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Updated: Feb 18, 2026
Kinetic Screening of Nuclease Activity using Nucleic Acid Probes
Published on: November 1, 2019
Streptococcus pyogenes nuclease A (SpnA) mediated virulence does not exclusively depend on nuclease activity
Callum Chalmers1, Adrina Hema J Khemlani1, Chae Ryeong Sohn1
1Department of Molecular Medicine & Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand.
Background:
Streptococcus pyogenes, or Group A Streptococcus (GAS), is a human pathogen that causes a wide range of diseases, including pharyngitis, necrotizing fasciitis and toxic shock syndrome. The bacterium produces a large arsenal of virulence factors, including the cell wall-anchored Streptococcus pyogenes nuclease A (SpnA), which facilitates immune evasion by degrading the DNA backbone of neutrophil extracellular traps. SpnA consists of a C-terminal endo/exonuclease domain and a N-terminal domain of unknown function.
Methods:
Recombinant SpnA mutants were generated by alanine conversion of selected residues that were predicted to play a role in the enzymatic activity and tested for their ability to degrade DNA. A GAS spnA deletion mutant was complemented with a plasmid-borne catalytic site mutant and analyzed for virulence in a Galleria mellonella (wax moth) infection model.
Results:
Several predicted residues were experimentally confirmed to play a role in SpnA enzymatic activity. These include Glu592, Arg696, His716, Asp767, Asn769, Asp810 and Asp842. Complementation of a GAS spnA deletion mutant with a spnA H716A mutant gene partially restored virulence in wax moth larvae, whereas complementation with the spnA wt gene completely restored activity. Furthermore, complementation with a secreted form of SpnA showed reduced virulence.
Conclusion:
Our results show that abolishing the enzymatic activity of SpnA only partially reduces virulence suggesting that SpnA has an additional virulence function, which might be located on the N-terminal domain. Furthermore, cell wall-anchoring of SpnA results in higher virulence compared to secreted SpnA, probably due to a higher local density of the enzyme.
Insights
Streptococcus pyogenes nuclease A (SpnA) enzymatic activity is crucial but not solely responsible for virulence. Cell wall anchoring of SpnA enhances its virulence, suggesting additional functions.
Area of Science:
- Microbiology
- Molecular Biology
- Pathogenesis
Background:
- Streptococcus pyogenes (Group A Streptococcus) is a significant human pathogen.
- It causes various diseases, including pharyngitis and necrotizing fasciitis.
- Streptococcus pyogenes nuclease A (SpnA) is a key virulence factor aiding immune evasion by degrading neutrophil extracellular traps.
Purpose of the Study:
- To investigate the role of SpnA enzymatic activity in Streptococcus pyogenes virulence.
- To identify key residues essential for SpnA's nuclease function.
- To explore potential non-enzymatic virulence functions of SpnA.
Main Methods:
- Site-directed mutagenesis was used to generate SpnA mutants.
- Enzymatic activity of SpnA mutants was assessed by DNA degradation assays.
- Virulence was evaluated using a Galleria mellonella infection model with complemented GAS mutants.
Main Results:
- Specific residues (Glu592, Arg696, His716, Asp767, Asn769, Asp810, Asp842) were confirmed to be critical for SpnA enzymatic activity.
- Ablating enzymatic activity (spnA H716A mutant) partially restored virulence in wax moth larvae.
- Cell wall-anchored SpnA exhibited higher virulence than secreted SpnA.
Conclusions:
- SpnA possesses virulence functions beyond its nuclease activity, potentially involving the N-terminal domain.
- Cell wall anchoring of SpnA contributes to increased virulence, likely due to localized enzyme concentration.
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