia ◽  
Uptake System ◽  
Heme Uptake ◽  
Leading Role ◽  
Methionine Residue ◽  
Reducing Conditions

As part of the infective process, Porphyromonas gingivalis must acquire heme which is indispensable for life and enables the microorganism to survive and multiply at the infection site. This oral pathogenic bacterium uses a newly discovered novel hmu heme uptake system with a leading role played by the HmuY hemophore-like protein, responsible for acquiring heme and increasing virulence of this periodontopathogen. We demonstrated that Prevotella intermedia produces two HmuY homologs, termed PinO and PinA. Both proteins were produced at higher mRNA and protein levels when the bacterium grew under low-iron/heme conditions. PinO and PinA bound heme, but preferentially under reducing conditions, and in a manner different from that of the P. gingivalis HmuY. The analysis of the three-dimensional structures confirmed differences between apo-PinO and apo-HmuY, mainly in the fold forming the heme-binding pocket. Instead of two histidine residues coordinating heme iron in P. gingivalis HmuY, PinO and PinA could use one methionine residue to fulfill this function, with potential support of additional methionine residue/s. The P. intermedia proteins sequestered heme only from the host albumin–heme complex under reducing conditions. Our findings suggest that HmuY-like family might comprise proteins subjected during evolution to significant diversification, resulting in different heme coordination modes. The newer data presented in this manuscript on HmuY homologs produced by P. intermedia sheds more light on the novel mechanism of heme uptake, could be helpful in discovering their biological function, and in developing novel therapeutic approaches.

Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

1998 ◽  
Vol 79 (01) ◽  
pp. 177-185 ◽  
Author(s):  
Ashia Siddiqua ◽  
Michael Wilkinson ◽  
Vijay Kakkar ◽  
Yatin Patel ◽  
Salman Rahman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Functional Characterization ◽  
Human Platelets ◽  
Western Blots ◽  
Activated Platelets ◽  
Reducing Conditions ◽  
Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

Radiographic Characterization of Enlarged Sternal Lymph Nodes in 71 Dogs and 13 Cats

2012 ◽  
Vol 48 (3) ◽  
pp. 176-181 ◽  
Author(s):  
Kelli Smith ◽  
Robert O'Brien
Keyword(s):  
Retrospective Study ◽  
Lymph Nodes ◽  
Statistical Difference ◽  
Inflammatory Diseases ◽  
Wide Spectrum ◽  
Etiologic Agent ◽  
Neoplastic Disease ◽  
Clinical Diagnoses ◽  
Average Size

In this retrospective study, radiographically enlarged sternal lymph nodes (LNs) were evaluated in 71 dogs and 13 cats for average size, location, and most representative radiographic view. Concurrent clinical diagnoses were also noted and grouped into one of three following categories: neoplastic, inflammatory, or hematologic. There were no statistically significant differences in LN size between lateral views within each species. Enlarged sternal LNs were more cranially positioned in dogs than cats. No statistical difference was noted between right and left laterals, as to on which projection the enlarged sterna lymph nodes was seen best. Neoplastic disease (78.9%) was the most prevalent condition seen in association with LN enlargement in dogs, followed by primary infectious or inflammatory diseases (14.1%) and various hematologic conditions (7.0%). In cats, neoplasia was also most common (69.2%), followed by inflammatory diseases (30.8%). No hematologic conditions were noted in cats. The most common etiologic agent seen concurrently with enlarged sternal LNs in both dogs (33.8%) and cats (38.5%) was malignant lymphoma. The results of this study provide a clinically useful representation of the average size and location of radiographically enlarged sternal LNs for dogs and cats. The diseases represented demonstrate the wide spectrum of potential causes of sternal lymphadenopathy.

scholarly journals Structural and functional characterization of engineered bifunctional fusion proteins of CD39 and CD73 ectonucleotidases

2021 ◽  
Vol 320 (1) ◽  
pp. C15-C29 ◽  
Author(s):  
Elizabeth H. Zhong ◽  
Carola Ledderose ◽  
Paola De Andrade Mello ◽  
Keiichi Enjyoji ◽  
Justin Mark Lunderberg ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
High Performance ◽  
Inflammatory Diseases ◽  
Functional Characterization ◽  
Bifunctional Enzymes ◽  
Human Acid ◽  
Inflammatory Processes ◽  
Single Polypeptide

Extracellular diphosphate and triphosphate nucleotides are released from activated or injured cells to trigger vascular and immune P2 purinergic receptors, provoking inflammation and vascular thrombosis. These metabokines are scavenged by ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1 or CD39). Further degradation of the monophosphate nucleoside end products occurs by surface ecto-5′-nucleotidase (NMPase) or CD73. These ectoenzymatic processes work in tandem to promote adenosinergic responses, which are immunosuppressive and antithrombotic. These homeostatic ectoenzymatic mechanisms are lost in the setting of oxidative stress, which exacerbates inflammatory processes. We have engineered bifunctional enzymes made up from ectodomains (ECDs) of CD39 and CD73 within a single polypeptide. Human alkaline phosphatase-ectodomain (ALP-ECD) and human acid phosphatase-ectodomain (HAP-ECD) fusion proteins were also generated, characterized, and compared with these CD39-ECD, CD73-ECD, and bifunctional fusion proteins. Through the application of colorimetrical functional assays and high-performance liquid chromatography kinetic assays, we demonstrate that the bifunctional ectoenzymes express high levels of CD39-like NTPDase activity and CD73-like NMPase activity. Chimeric CD39-CD73-ECD proteins were superior in converting triphosphate and diphosphate nucleotides into nucleosides when compared with ALP-ECD and HAP-ECD. We also note a pH sensitivity difference between the bifunctional fusion proteins and parental fusions, as well as ectoenzymatic property distinctions. Intriguingly, these innovative reagents decreased platelet activation to exogenous agonists in vitro. We propose that these chimeric fusion proteins could serve as therapeutic agents in inflammatory diseases, acting to scavenge proinflammatory ATP and also generate anti-inflammatory adenosine.

scholarly journals Tannerella forsythia Tfo belongs to Porphyromonas gingivalis HmuY-like family of proteins but differs in heme-binding properties

2018 ◽  
Vol 38 (5) ◽  
Author(s):  
Marcin Bielecki ◽  
Svetlana Antonyuk ◽  
Richard W. Strange ◽  
John W. Smalley ◽  
Paweł Mackiewicz ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Phylogenetic Analyses ◽  
3D Structure ◽  
Binding Pocket ◽  
Etiologic Agent ◽  
Heme Iron ◽  
Heme Binding ◽  
Binding Properties ◽  
Tannerella Forsythia ◽  
Histidine Residues

Porphyromonas gingivalis is considered the principal etiologic agent and keystone pathogen of chronic periodontitis. As an auxotrophic bacterium, it must acquire heme to survive and multiply at the infection site. P. gingivalis HmuY is the first member of a novel family of hemophore-like proteins. Bacterial heme-binding proteins usually use histidine-methionine or histidine-tyrosine residues to ligate heme-iron, whereas P. gingivalis HmuY uses two histidine residues. We hypothesized that other ‘red complex’ members, i.e. Tannerella forsythia and Treponema denticola might utilize similar heme uptake mechanisms to the P. gingivalis HmuY. Comparative and phylogenetic analyses suggested differentiation of HmuY homologs and low conservation of heme-coordinating histidine residues present in HmuY. The homologs were subjected to duplication before divergence of Bacteroidetes lineages, which could facilitate evolution of functional diversification. We found that T. denticola does not code an HmuY homolog. T. forsythia protein, termed as Tfo, binds heme, but preferentially in the ferrous form, and sequesters heme from the albumin–heme complex under reducing conditions. In agreement with that, the 3D structure of Tfo differs from that of HmuY in the folding of heme-binding pocket, containing two methionine residues instead of two histidine residues coordinating heme in HmuY. Heme binding to apo-HmuY is accompanied by movement of the loop carrying the His166 residue, closing the heme-binding pocket. Molecular dynamics simulations (MD) demonstrated that this conformational change also occurs in Tfo. In conclusion, our findings suggest that HmuY-like family might comprise proteins subjected during evolution to significant diversification, resulting in different heme-binding properties.

scholarly journals Phenotypical and Functional Characterization of Neutrophils in Two Pyrin-Associated Auto-inflammatory Diseases

2021 ◽  
Author(s):  
Bert Malengier-Devlies ◽  
Mieke Metzemaekers ◽  
Mieke Gouwy ◽  
Erika Van Nieuwenhove ◽  
Albrecht Betrains ◽  
...  
Keyword(s):  
Inflammatory Diseases ◽  
Functional Characterization
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