The Role of Sortase A in the Maintenance of Cell Surface Properties and Cell Hardiness in Streptococcus pyogenes AP53

Sortases play important roles in covalently anchoring bacterial virulence factors to the cell wall. Sortase A (srtA), the most ubiquitous sortase in Gram-positive bacteria, is regarded as ‘the housekeeping sortase’ due to its role in anchoring many proteins involved in adhesion, immune invasion, internalization, and phage recognition. The activity of SrtA is specific to cell surface proteins with a C-terminal LPXTG-motif followed by a hydrophobic region and a positively charged tail. Many of the cell surface virulence factors in Group A Streptococcus pyogenes (GAS), including Plasminogen-binding M-protein (PAM), have a LPXTG-motif and are anchored to the cell wall by srtA. While the role of srtA in the presentation of virulence factors on the cell wall has been recognized, the ability of these virulence factors to present themselves and maintain function without srtA has not been well studied. The goal of this research is to investigate how LPXTG-motif surface proteins anchor and present themselves without srtA, what effects this has on their function, and what subsequent effects these changes have on cell hardiness and virulence.

A gene deletion mutant for srtA was made in GAS strain AP53 and compared with the parent strain for the investigations described. The cell wall and cell membrane were isolated via phage lysin C (plyC) and centrifugation and examined for PAM content by Western blotting. Whole cell ELISAs were also employed to investigate the presence and presentation of proteins on the cell surface. The results obtained from these assays demonstrate in the ΔsrtA mutant the retention of LPXTG-motif proteins in the cell membrane and that the presentation of these proteins to the environment remains unaltered. The functionality of these proteins was investigated via the ability of the cells to bind and activate human plasminogen (hPg). In the absence of srtA, the cells were found to retain their ability to bind hPg via PAM and activate hPg in the presence of the secreted virulence factor streptokinase. The viability and environmental hardiness of the cells were investigated via growth assays to observe their ability to tolerate high salt, antibiotics, and neutrophil killing and their ability to form adherent biofilms via crystal violet staining. The cells without srtA were not significantly different in their ability to grow under favorable conditions, but they were found to have increased susceptibilities to high salt and antibiotics, indicating a disruption in their membrane integrity. The ΔsrtA mutant was also found to have a reduced survivability against direct neutrophil exposure and a reduced ability to form adherent biofilms, indicating that covalent anchoring of LPXTG-motif proteins is necessary for full functionality in neutrophil evasion and cell adherence. Overall, the results of this study suggest the existence of an alternative mechanism for cell surface protein expression and the necessity for srtA activity for bacterial viability and hardiness.