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.

Sorting a Staphylococcus aureus phage display library against ex vivo biomaterial

2004 ◽  
Vol 53 (10) ◽  
pp. 945-951 ◽  
Author(s):  
Joakim Bjerketorp ◽  
Anna Rosander ◽  
Martin Nilsson ◽  
Karin Jacobsson ◽  
Lars Frykberg
Keyword(s):  
Staphylococcus Aureus ◽  
Phage Display ◽  
Ex Vivo ◽  
Phage Display Library

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 Evaluation of a Novel Chimeric B Cell Epitope-Based Vaccine against Mastitis Induced by Either Streptococcus agalactiae or Staphylococcus aureus in Mice

2011 ◽  
Vol 18 (6) ◽  
pp. 893-900 ◽  
Author(s):  
Haiyang Xu ◽  
Changmin Hu ◽  
Rui Gong ◽  
Yingyu Chen ◽  
Ningning Ren ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
B Cell ◽  
Streptococcus Agalactiae ◽  
Histopathological Examination ◽  
Cell Epitope ◽  
Mammary Glands ◽  
Universal Vaccine ◽  
Content Type ◽  
Immunization Group ◽  
B Cell Epitope

ABSTRACTTo construct a universal vaccine against mastitis induced by eitherStreptococcus agalactiaeorStaphylococcus aureus, the B cell epitopes of the surface immunogenic protein (Sip) fromS. agalactiaeand clumping factor A (ClfA) fromS. aureuswere analyzed and predicted.sip-clfA, a novel chimeric B cell epitope-based gene, was obtained by overlap PCR, and then the recombinant Sip-ClfA (rSip-ClfA) was expressed and purified. rSip-ClfA and inactivatedS. agalactiaeandS. aureuswere formulated into different vaccines with mineral oil as the adjuvant and evaluated in mouse models. The rSip-ClfA vaccination induced immunoglobulin G (IgG) titers higher than those seen in groups immunized with inactivated bacteria. Furthermore, the response to rSip-ClfA immunization was characterized as having a dominant IgG1 subtype, whereas both bacterial immunizations produced similar levels of IgG1 and IgG2a. The antiserum capacities for opsonizing adhesion and phagocytosis were significantly greater in the rSip-ClfA immunization group than in the killed-bacterium immunization groups (P< 0.05). The immunized lactating mice were challenged with eitherS. agalactiaeorS. aureusvia the intramammary route. At 24 h postinfection, the numbers of bacteria recovered from the mammary glands in the rSip-ClfA group were >5-fold lower than those in both inactivated-bacterium groups (P< 0.01). Histopathological examination of the mammary glands showed that rSip-ClfA immunization provided better protection of mammary gland tissue integrity against bothS. agalactiaeandS. aureuschallenges. Thus, the recombinant protein rSip-ClfA would be a promising vaccine candidate against mastitis induced by eitherS. agalactiaeorS. aureus.

scholarly journals Staphylococcal Protein A Contributes to Persistent Colonization of Mice withStaphylococcus aureus

2018 ◽  
Vol 200 (9) ◽  
Author(s):  
Yan Sun ◽  
Carla Emolo ◽  
Silva Holtfreter ◽  
Siouxsie Wiles ◽  
Barry Kreiswirth ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
B Cell ◽  
Neutralizing Antibodies ◽  
Protein A ◽  
Staphylococcal Protein A ◽  
Content Type ◽  
Staphylococcal Protein ◽  
Host Immune Responses ◽  
Increased Risk ◽  
Drug Resistant Strains

ABSTRACTStaphylococcus aureuspersistently colonizes the nasopharynx in humans, which increases the risk for invasive diseases, such as skin infection and bacteremia. Nasal colonization triggers IgG responses against staphylococcal surface antigens; however, these antibodies cannot prevent subsequent colonization or disease. Here, we describeS. aureusWU1, a multilocus sequence type 88 (ST88) isolate that persistently colonizes the nasopharynx in mice. We report that staphylococcal protein A (SpA) is required for persistence ofS. aureusWU1 in the nasopharynx. Compared to animals colonized by wild-typeS. aureus, mice colonized with the Δspavariant mount increased IgG responses against staphylococcal colonization determinants. Immunization of mice with a nontoxigenic SpA variant, which cannot cross-link B cell receptors and divert antibody responses, elicits protein A-neutralizing antibodies that promote IgG responses against colonizingS. aureusand diminish pathogen persistence.IMPORTANCEStaphylococcus aureuspersistently colonizes the nasopharynx in about one-third of the human population, thereby promoting community- and hospital-acquired infections. Antibiotics are currently used for decolonization of individuals at increased risk of infection. However, the efficacy of antibiotics is limited by recolonization and selection for drug-resistant strains. Here, we propose a model of how staphylococcal protein A (SpA), a B cell superantigen, modifies host immune responses during colonization to support continued persistence ofS. aureusin the nasopharynx. We show that this mechanism can be thwarted by vaccine-induced anti-SpA antibodies that promote IgG responses against staphylococcal antigens and diminish colonization.

scholarly journals A Putative Cro-Like Repressor Contributes to Arylomycin Resistance in Staphylococcus aureus

2015 ◽  
Vol 59 (6) ◽  
pp. 3066-3074 ◽  
Author(s):  
Arryn Craney ◽  
Floyd E. Romesberg
Keyword(s):  
Staphylococcus Aureus ◽  
Ex Vivo ◽  
Transcriptional Regulator ◽  
Signal Peptidase ◽  
Health Concern ◽  
Resistant Bacteria ◽  
Type I ◽  
Wall Teichoic Acid ◽  
Content Type ◽  
Wide Range

ABSTRACTAntibiotic-resistant bacteria are a significant public health concern and motivate efforts to develop new classes of antibiotics. One such class of antibiotics is the arylomycins, which target type I signal peptidase (SPase), the enzyme responsible for the release of secreted proteins from their N-terminal leader sequences. Despite the essentiality, conservation, and relative accessibility of SPase, the activity of the arylomycins is limited against some bacteria, including the important human pathogenStaphylococcus aureus. To understand the origins of the limited activity againstS. aureus, we characterized the susceptibility of a panel of strains to two arylomycin derivatives, arylomycin A-C16and its more potent analog arylomycin M131. We observed a wide range of susceptibilities to the two arylomycins and found that resistant strains were sensitized by cotreatment with tunicamycin, which inhibits the first step of wall teichoic acid synthesis. To further understand howS. aureusresponds to the arylomycins, we profiled the transcriptional response ofS. aureusNCTC 8325 to growth-inhibitory concentrations of arylomycin M131 and found that it upregulates the cell wall stress stimulon (CWSS) and an operon consisting of a putative transcriptional regulator and three hypothetical proteins. Interestingly, we found that mutations in the putative transcriptional regulator are correlated with resistance, and selection for resistanceex vivodemonstrated that mutations in this gene are sufficient for resistance. The results begin to elucidate howS. aureuscopes with secretion stress and how it evolves resistance to the inhibition of SPase.

scholarly journals A Multiepitope Fusion Antigen Elicits Neutralizing Antibodies against Enterotoxigenic Escherichia coli and Homologous Bovine Viral Diarrhea VirusIn Vitro

2013 ◽  
Vol 20 (7) ◽  
pp. 1076-1083 ◽  
Author(s):  
Emad A. Hashish ◽  
Chengxian Zhang ◽  
Xiaosai Ruan ◽  
David E. Knudsen ◽  
Christopher C. Chase ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Viral Infection ◽  
Neutralizing Antibodies ◽  
Cell Epitope ◽  
Enterotoxigenic Escherichia Coli ◽  
Bovine Viral Diarrhea ◽  
Content Type ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Fusion Antigen

ABSTRACTDiarrhea is one of the most important bovine diseases. EnterotoxigenicEscherichia coli(ETEC) and bovine viral diarrhea virus (BVDV) are the major causes of diarrhea in calves and cattle. ETEC expressing K99 (F5) fimbriae and heat-stable type Ia (STa) toxin are the leading bacteria causing calf diarrhea, and BVDV causes diarrhea and other clinical illnesses in cattle of all ages. It is reported that maternal immunization with K99 fimbrial antigens provides passive protection to calves against K99 fimbrial ETEC and that BVDV major structural protein E2 elicits antibodies neutralizing against BVDV viral infection. Vaccines inducing anti-K99 and anti-STa immunity would protect calves more effectively against ETEC diarrhea, and those also inducing anti-E2 neutralizing antibodies would protect calves and cattle against diarrhea caused by both ETEC and BVDV. In this study, we used the ETEC K99 major subunit FanC as a backbone, genetically embedded the STa toxoid STaP12Fand the most-antigenic B-cell epitope and T-cell epitope predicted from the BVDV E2 glycoprotein into FanC for the multivalent antigen FanC-STa-E2, and examined immunogenicity of this multivalent antigen to assess vaccine potential against bovine diarrhea. Mice intraperitoneally (i.p.) immunized with this multivalent antigen developed anti-K99, anti-STa, and anti-BVDV antibodies. Moreover, elicited antibodies showed neutralization activities, as they inhibited adherence of K99 fimbrialE. coli, neutralized STa toxin, and prevented homologous BVDV viral infectionin vitro. Results from this study suggest that this multiepitope fusion antigen can potentially be developed as a vaccine for broad protection against bovine diarrhea and that the multiepitope fusion strategy may be generally applied for multivalent vaccine development against heterogeneous pathogens.

scholarly journals Antimicrobial Activity against Intraosteoblastic Staphylococcus aureus

2015 ◽  
Vol 59 (4) ◽  
pp. 2029-2036 ◽  
Author(s):  
Florent Valour ◽  
Sophie Trouillet-Assant ◽  
Natacha Riffard ◽  
Jason Tasse ◽  
Sacha Flammier ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Ex Vivo ◽  
Joint Infection ◽  
Treatment Strategies ◽  
Bactericidal Effect ◽  
Infection Model ◽  
Content Type ◽  
Intracellular Activity ◽  
Mg63 Cells ◽  
Choice Of Treatment

ABSTRACTAlthoughStaphylococcus aureuspersistence in osteoblasts, partly as small-colony variants (SCVs), can contribute to bone and joint infection (BJI) relapses, the intracellular activity of antimicrobials is not currently considered in the choice of treatment strategies for BJI. Here, antistaphylococcal antimicrobials were evaluated for their intraosteoblastic activity and their impact on the intracellular emergence of SCVs in anex vivoosteoblast infection model. Osteoblastic MG63 cells were infected for 2 h with HG001S. aureus. After killing the remaining extracellular bacteria with lysostaphin, infected cells were incubated for 24 h with antimicrobials at the intraosseous concentrations reached with standard therapeutic doses. Intracellular bacteria and SCVs were then quantified by plating cell lysates. A bactericidal effect was observed with fosfomycin, linezolid, tigecycline, oxacillin, rifampin, ofloxacin, and clindamycin, with reductions in the intracellular inocula of −2.5, −3.1, −3.9, −4.2, −4.9, −4.9, and −5.2 log10CFU/100,000 cells, respectively (P< 10−4). Conversely, a bacteriostatic effect was observed with ceftaroline and teicoplanin, whereas vancomycin and daptomycin had no significant impact on intracellular bacterial growth. Ofloxacin, daptomycin, and vancomycin significantly limited intracellular SCV emergence. Overall, ofloxacin was the only molecule to combine an excellent intracellular activity while limiting the emergence of SCVs. These data provide a basis for refining the choice of antibiotics to prioritise in the management of BJI, justifying the combination of a fluoroquinolone for its intracellular activity with an anti-biofilm molecule, such as rifampin.

scholarly journals Clumping Factor B Promotes Adherence of Staphylococcus aureus to Corneocytes in Atopic Dermatitis

2017 ◽  
Vol 85 (6) ◽  
Author(s):  
Orla M. Fleury ◽  
Maeve A. McAleer ◽  
Cécile Feuillie ◽  
Cécile Formosa-Dague ◽  
Emily Sansevere ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Atopic Dermatitis ◽  
Clonal Complex ◽  
Ex Vivo ◽  
Nasal Carriage ◽  
Binding Activity ◽  
Clinical Strain ◽  
Healthy Children ◽  
Content Type ◽  
Force Microscopy

ABSTRACT Staphylococcus aureus skin infection is a frequent and recurrent problem in children with the common inflammatory skin disease atopic dermatitis (AD). S. aureus colonizes the skin of the majority of children with AD and exacerbates the disease. The first step during colonization and infection is bacterial adhesion to the cornified envelope of corneocytes in the outer layer, the stratum corneum. Corneocytes from AD skin are structurally different from corneocytes from normal healthy skin. The objective of this study was to identify bacterial proteins that promote the adherence of S. aureus to AD corneocytes. S. aureus strains from clonal complexes 1 and 8 were more frequently isolated from infected AD skin than from the nasal cavity of healthy children. AD strains had increased ClfB ligand binding activity compared to normal nasal carriage strains. Adherence of single S. aureus bacteria to corneocytes from AD patients ex vivo was studied using atomic force microscopy. Bacteria expressing ClfB recognized ligands distributed over the entire corneocyte surface. The ability of an isogenic ClfB-deficient mutant to adhere to AD corneocytes compared to that of its parent clonal complex 1 clinical strain was greatly reduced. ClfB from clonal complex 1 strains had a slightly higher binding affinity for its ligand than ClfB from strains from other clonal complexes. Our results provide new insights into the first step in the establishment of S. aureus colonization in AD patients. ClfB is a key adhesion molecule for the interaction of S. aureus with AD corneocytes and represents a target for intervention.

scholarly journals Nonredundant Roles of Interleukin-17A (IL-17A) and IL-22 in Murine Host Defense against Cutaneous and Hematogenous Infection Due to Methicillin-Resistant Staphylococcus aureus

2015 ◽  
Vol 83 (11) ◽  
pp. 4427-4437 ◽  
Author(s):  
Liana C. Chan ◽  
Siyang Chaili ◽  
Scott G. Filler ◽  
Kevin Barr ◽  
Huiyuan Wang ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
T Cell ◽  
Host Defense ◽  
Neutralizing Antibodies ◽  
Neutrophil Infiltration ◽  
Renal Tissue ◽  
Methicillin Resistant ◽  
Content Type ◽  
Interleukin 17A ◽  
Mrsa Infection

ABSTRACTStaphylococcus aureusis the leading cause of skin and skin structure infections (SSSI) in humans. Moreover, the high frequency of recurring SSSI due toS. aureus, particularly methicillin-resistantS. aureus(MRSA) strains, suggests that infection induces suboptimal anamnestic defenses. The present study addresses the hypothesis that interleukin-17A (IL-17A) and IL-22 play distinct roles in immunity to cutaneous and invasive MRSA infection in a mouse model of SSSI. Mice were treated with specific neutralizing antibodies against IL-17A and/or IL-22 and infected with MRSA, after which the severity of infection and host immune response were determined. Neutralization of either IL-17A or IL-22 reduced T cell and neutrophil infiltration and host defense peptide elaboration in lesions. These events corresponded with increased abscess severity, MRSA viability, and CFU density in skin. Interestingly, combined inhibition of IL-17A and IL-22 did not worsen abscesses but did increase gamma interferon (IFN-γ) expression at these sites. The inhibition of IL-22 led to a reduction in IL-17A expression, but not vice versa. These results suggest that the expression of IL-17A is at least partially dependent on IL-22 in this model. Inhibition of IL-17A but not IL-22 led to hematogenous dissemination to kidneys, which correlated with decreased T cell infiltration in renal tissue. Collectively, these findings indicate that IL-17A and IL-22 have complementary but nonredundant roles in host defense against cutaneous versus hematogenous infection. These insights may support targeted immune enhancement or other novel approaches to address the challenge of MRSA infection.

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