Functional analysis of the N-terminal domain of Streptococcus pyogenes nuclease A (SpnA) in Group A Streptococcus

Abstract

Streptococcus pyogenes, also known as Group A Streptococcus (GAS) is an obligate human pathogen that is responsible for a spectrum of diseases. The virulence of this pathogen is largely dependent on the array of virulence factors that it expresses. Immune evasion is an important aspect for establishing a successful infection. GAS is known to express four types of DNases that degrade neutrophil extracellular traps (NETs), thereby facilitating the pathogen’s escape from the innate immune system. SpnA is a novel streptococcal protein that was only recently characterised. SpnA is the only cell wall-anchored DNase in GAS and has been shown by a previous study to be protective of GAS and a Lactococcus lactis gain-of-function mutant in human whole blood. However, a GAS mutant that expressed a nuclease-inactive SpnA (H716A) displayed similar survival in whole blood as the wild type GAS. Furthermore, preliminary studies also showed binding of SpnA to white blood cells subpopulations. Due to these observations, SpnA was hypothesised to have additional functions to its nuclease activity possibly contributed by the N-terminal domain of SpnA. The purpose of this project was to analyse the functional properties of the N-terminal domain. A series of recombinant SpnA proteins were produced in Escherichia coli and used in blood killing assays to determine if the rSpnA C- and N-terminal domain proteins were protective for L. lactis. Results from the whole blood killing assays were inconclusive due to experimental complications. In response to this, a L. lactis gain-of-function mutant that heterologously expresses the SpnA N-terminal domain was generated and would be used in future blood killing assays as a better representative to study the function of the SpnA Nterminal domain. In addition, the rSpnA proteins were also used in flow cytometry with white blood cells to assess if binding occurred. The rSpnA C-terminal domain was found to bind to granulocytes and monocytes, while the N-terminal domain was shown to mainly bind to monocytes. This indicated the binding of SpnA to granulocytes and monocytes may serve as another attribute to GAS virulence.