scholarly journals Unbiased Identification of Immunogenic Staphylococcus aureus Leukotoxin B-Cell Epitopes

2020 ◽  
Vol 88 (4) ◽  
Author(s):  
David N. Hernandez ◽  
Kayan Tam ◽  
Bo Shopsin ◽  
Emily E. Radke ◽  
Pegah Kolahi ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Phage Display ◽  
B Cell ◽  
Three Dimensional ◽  
Amino Acid Side Chain ◽  
Gene Fragment ◽  
B Cell Epitopes ◽  
Content Type ◽  
Amino Acid Peptide

ABSTRACT Unbiased identification of individual immunogenic B-cell epitopes in major antigens of a pathogen remains a technology challenge for vaccine discovery. We therefore developed a platform for rapid phage display screening of deep recombinant libraries consisting of as few as one major pathogen antigen. Using the bicomponent pore-forming leukocidin (Luk) exotoxins of the major pathogen Staphylococcus aureus as a prototype, we randomly fragmented and separately ligated the hemolysin gamma A (HlgA) and LukS genes into a custom-built phage display system, termed pComb-Opti8. Deep sequence analysis of barcoded amplimers of the HlgA and LukS gene fragment libraries demonstrated that biopannng against a cross-reactive anti-Luk monoclonal antibody (MAb) recovered convergent molecular clones with short overlapping homologous sequences. We thereby identified an 11-amino-acid sequence that is highly conserved in four Luk toxin subunits and is ubiquitous in representation within S. aureus clinical isolates. The isolated 11-amino-acid peptide probe was predicted to retain the native three-dimensional (3D) conformation seen within the Luk holotoxin. Indeed, this peptide was recognized by the selecting anti-Luk MAb, and, using mutated peptides, we showed that a particular amino acid side chain was essential for these interactions. Furthermore, murine immunization with this peptide elicited IgG responses that were highly reactive with both the autologous synthetic peptide and the full-length Luk toxin homologues. Thus, using a gene fragment- and phage display-based pipeline, we have identified and validated immunogenic B-cell epitopes that are cross-reactive between members of the pore-forming leukocidin family. This approach could be harnessed to identify novel epitopes for a much-needed S. aureus-protective subunit vaccine.

scholarly journals Convergent Evolution of Neutralizing Antibodies to Staphylococcus aureus γ-Hemolysin C That Recognize an Immunodominant Primary Sequence-Dependent B-Cell Epitope

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
David N. Hernandez ◽  
Kayan Tam ◽  
Bo Shopsin ◽  
Emily E. Radke ◽  
Karen Law ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Phage Display ◽  
B Cell ◽  
Neutralizing Antibodies ◽  
Ex Vivo ◽  
Cell Epitope ◽  
Phage Display Library ◽  
Structural Basis ◽  
Peptide Fragments ◽  
Content Type

ABSTRACT Staphylococcus aureus infection is a major public health threat in part due to the spread of antibiotic resistance and repeated failures to develop a protective vaccine. Infection is associated with production of virulence factors that include exotoxins that attack host barriers and cellular defenses, such as the leukocidin (Luk) family of bicomponent pore-forming toxins. To investigate the structural basis of antibody-mediated functional inactivation of Luk toxins, we generated a panel of murine monoclonal antibodies (MAbs) that neutralize host cell killing by the γ-hemolysin HlgCB. By biopanning these MAbs against a phage-display library of random Luk peptide fragments, we identified a small subregion within the rim domain of HlgC as the epitope for all the MAbs. Within the native holotoxin, this subregion folds into a conserved β-hairpin structure, with exposed key residues, His252 and Tyr253, required for antibody binding. On the basis of the phage-display results and molecular modeling, a 15-amino-acid synthetic peptide representing the minimal epitope on HlgC (HlgC241-255) was designed, and preincubation with this peptide blocked antibody-mediated HIgCB neutralization. Immunization of mice with HlgC241-255 or the homologous LukS246-260 subregion peptide elicited serum antibodies that specifically recognized the native holotoxin subunits. Furthermore, serum IgG from patients who were convalescent for invasive S. aureus infection showed neutralization of HlgCB toxin activity ex vivo, which recognized the immunodominant HlgC241-255 peptide and was dependent on His252 and Tyr253 residues. We have thus validated an efficient, rapid, and scalable experimental workflow for identification of immunodominant and immunogenic leukotoxin-neutralizing B-cell epitopes that can be exploited for new S. aureus-protective vaccines and immunotherapies.

scholarly journals Immunisation With Immunodominant Linear B Cell Epitopes Vaccine of Manganese Transport Protein C Confers Protection against Staphylococcus aureus Infection

PLoS ONE ◽  
2016 ◽  
Vol 11 (2) ◽  
pp. e0149638 ◽  
Author(s):  
Hui-Jie Yang ◽  
Jin-Yong Zhang ◽  
Chao Wei ◽  
Liu-Yang Yang ◽  
Qian-Fei Zuo ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
B Cell ◽  
Protein C ◽  
Transport Protein ◽  
B Cell Epitopes ◽  
Aureus Infection ◽  
Manganese Transport ◽  
Linear B

Prediction of linear B-cell epitopes using amino acid pair antigenicity scale

Amino Acids ◽  
2007 ◽  
Vol 33 (3) ◽  
pp. 423-428 ◽  
Author(s):  
J. Chen ◽  
H. Liu ◽  
J. Yang ◽  
K.-C. Chou
Keyword(s):  
Amino Acid ◽  
B Cell ◽  
Amino Acid Pair ◽  
B Cell Epitopes ◽  
Linear B

Phage Display Selection, Analysis, and Prediction of B Cell Epitopes

2009 ◽  
Vol 86 (1) ◽  
Author(s):  
Natalia Tarnovitski Freund ◽  
David Enshell‐Seijffers ◽  
Jonathan M. Gershoni
Keyword(s):  
Phage Display ◽  
B Cell ◽  
B Cell Epitopes ◽  
Selection Analysis

scholarly journals Susceptibility of Methicillin-Resistant Staphylococcus aureus to Five Quinazolinone Antibacterials

2019 ◽  
Vol 64 (1) ◽  
Author(s):  
Sara Ceballos ◽  
Choon Kim ◽  
Yuanyuan Qian ◽  
Shahriar Mobashery ◽  
Mayland Chang ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Binding Proteins ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Methicillin Resistant ◽  
Content Type ◽  
Penicillin Binding Proteins

ABSTRACT The in vitro activities of five quinazolinone antibacterials, compounds Q1 to Q5, were tested against 210 strains of methicillin-resistant Staphylococcus aureus (MRSA). The MIC50/MIC90 values (in μg/ml) were as follows: Q1, 0.5/2; Q2, 1/4; Q3, 2/4; Q4, 0.06/0.25; and Q5, 0.125/0.5. Several strains with high MIC values (from 8 to >32 μg/ml) for some of these compounds exhibited amino acid changes in the penicillin-binding proteins, which are targeted by these antibacterials.

scholarly journals Prediction of B-Cell Epitopes in Listeriolysin O, a Cholesterol Dependent Cytolysin Secreted by Listeria monocytogenes

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Morris S. Jones ◽  
J. Mark Carter
Keyword(s):  
Monoclonal Antibodies ◽  
Listeria Monocytogenes ◽  
B Cell ◽  
Three Dimensional ◽  
Listeriolysin O ◽  
Molecular Models ◽  
B Cell Epitopes ◽  
Discontinuous Epitopes ◽  
Pathogenic Effects ◽  
Domain 4

Listeria monocytogenes is a gram-positive, foodborne bacterium responsible for disease in humans and animals. Listeriolysin O (LLO) is a required virulence factor for the pathogenic effects of L. monocytogenes. Bioinformatics revealed conserved putative epitopes of LLO that could be used to develop monoclonal antibodies against LLO. Continuous and discontinuous epitopes were located by using four different B-cell prediction algorithms. Three-dimensional molecular models were generated to more precisely characterize the predicted antigenicity of LLO. Domain 4 was predicted to contain five of eleven continuous epitopes. A large portion of domain 4 was also predicted to comprise discontinuous immunogenic epitopes. Domain 4 of LLO may serve as an immunogen for eliciting monoclonal antibodies that can be used to study the pathogenesis of L. monocytogenes as well as develop an inexpensive assay.

scholarly journals Three-Dimensional In Vitro Staphylococcus aureus Abscess Communities Display Antibiotic Tolerance and Protection from Neutrophil Clearance

2020 ◽  
Vol 88 (11) ◽  
Author(s):  
Marloes I. Hofstee ◽  
Martijn Riool ◽  
Igors Terjajevs ◽  
Keith Thompson ◽  
Martin J. Stoddart ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Immune Cells ◽  
Early Stage ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Maximum Size ◽  
Human Neutrophils ◽  
Content Type

ABSTRACT Staphylococcus aureus is a prominent human pathogen in bone and soft-tissue infections. Pathophysiology involves abscess formation, which consists of central staphylococcal abscess communities (SACs), surrounded by a fibrin pseudocapsule and infiltrating immune cells. Protection against the ingress of immune cells such as neutrophils, or tolerance to antibiotics, remains largely unknown for SACs and is limited by the lack of availability of in vitro models. We describe a three-dimensional in vitro model of SACs grown in a human plasma-supplemented collagen gel. The in vitro SACs reached their maximum size by 24 h and elaborated a fibrin pseudocapsule, as confirmed by electron and immunofluorescence microscopy. The in vitro SACs tolerated 100× the MIC of gentamicin alone and in combination with rifampin, while planktonic controls and mechanically dispersed SACs were efficiently killed. To simulate a host response, SACs were exposed to differentiated PLB-985 neutrophil-like (dPLB) cells and to primary human neutrophils at an early stage of SAC formation or after maturation at 24 h. Both cell types were unable to clear mature in vitro SACs, but dPLB cells prevented SAC growth upon early exposure before pseudocapsule maturation. Neutrophil exposure after plasmin pretreatment of the SACs resulted in a significant decrease in the number of bacteria within the SACs. The in vitro SAC model mimics key in vivo features, offers a new tool to study host-pathogen interactions and drug efficacy assessment, and has revealed the functionality of the S. aureus pseudocapsule in protecting the bacteria from host phagocytic responses and antibiotics.

scholarly journals MixedChlamydia trachomatisPeptide Antigens Provide a Specific and Sensitive Single-Well Colorimetric Enzyme-Linked Immunosorbent Assay for Detection of Human Anti-C. trachomatisAntibodies

mSphere ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
K. Shamsur Rahman ◽  
Toni Darville ◽  
Harold C. Wiesenfeld ◽  
Sharon L. Hillier ◽  
Bernhard Kaltenboeck
Keyword(s):  
B Cell ◽  
High Specificity ◽  
Cross Reactivity ◽  
Specific Detection ◽  
B Cell Epitopes ◽  
Content Type ◽  
Peptide Antigens ◽  
Specificity And Sensitivity ◽  
Single Well ◽  
Individual Peptide

ABSTRACTSensitive and specific detection of anti-Chlamydia trachomatisantibodies in standard enzyme-linked immunosorbent assays (ELISAs) is compromised by cross-reactivity and poor sensitivity of classicalC.trachomatisantigens. Previously, we discovered 48 strongly reactive peptide antigens ofC. trachomatis-specific B-cell epitopes from 21 immunodominant proteins. By comprehensive individual testing of 11 top-ranked peptide antigens, we found very high sensitivity and specificity for detection of anti-C. trachomatisantibodies in chemiluminescent ELISAs. The current study established a labor-saving colorimetric ELISA by using a mixture of 12 strongly reactiveC. trachomatispeptide antigens (Ctr Mix1) in a single well/serum rather than assaying reactivity to each individual peptide. For performance evaluation, we used a simulated population of 212 anti-C. trachomatisantibody-positive and -negative sera from 125 women with NAAT-confirmed activeC. trachomatisinfection and from 87 healthy women at low risk forC. trachomatisinfection. In comparison to a composite reference standard (CRS) for anti-C. trachomatisantibody status, the Ctr Mix1 IgG ELISA achieved 93.9% sensitivity, significantly superior to the 49% to 79% sensitivities of four commercial anti-C. trachomatisIgG ELISAs, and 98% specificity of all tested assays. Compared to the labor-intensive individual peptide testing, this mixed peptide ELISA retained high specificity with only marginal, ∼5% sensitivity loss. By ROC-AUC, likelihood ratio, and predictive value analyses, the Ctr Mix1 ELISA performed satisfactorily at 10% to 75% prevalence range of anti-C. trachomatisantibodies but significantly better than commercial ELISAs. Thus, the labor-saving mixed peptide colorimetric ELISA format provides simultaneously high specificity and sensitivity for detection of anti-C. trachomatisantibodies.IMPORTANCEFor detection of anti-C. trachomatisantibodies by serological assays, use of classical chlamydial antigens results in high cross-reactivity and poor sensitivity. Previously, we discovered 48 strongly reactive peptide antigens ofC. trachomatis-specific B-cell epitopes from 21 immunodominant proteins, and individual testing and combined scoring of 5 to 11 peptide antigens provided highly sensitive and specific detection of anti-C. trachomatisantibodies in chemiluminescent ELISAs. To simplify this method, this study established a single-well labor-saving colorimetric ELISA using a mixture of 12 strongly reactiveC. trachomatispeptide antigens (Ctr Mix1) for detection of anti-C. trachomatisantibodies. This Ctr Mix1 ELISA (94% sensitivity and 98% specificity) outperformed 4 commercial ELISAs (49% to 79% sensitivity and 98% specificity). This ELISA can be easily implemented and commercialized, with convenient setup for use in nonspecialized laboratories. Thus, this mixed peptide assay with superior specificity and sensitivity will improve serodiagnosis ofC. trachomatisinfections.

Using random forest to classify linear B-cell epitopes based on amino acid properties and molecular features

Biochimie ◽  
2014 ◽  
Vol 103 ◽  
pp. 1-6 ◽  
Author(s):  
Jian-Hua Huang ◽  
Ming Wen ◽  
Li-Juan Tang ◽  
Hua-Lin Xie ◽  
Liang Fu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Random Forest ◽  
B Cell ◽  
B Cell Epitopes ◽  
Acid Properties ◽  
Molecular Features ◽  
Amino Acid Properties ◽  
Linear B

scholarly journals Identification ofypqPas a New Bacillus subtilis Biofilm Determinant That Mediates the Protection of Staphylococcus aureus against Antimicrobial Agents in Mixed-Species Communities

2014 ◽  
Vol 81 (1) ◽  
pp. 109-118 ◽  
Author(s):  
Pilar Sanchez-Vizuete ◽  
Dominique Le Coq ◽  
Arnaud Bridier ◽  
Jean-Marie Herry ◽  
Stéphane Aymerich ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Bacillus Subtilis ◽  
Extracellular Polymeric Substances ◽  
Antimicrobial Agents ◽  
Three Dimensional ◽  
Laboratory Strain ◽  
Spatial Arrangement ◽  
Content Type ◽  
Microbial Life ◽  
Multispecies Biofilms

ABSTRACTIn most habitats, microbial life is organized in biofilms, three-dimensional edifices sustained by extracellular polymeric substances that enable bacteria to resist harsh and changing environments. Under multispecies conditions, bacteria can benefit from the polymers produced by other species (“public goods”), thus improving their survival under toxic conditions. A recent study showed that aBacillus subtilishospital isolate (NDmed) was able to protectStaphylococcus aureusfrom biocide action in multispecies biofilms. In this work, we identifiedypqP, a gene whose product is required in NDmed for thick-biofilm formation on submerged surfaces and for resistance to two biocides widely used in hospitals. NDmed andS. aureusformed mixed biofilms, and both their spatial arrangement and pathogen protection were mediated by YpqP. FunctionalypqPis present in other naturalB. subtilisbiofilm-forming isolates. However, the gene is disrupted by the SPβ prophage in the weak submerged-biofilm-forming strains NCIB3610 and 168, which are both less resistant than NDmed to the biocides tested. Furthermore, in a 168 laboratory strain cured of the SPβ prophage, the reestablishment of a functionalypqPgene led to increased thickness and resistance to biocides of the associated biofilms. We therefore propose that YpqP is a new and important determinant ofB. subtilissurface biofilm architecture, protection against exposure to toxic compounds, and social behavior in bacterial communities.

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