Characterization of recombinant wild-type and nontoxigenic protein A from Staphylococcus pseudintermedius

Staphylococcus pseudintermedius is an opportunistic pathogen that is the major cause of pyoderma affecting dogs. Conventional antimicrobial treatment for infections caused by this organism have failed in recent years due to widespread resistance and alternative treatment strategies are a high priority. Protein A encoded in Staphylococcus aureus by spa protects the bacterium by binding IgG and acts as a superantigen. Staphylococcus pseudintermedius possess two genes orthologous to S. aureus spa, spsP, and spsQ.

SpsQ and SpsQ-M, a non-toxigenic SpsQ, were cloned and expressed as recombinant proteins and their cytotoxic effect on canine B cells was measured. The neutralizing ability of antibody raised against them in clinically healthy dogs was evaluated.

S. pseudintermedius SpsQ induced apoptosis of canine B cells. Specific amino acid substitutions diminished SpsQ-M binding to immunoglobulin and its super-antigenic activity, while its antigenicity was maintained. This recombinant, non-toxigenic S. pseudintermedius SpsQ stimulated the production of antibodies in dogs that specifically reacted with SpsQ and greatly diminished its cytotoxic effect on canine B cells.

The production of neutralizing antibody suggests that attenuated, non-toxic SpsQ produced in this study is a good candidate for inclusion in a vaccine for use in the treatment and prevention of S. pseudintermedius infections.

SpA: Staphylococcus aureus protein A; SpsP: Staphylococcus pseudintermedius protein A; SpsQ: Staphylococcus pseudintermedius protein A; SpsQ-M: attenuated Staphylococcus pseudintermedius protein A; MRSP: methicillin resistant Staphylococcus pseudintermedius; IgA: immunoglobulin A; IgG: immunoglobulin G; IgM: immunoglobulin M; VH: variable region of immunoglobulin heavy chain; IgBD: immunoglobulin binding domains; MFI: mean fluorescent intensity; SEM: standard error of the mean; PBMC: Peripheral blood mononuclear cells; CD21: complement receptor type 2; ST: Sequence type; OD: Optical density; ORF: open reading frame; PBS: Phosphate buffered saline; Tween 20: Polyethylene glycol sorbitan monolaurate 20; HRP: horseradish peroxidase; TMB- 3,3',5,5'-Tetramethylbenzidine.