Functional and Structural Characterisation of Streptococcus pyogenes Nuclease A (SpnA)

Abstract

Streptococcus pyogenes, or Group A streptococcus (GAS) is a major human pathogen that is responsible for a wide range of diseases. GAS produces a large arsenal of virulence factors that interferes with the host immune system. Streptococcus pyogenes nuclease A (SpnA) is a recently discovered DNase that plays a role in virulence as shown in a mouse infection model. SpnA is the only cell wall-anchored DNase identified in GAS thus far. The 910-amino acid protein is predicted to contain a N-terminal signal peptide that is followed by three oligonucleotide binding (OB) domains. A predicted exo/endonuclease nuclease is located in the C-terminus with a conserved Gram-positive sortase cell wall anchoring motif, LPKTG, and a cell wall spanning domain. The C-terminal nuclease domain alone is enzymatically inactive. SpnA requires at least two out of the three OB-domains in the N-terminus to be nuclease active. The N-terminal OB-domains are involved in DNA binding as a large amount of DNA is found bound to the peptide. Using a combination of a spnA-knockout mutant and a Lactococcus lactis gain-of-function mutant, this study has shown that SpnA promotes bacterial survival in human whole blood and in neutrophil killing assays. The protective effect of SpnA on bacterial cells against killing by blood and NETs is, at least partially, due to its destructive nuclease activity on NETs. SpnA expression is found on the cell surface of GAS as well as in the culture supernatant. Heterologous expression of secreted SpnA in L. lactis reveals that the protein is able to protect bacterial cell against killing by blood in both secreted and cell surface anchored forms. A zebrafish embryo infection model is able to present a significant difference between the virulence between GAS wildtype and the spnA-knockout. Structural prediction of SpnA’s nuclease domain based on the structure of bovine pancreatic DNase I (BP DNase I) reveals several conserved catalytic activity/Mg2+ binding/substrate-binding sites between the two proteins. Site directed alanine mutagesis at these conserved functional residues in SpnA can significantly reduce the nucleolytic activities of the protein. One of the nuclease inactive mutants, SpnA_H716A, appears to inhibit bacterial killing by blood, suggesting SpnA may possess mechanisms other than the nuclease activity that contribute to the overall virulence of GAS. With the results from this study, we have a more comprehensive understanding in the complex interaction between GAS and its human host. Characterising these pathogen-host interactions may assist in the identification of novel therapeutic targets against GAS infection.