Expression and function of protein A in Staphylococcus pseudintermedius

Abstract

Staphylococcus pseudintermedius is an opportunistic pathogen in dogs and the most frequent cause of canine pyoderma. Protein A, a potent virulence factor in S. aureus is encoded by the spa gene. S. pseudintermedius possesses genes seemingly analogous to spa, but the expression and the characteristics of their products have not been directly determined. The purpose of this study was to test isolates from major clonal groups for the presence of spa gene orthologs, quantitate their expression levels, and to characterize protein A in S. pseudintermedius. From the data, it was observed that S. pseudintermedius isolates express genes analogous to spa in S. aureus. Isolates representing major clonal populations in the United States and Europe, ST68 and ST71 respectively, bound significantly higher amounts of canine IgG than isolates with other genetic backgrounds, suggesting that these isolates have a higher density of protein A on their surface. Also, canine IgG bound to protein A on S. pseudintermedius via its Fc region similar to protein A from S. aureus. The mRNA profile differed based on the bacterial sequence types and correlated to the density of protein A on the bacterial surface. Protein A was also found to be secreted during the exponential growth phase. Phagocytosis experiments with S. pseudintermedius show that blocking of protein A enhanced phagocytosis in whole blood, neutrophils and in DH82 canine macrophage-like cell line. Taken together, the results demonstrate that S. pseudintermedius produces protein A that shares S. aureus protein A's ability to bind the Fc region of immunoglobulins and may serve as a potential virulence factor by evading the host immune system.

Keywords: Fc-binding; Protein A; Staphylococcus pseudintermedius; immune evasion; vaccine; virulence.

Figure 1.

Expression levels of spsQ in S. pseudintermedius. qPCR was performed to measure the amount of spsQ mRNA in log phase bacterial cells. Relative gene expression was calculated using the 2^−ΔΔCT method and expressed as fold change. 16S rRNA was used as the endogenous control. The values represent average from three independent experiments. (*P < 0.05 was considered significant). ^#spsQ mRNA levels in isolates NA12, NA45, KM241, 08–1791, 08–1721, 07–1447 and 57395 were below the detection limits of the assay.

Figure 2.

Binding of canine IgG to protein A on S. pseudintermedius (dose-response). A dose-response effect observed when varying concentrations of canine IgG ranging from 10 μg/ml-1000 μg/ml was reacted with S. pseudintermedius. The values represent average from three independent experiments. (P < 0.05 was considered significant). Data for isolates representing ST 68 (06-3228), ST 71 (08-1661) and ST 84 (NA45) have been presented.

Figure 3.

Canine IgG recognizes and binds to protein A on S. pseudintermedius. Isolates representing various sequence types of S. pseudintermedius were reacted with 100μg/ml of canine IgG. The amount of binding was determined by flow cytometry and expressed as percentage bound relative to the positive control S. aureus Cowan 1 strain. The values represent average from three independent experiments. (*P < 0.05 was considered significant).

Figure 4.

Protein A in S. pseudintermedius binds to canine IgG primarily via its Fc region. S. pseudintermedius isolates were treated with IgG and its papain digestion fragments, Fab and Fc. The amount of binding was determined by flow cytometry. The values represent average from three independent experiments. (*P < 0.05 was considered significant). Data for isolates representing ST 68 (06-3228), ST 71 (08-1661) and ST 84 (NA45) are presented.

Figure 5.

Protein A on the surface of S. pseudintermedius. The density of cell wall-associated protein A in various strains of S. pseudintermedius was measured using FITC-conjugated anti-protein A antibody by flow cytometry. The values represent the average from three independent experiments. (*P < 0.05 was considered significant).

Figure 6.

Binding of anti-protein A antibody prevents the binding of canine Fc to protein A on S. pseudintermedius. Bacterial isolates were incubated with an excess of chicken anti-protein A antibody prior to the addition of canine Fc. There was at least 50% reduction in the binding of canine Fc because of anti-protein A antibody binding. The values represent average from three independent experiments. (*P < 0.05 was considered significant). Data for isolates representing ST 68 (06-3228), ST 71 (08-1661) and ST 84 (NA45) are presented.

Figure 7.

Production of extracellular protein A in S. pseudintermedius. The amount of extracellular protein A was measured using an antigen-capture ELISA. Commercially available protein A from S. aureus was used to generate the standard curve. The values represent the average from three independent experiments. (*P < 0.05 was considered significant).

Figure 8.

Phagocytosis of S. pseudintermedius. The involvement of protein A in immune evasion was demonstrated by phagocytosis using pHrodo™ Red-labeled 08–1661 in whole blood, neutrophils and DH82 cells. Pre-incubation of bacteria with anti-protein A antibody enhanced phagocytosis (red). The values represent average from three independent experiments. (*P < 0.05 was considered significant). NS – not significant.