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Infection and Immunity Sep 2019Multidrug-resistant enterococci are major causes of hospital-acquired infections. Immunotherapy with monoclonal antibodies (MAbs) targeting bacterial antigens would be a...
Multidrug-resistant enterococci are major causes of hospital-acquired infections. Immunotherapy with monoclonal antibodies (MAbs) targeting bacterial antigens would be a valuable treatment option in this setting. Here, we describe the development of two MAbs through hybridoma technology that target antigens from the most clinically relevant enterococcal species. Diheteroglycan (DHG), a well-characterized capsular polysaccharide of , and the secreted antigen A (SagA), an immunogenic protein from , are both immunogens that have been proven to raise opsonic and cross-reactive antibodies against enterococcal strains. For this purpose, a conjugated form of the native DHG with SagA was used to raise the antibodies in mice, while enzyme-linked immunosorbent assay and opsonophagocytic assay were combined in the selection process of hybridoma cells producing immunoreactive and opsonic antibodies targeting the selected antigens. From this process, two highly specific IgG1(κ) MAbs were obtained, one against the polysaccharide (DHG.01) and one against the protein (SagA.01). Both MAbs exhibited good opsonic killing against the target bacterial strains: DHG.01 showed 90% killing against type 2, and SagA.01 showed 40% killing against 11231/6. In addition, both MAbs showed cross-reactivity toward other and strains. The sequences from the variable regions of the heavy and light chains were reconstructed in expression vectors, and the activity of the MAbs upon expression in eukaryotic cells was confirmed with the same immunological assays. In summary, we identified two opsonic MAbs against enterococci which could be used for therapeutic or prophylactic approaches against enterococcal infections.
Topics: Animals; Antibodies, Monoclonal; Antigens, Bacterial; Bacterial Capsules; Drug Resistance, Microbial; Enterococcus faecalis; Enterococcus faecium; Immunotherapy; Mice; Opsonin Proteins; Polysaccharides
PubMed: 31285252
DOI: 10.1128/IAI.00276-19 -
Microbes and Infection Mar 2005Recently we described a novel bacteriophage-encoded pathogenicity island in Staphylococcus aureus that harbors a number of virulence factors that are all involved in the...
Recently we described a novel bacteriophage-encoded pathogenicity island in Staphylococcus aureus that harbors a number of virulence factors that are all involved in the evasion of innate immunity. Here we describe a mechanism by which staphylokinase (SAK), frequently present on this pathogenicity island, interferes with innate immune defenses: SAK is anti-opsonic. By activating human plasminogen (PLG) into plasmin (PL) at the bacterial surface, it creates bacterium-bound serine protease activity that leads to degradation of two major opsonins: human immunoglobulin G (IgG) and human C3b. Incubation of opsonized bacteria with PLG and SAK resulted in removal of anti-staphylococcal IgGs and C3b from the bacterial surface. In phagocytosis assays this proved to be a very efficient mechanism to reduce the opsonic activity of human IgG and serum. The fact that SAK activates human PLG at the bacterial surface and removes IgG as well as C3b makes this protein a unique anti-opsonic molecule.
Topics: Complement C3b; Enzyme Activation; Fibrinolysin; Humans; Immunoglobulin G; Metalloendopeptidases; Opsonin Proteins; Plasminogen; Protein Binding; Staphylococcus aureus
PubMed: 15792635
DOI: 10.1016/j.micinf.2004.12.014 -
The Journal of Biological Chemistry Sep 2022Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is a human granulocyte receptor mediating the efficient phagocytosis of a subset of human-restricted...
Carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) is a human granulocyte receptor mediating the efficient phagocytosis of a subset of human-restricted bacterial pathogens. Its function depends on phosphorylation of a tyrosine-based sequence motif, but the enzyme(s) responsible for reversing this modification are unclear. Here, we identify the receptor-type protein tyrosine phosphatase PTPRJ as a negative regulator of CEACAM3-mediated phagocytosis. We show depletion of PTPRJ results in a gain-of-function phenotype, while overexpression of a constitutively active PTPRJ phosphatase strongly reduces bacterial uptake via CEACAM3. We also determined that recombinant PTPRJ directly dephosphorylates the cytoplasmic tyrosine residues of purified full-length CEACAM3 and recognizes synthetic CEACAM3-derived phosphopeptides as substrates. Dephosphorylation of CEACAM3 by PTPRJ is also observed in intact cells, thereby limiting receptor-initiated cytoskeletal re-arrangements, lamellipodia formation, and bacterial uptake. Finally, we show that human phagocytes deficient for PTPRJ exhibit exaggerated lamellipodia formation and enhanced opsonin-independent phagocytosis of CEACAM3-binding bacteria. Taken together, our results highlight PTPRJ as a bona fide negative regulator of CEACAM3-initiated phagocyte functions, revealing a potential molecular target to limit CEACAM3-driven inflammatory responses.
Topics: Carcinoembryonic Antigen; Granulocytes; Humans; Opsonin Proteins; Phagocytosis; Phosphopeptides; Receptor-Like Protein Tyrosine Phosphatases, Class 3
PubMed: 35850306
DOI: 10.1016/j.jbc.2022.102269 -
The Journal of Experimental Medicine Jan 2000Infections with gram-positive bacteria are a major cause of morbidity and mortality in humans. Opsonin-dependent phagocytosis plays a major role in protection against...
Infections with gram-positive bacteria are a major cause of morbidity and mortality in humans. Opsonin-dependent phagocytosis plays a major role in protection against and recovery from gram-positive infections. Inborn and acquired defects in opsonin generation and/or recognition by phagocytes are associated with an increased susceptibility to bacterial infections. In contrast, the physiological significance of opsonin-independent phagocytosis is unknown. Type I and II class A scavenger receptors (SR-AI/II) recognize a variety of polyanions including bacterial cell wall products such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), suggesting a role for SR-AI/II in innate immunity to bacterial infections. Here, we show that SR-AI/II-deficient mice (MSR-A(-/-)) are more susceptible to intraperitoneal infection with a prototypic gram-positive pathogen, Staphylococcus aureus, than MSR-A(+/+) control mice. MSR-A(-/-) mice display an impaired ability to clear bacteria from the site of infection despite normal killing of S. aureus by neutrophils and die as a result of disseminated infection. Opsonin-independent phagocytosis of gram-positive bacteria by MSR-A(-/-) macrophages is significantly decreased although their phagocytic machinery is intact. Peritoneal macrophages from control mice phagocytose a variety of gram-positive bacteria in an SR-AI/II-dependent manner. Our findings demonstrate that SR-AI/II mediate opsonin-independent phagocytosis of gram-positive bacteria, and provide the first evidence that opsonin-independent phagocytosis plays a critical role in host defense against bacterial infections in vivo.
Topics: Animals; Gram-Positive Bacterial Infections; Lipopolysaccharides; Macrophages; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Knockout; Opsonin Proteins; Phagocytosis; Receptors, Immunologic; Receptors, Lipoprotein; Receptors, Scavenger; Scavenger Receptors, Class A; Scavenger Receptors, Class B; Staphylococcal Infections; Teichoic Acids
PubMed: 10620613
DOI: 10.1084/jem.191.1.147 -
The Journal of Cell Biology Aug 1973Kinetic analysis of the initial ingestion rate of albumin-coated paraffin oil particles by human granulocytes and rabbit alveolar macrophages was undertaken to study the...
Kinetic analysis of the initial ingestion rate of albumin-coated paraffin oil particles by human granulocytes and rabbit alveolar macrophages was undertaken to study the mechanism of action of cations and of heat-labile opsonin on engulfment. The rate of uptake of the particles was stimulated by Ca(++), Mg(++), Mn(++), or Co(++). At high concentrations (> 20 mM) Ca(++) and Mg(++) inhibited the rate of ingestion. Treatment of the particles with fresh serum (heat-labile opsonin) also stimulated the rate of ingestion. (125)I-labeled C3 was bound to the particles during opsonization. C3-deficient human serum lacked opsonic activity, which was restored by addition of purified C3. Normal, C2-deficient, and hereditary angioneurotic edema sera had equivalent opsonic activity. The serum opsonic activity thus involved C3 fixation to the particles by means of the properdin system. Although Mg(++) and heat-labile opsonin both accelerated the maximal rates of ingestion of the particles, neither altered the particle concentrations associated with one-half maximal ingestion rates. Opsonization of the particles markedly diminished the concentrations of divalent cations causing both stimulatory and inhibitory effects on ingestion rates and altered the shapes of the cation activation curves. (45)Ca was not bound to the particles during opsonization. The results are consistent with a mechanism whereby divalent cations and heat-labile opsonin activate ingestion by stimulating the work of engulfment rather than by merely enhancing cell-particle affinity, and whereby heat-labile opsonin acts by potentiating the effects of divalent cations.
Topics: Angioedema; Animals; Calcium; Cobalt; Complement System Proteins; Drug Synergism; Humans; Iodine Isotopes; Kinetics; Leukocytes; Macrophages; Magnesium; Manganese; Opsonin Proteins; Phagocytosis; Rabbits; Serum Albumin
PubMed: 4738105
DOI: 10.1083/jcb.58.2.346 -
Pathogens and Disease Aug 2017Here, we describe the application of an 'artificial opsonin' to stimulate the innate immune response against Gram-positive bacteria. The artificial opsonin comprises a...
Here, we describe the application of an 'artificial opsonin' to stimulate the innate immune response against Gram-positive bacteria. The artificial opsonin comprises a poly(L-lysine)-graft-poly(ethylene glycol) backbone displaying multiple copies of vancomycin and human IgG-Fc. The vancomycin targets bacteria by recognizing d-Ala-d-Ala-terminated peptides present in the bacterial cell wall. The human IgG-Fc antibody fragments serve as phagocyte recognition moieties that recognize the Fcγ cell surface receptors expressed by professional human phagocytes. Staphylococcus epidermidis RP62A, a biofilm-forming, methicillin-resistant strain, was utilized to investigate the effects of opsonization on phagocytosis, oxidative burst and IL-8 chemokine production by human neutrophils. Results show that opsonization of S. epidermidis RP62A with the artificial opsonin resulted in an ∼2-fold increase in neutrophil phagocytosis. Analysis of the cell supernatant found a 2- to 3-fold increase in neutrophil IL-8 secretion. The neutrophil oxidative burst was investigated using the oxidation-sensitive fluorophore dihydrorhodamine-123. Bacterial opsonization resulted in a 20% increase in fluorescence intensity, indicating a significant increase in the production of reactive oxygen species by the neutrophils. These studies suggest that artificial opsonins may be a novel immunostimulation therapeutic strategy to control infections caused by Gram-positive bacteria, particularly those that are known to be immune evasive and/or antibiotic resistant.
Topics: Humans; Immunity, Innate; Immunoconjugates; Immunoglobulin Fc Fragments; Immunoglobulin G; Interleukin-8; Neutrophils; Opsonin Proteins; Phagocytosis; Polyethylene Glycols; Polylysine; Primary Cell Culture; Reactive Oxygen Species; Respiratory Burst; Staphylococcus epidermidis; Vancomycin
PubMed: 28859309
DOI: 10.1093/femspd/ftx075 -
Clinical and Diagnostic Laboratory... Jan 2005
Review
Topics: Adult; Animals; Antibodies, Bacterial; Biological Assay; Cell Differentiation; Female; HL-60 Cells; Humans; Mice; Opsonin Proteins; Pneumococcal Vaccines; Streptococcus pneumoniae
PubMed: 15642980
DOI: 10.1128/CDLI.12.1.19-27.2005 -
Clinical and Vaccine Immunology : CVI Feb 2006
Review
Topics: Animals; Antibodies, Bacterial; Cell Line; Humans; In Vitro Techniques; Opsonin Proteins; Phagocytosis; Pneumococcal Vaccines
PubMed: 16467321
DOI: 10.1128/CVI.13.2.165-169.2006 -
Cytometry Dec 1998A one-step flow cytometric (FCM) assay has been developed to quantify both opsonin- and antigen-dependent phagocytosis and intraphagocyte oxidative burst responses....
A one-step flow cytometric (FCM) assay has been developed to quantify both opsonin- and antigen-dependent phagocytosis and intraphagocyte oxidative burst responses. Meningococcal outer membrane structures (OMV) were adsorbed to fluorescent polystyrene beads, opsonized with serum, and exposed to leukocytes. FCM parameters of phagocytosis were evaluated in combinations with oxidative burst indicators. Rhodamine-123 was the most sensitive indicator and was compatible with quantitation of phagocytosis. The phagocytosis and oxidative burst responses induced by OMV beads were dependent on both antigens and opsonins. Increased human opsonic responses against OMV were induced during clinical meningococcal disease. A dissociation was noted between phagocytosis and oxidative burst in individual cells, indicating that functional opsonins against OMV components may differ in their ability to stimulate phagocytosis and oxidative burst responses. The method facilitates evaluation of purified bacterial structures as mediators of opsonin-dependent phagocytosis and intracellular oxidative microbicidal mechanisms, which is of interest in the complex process of selecting bacterial antigens as constituents of certain vaccines.
Topics: Adult; Antibodies, Bacterial; Antigens, Bacterial; Ethidium; Flow Cytometry; Fluorescence; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Light Signal Transduction; Luminescent Measurements; Neisseria meningitidis; Opsonin Proteins; Phagocytosis; Respiratory Burst
PubMed: 9845434
DOI: 10.1002/(sici)1097-0320(19981201)33:4<406::aid-cyto3>3.0.co;2-l -
Bulletin of the World Health... 1981Phagocytic host defence mechanisms require both normally functioning cells and humoral factors. For example, activated complement components and/or specific...
Phagocytic host defence mechanisms require both normally functioning cells and humoral factors. For example, activated complement components and/or specific immunoglobulin are essential for effective ingestion and killing of bacteria by neutrophils, and complement is especially important early in infection, before specific antibody has been produced. Abnormalities of serum complement have previously been reported in malnutrition, and the present study investigated the levels of serum opsonins in children with protein-energy malnutrition (PEM).Opsonic activity for Escherichia coli and Staphylococcus aureus was depressed in acute PEM patients, but recovered to higher levels with treatment. This depression was detected only when low concentrations of serum (10-20 ml/litre) were used. Marked and persistent opsonin deficiencies were associated with poor clinical response. Reduced opsonic activity may adversely affect host defence mechanisms and contribute to morbidity and mortality from pyogenic infections in PEM. Replacement therapy with fresh or fresh frozen plasma might restore opsonic activity in these patients and reduce the risk of septicaemia and its attendant high mortality.
Topics: Child; Child, Preschool; Guatemala; Humans; Infant; Opsonin Proteins; Phagocytosis; Protein-Energy Malnutrition
PubMed: 6802507
DOI: No ID Found