scholarly journals Nicotiana spp. for the Expression and Purification of Functional IgG3 Antibodies Directed Against the Staphylococcus aureus Alpha Toxin

2021 ◽  
Vol 3 ◽  
Author(s):  
P. Opdensteinen ◽  
S. Meyer ◽  
J. F. Buyel
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Capacity ◽  
Protein A ◽  
Adaptive Immune Response ◽  
Hybridoma Cells ◽  
Alpha Toxin ◽  
In Planta ◽  
Affinity Ligand ◽  
Igg3 Subclass ◽  
Drug Resistant Strains

Immunoglobulin subclass IgG1 is bound and neutralized effectively by Staphylococcus aureus protein A, allowing the bacterium to evade the host’s adaptive immune response. In contrast, the IgG3 subclass is not bound by protein A and can be used to treat S. aureus infections, including drug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA). However, the yields of recombinant IgG3 are generally low because this subclass is prone to degradation, and recovery is hindered by the inability to use protein A as an affinity ligand for antibody purification. Here, we investigated plants (Nicotiana spp.) as an alternative to microbes and mammalian cell cultures for the production of an IgG3 antibody specific for the S. aureus alpha toxin. We targeted recombinant IgG3 to different subcellular compartments and tested different chromatography conditions to improve recovery and purification. Finally, we tested the antigen-binding capacity of the purified antibodies. The highest IgG3 levels in planta (>130 mg kg−1 wet biomass) were achieved by targeting the endoplasmic reticulum or apoplast. Although the purity of IgG3 exceeded 95% following protein G chromatography, product recovery requires further improvement. Importantly, the binding affinity of the purified antibodies was in the nanomolar range and thus comparable to previous studies using murine hybridoma cells as the production system.

scholarly journals Corneal virulence of Staphylococcus aureus: roles of alpha-toxin and protein A in pathogenesis.

1994 ◽  
Vol 62 (6) ◽  
pp. 2478-2482 ◽  
Author(s):  
M C Callegan ◽  
L S Engel ◽  
J M Hill ◽  
R J O'Callaghan
Keyword(s):  
Staphylococcus Aureus ◽  
Protein A ◽  
Alpha Toxin

scholarly journals mgr, a Novel Global Regulator in Staphylococcus aureus

2003 ◽  
Vol 185 (13) ◽  
pp. 3703-3710 ◽  
Author(s):  
Thanh T. Luong ◽  
Steven W. Newell ◽  
Chia Y. Lee
Keyword(s):  
Staphylococcus Aureus ◽  
Capsular Polysaccharide ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Binding Motif ◽  
Transcriptional Level ◽  
Virulence Determinants ◽  
Alpha Toxin ◽  
Extracellular Protein Production

ABSTRACT The virulence determinants of Staphylococcus aureus are coordinately controlled by several unlinked chromosomal loci. Here, we report the identification of CYL5614, derived from strain Becker, with a mutation that affects the expression of type 8 capsular polysaccharide (CP8), nuclease, alpha-toxin, coagulase, protease, and protein A. This novel locus, named mgr, was linked by transposon Tn917 and mapped by three-factorial transduction crosses. The region containing the mgr locus was cloned and sequenced. Deletion mutagenesis and genetic complementation showed that the locus consisted of one gene, mgrA. Interestingly, mgrA-null mutants exhibited a phenotype opposite to that of CYL5614. This was due to a T-to-C mutation upstream of mgrA that resulted in a four- to eightfold increase in mgrA transcription in strain CYL5614. Thus, these results indicate that mgrA is an activator of CP8 and nuclease but a repressor of alpha-toxin, coagulase, protease, and protein A. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that the mgr locus profoundly affected extracellular protein production, suggesting that the locus may regulate many other genes as well. The translated MgrA protein has a region of significant homology, which includes the helix-turn-helix DNA-binding motif, with the Escherichia coli MarR family of transcriptional regulators. Northern slot blot analyses suggested that mgr affected CP8, alpha-toxin, nuclease, and protein A at the transcriptional level.

scholarly journals Staphylococcus aureus Protein A Promotes Immune Suppression

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Scott D. Kobayashi ◽  
Frank R. DeLeo
Keyword(s):  
Staphylococcus Aureus ◽  
B Cell ◽  
Immune Evasion ◽  
Bacterial Infections ◽  
Binding Capacity ◽  
Protein A ◽  
Strong Support ◽  
The United States ◽  
Content Type

ABSTRACTStaphylococcus aureusis a prominent cause of human infections worldwide and is notorious for its ability to acquire resistance to antibiotics. Methicillin-resistantS. aureus(MRSA), in particular, is endemic in hospitals and is the most frequent cause of community-associated bacterial infections in the United States. Inasmuch as treatment options for severe MRSA infections are limited, there is need for a vaccine that protects against such infections. However, recent efforts to generate a staphylococcal vaccine have met with little success in human clinical trials. These failures are somewhat puzzling, since the vaccine antigens tested promote opsonophagocytosisin vitroand confer protection in animal infection models. One possibility is that the pathogen inhibits (and/or fails to elicit) the development of protective immunity in humans. Indeed,S. aureusproduces numerous molecules that can potentially promote immune evasion, including protein A (SpA), an immunoglobulin (Ig)-binding protein present on the bacterial surface and freely secreted into the extracellular environment. SpA binds the Fc region of antibody and the Fab regions of the B-cell receptor, processes that are known to block opsonophagocytosis and cause B-cell deathin vitro. In a recent study, Falugi et al. [F. Falugi, H. K. Kim, D. M. Missiakas, and O. Schneewind, mBio 4(5):e00575-13, 2013] showed that vaccination withspamutantS. aureusstrains lacking antibody Fc- and/or Fab-binding capacity protects against subsequent challenge with the USA300 epidemic strain. The findings provide strong support for the idea that SpA promotesS. aureusimmune evasionin vivoand form the foundation for a new approach in our efforts to develop a vaccine that prevents severeS. aureusinfections.

scholarly journals Staphylococcal Surface Display of Immunoglobulin A (IgA)- and IgE-Specific In Vitro-Selected Binding Proteins (Affibodies) Based on Staphylococcus aureus Protein A

1999 ◽  
Vol 65 (9) ◽  
pp. 4134-4140 ◽  
Author(s):  
Elin Gunneriusson ◽  
Patrik Samuelson ◽  
Jenny Ringdahl ◽  
Hans Grönlund ◽  
Per-Åke Nygren ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Surface ◽  
Binding Capacity ◽  
Expression System ◽  
Surface Display ◽  
Protein A ◽  
Surface Proteins ◽  
Phage Library ◽  
Protein Chemistry ◽  
Ige Binding

ABSTRACT An expression system designed for cell surface display of hybrid proteins on Staphylococcus carnosus has been evaluated for the display of Staphylococcus aureus protein A (SpA) domains, normally binding to immunoglobulin G (IgG) Fc but here engineered by combinatorial protein chemistry to yield SpA domains, denoted affibodies, with new binding specificities. Such affibodies, with human IgA or IgE binding activity, have previously been selected from a phage library, based on an SpA domain. In this study, these affibodies have been genetically introduced in monomeric or dimeric forms into chimeric proteins expressed on the surface of S. carnosus by using translocation signals from aStaphylococcus hyicus lipase construct together with surface-anchoring regions of SpA. The recombinant surface proteins, containing the IgA- or IgE-specific affibodies, were demonstrated to be expressed as full-length proteins, localized and properly exposed at the cell surface of S. carnosus. Furthermore, these chimeric receptors were found to be functional, since recombinantS. carnosus cells were shown to have gained IgA and IgE binding capacity, respectively. In addition, a positive effect in terms of IgA and IgE reactivity was observed when dimeric versions of the affibodies were present. Potential applications for recombinant bacteria with redirected binding specificity in their surface proteins are discussed.

scholarly journals Staphylococcus aureus vaccines: Deviating from the carol

2016 ◽  
Vol 213 (9) ◽  
pp. 1645-1653 ◽  
Author(s):  
Dominique Missiakas ◽  
Olaf Schneewind
Keyword(s):  
Staphylococcus Aureus ◽  
Protein A ◽  
Invasive Disease ◽  
Phagocytic Cells ◽  
Subunit Vaccines ◽  
Clinical Endpoints ◽  
Patients At Risk ◽  
Resistant Strains ◽  
Opsonophagocytic Killing ◽  
Drug Resistant Strains

Staphylococcus aureus, a commensal of the human nasopharynx and skin, also causes invasive disease, most frequently skin and soft tissue infections. Invasive disease caused by drug-resistant strains, designated MRSA (methicillin-resistant S. aureus), is associated with failure of antibiotic therapy and elevated mortality. Here we review polysaccharide-conjugate and subunit vaccines that were designed to prevent S. aureus infection in patients at risk of bacteremia or surgical wound infection but failed to reach their clinical endpoints. We also discuss vaccines with ongoing trials for combinations of polysaccharide-conjugates and subunits. S. aureus colonization and invasive disease are not associated with the development of protective immune responses, which is attributable to a large spectrum of immune evasion factors. Two evasive strategies, assembly of protective fibrin shields via coagulases and protein A–mediated B cell superantigen activity, are discussed as possible vaccine targets. Although correlates for protective immunity are not yet known, opsonophagocytic killing of staphylococci by phagocytic cells offers opportunities to establish such criteria.

scholarly journals Impacts of sarA and agr in Staphylococcus aureus Strain Newman on Fibronectin-Binding Protein A Gene Expression and Fibronectin Adherence Capacity In Vitro and in Experimental Infective Endocarditis

2004 ◽  
Vol 72 (3) ◽  
pp. 1832-1836 ◽  
Author(s):  
Yan-Qiong Xiong ◽  
Arnold S. Bayer ◽  
Michael R. Yeaman ◽  
Willem van Wamel ◽  
Adhar C. Manna ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Capacity ◽  
Protein A ◽  
Aureus Strain ◽  
Global Regulators ◽  
Fibronectin Binding Protein ◽  
Regulatory Loci ◽  
Fibronectin Binding

ABSTRACT We investigated the impacts of sarA and agr on fnbA expression and fibronectin-binding capacity in Staphylococcus aureus in vitro and in experimental endocarditis. Although sarA up-regulated and agr down-regulated both fnbA expression and fibronectin binding in vitro and in vivo, fnbA expression was positively regulated in the absence of both global regulators. Thus, additional regulatory loci contribute to fnbA regulation and fibronectin-binding capacities in S. aureus.

scholarly journals The first report on the sortase-mediated display of bioactive protein A from Staphylococcus aureus (SpA) on the surface of the vegetative form of Bacillus subtilis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Samira Ghaedmohammadi ◽  
Gholamreza Ahmadian
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Bacillus Subtilis ◽  
Binding Capacity ◽  
Surface Display ◽  
Protein A ◽  
Production Costs ◽  
Rabbit Serum ◽  
Experimental Conditions ◽  
Binding Domains

AbstractProtein A (SpA) is one of the most important Staphylococcus aureus cell wall proteins. It includes five immunoglobulin (Ig)-binding domains which can bind to immune complexes through the Fc region of immunoglobulins. The binding of SpA to the polymeric supports can be used to prepare affinity chromatography resins, which are useful for immunoprecipitation (IP) of antibodies. Protein A is also used to purify many anti-cancer antibodies. In this study, SpA was displayed on the surface of Bacillus subtilis cells using a sortase-mediated system to display the target protein to the B. subtilis cell wall. A series of plasmids consisting of cassettes for cell wall-directed protein A as well as negative controls were constructed and transformed into B. subtilis WASD (wprA sigD) cells. SDS-PAGE, western blot, flow cytometry, functional IgG purification assay, and a modified ELISA assay were used to confirm the surface display of SpA and evaluate its function. Semi-quantitative ELISA results showed that the binding capacity of lyophilized Bs-SpA is 100 μg IgG from rabbit serum per 1 mg of cells under optimal experimental conditions. Low production costs, optimal performance, and the use of a harmless strain compared to a similar commercial product predict the possible use of SpA immobilization technology in the future.

scholarly journals Regulation of agr-Dependent Virulence Genes in Staphylococcus aureus by RNAIII from Coagulase-Negative Staphylococci

1998 ◽  
Vol 180 (12) ◽  
pp. 3181-3186 ◽  
Author(s):  
Karin Tegmark ◽  
Eva Morfeldt ◽  
Staffan Arvidson
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Surface ◽  
Serine Protease ◽  
Protein A ◽  
Surface Proteins ◽  
Open Reading Frames ◽  
Alpha Toxin ◽  
Coagulase Negative Staphylococci ◽  
Cell Surface Proteins ◽  
Genes Encoding

ABSTRACT Many of the genes coding for extracellular toxins, enzymes, and cell surface proteins in Staphylococcus aureus are regulated by a 510-nucleotide (nt) RNA molecule, RNAIII. Transcription of genes encoding secreted toxins and enzymes, includinghla (alpha-toxin), saeB (enterotoxin B),tst (toxic shock syndrome toxin 1), and ssp(serine protease), is stimulated, while transcription of genes encoding cell surface proteins, like spa (protein A) andfnb (fibronectin binding proteins), is repressed. Besides being a regulator, RNAIII is also an mRNA coding for staphylococcal delta-lysin. We have identified RNAIII homologs in three different coagulase-negative staphylococci (CoNS), i.e., Staphylococcus epidermidis, Staphylococcus simulans, andStaphylococcus warneri. RNAIII from these CoNS turned out to be very similar to that of S. aureus and contained open reading frames encoding delta-lysin homologs. Though a number of big insertions and/or deletions have occurred, mainly in the 5′ half of the molecules, the sequences show a high degree of identity, especially in the first 50 and last 150 nt. The CoNS RNAIII had the ability to completely repress transcription of protein A in an RNAIII-deficientS. aureus mutant and the ability to stimulate transcription of the alpha-toxin and serine protease genes. However, the stimulatory effect was impaired compared to that of S. aureus RNAIII, suggesting that these regulatory functions are independent. By creating S. epidermidis-S. aureus RNAIII hybrids, we could also show that both the 5′ and 3′ halves of the RNAIII molecule are involved in the transcriptional regulation of alpha-toxin and serine protease mRNAs in S. aureus.

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