Figure 1: Kaplan-Meier survival curves of wax worms infected with labelled dose (CFUs) of GAS SpnA variants over 3 days **** P<0.05 (One-way ANOVA)
A methyl green assay allows quantification of DNAse activity by measuring the change in absorbance as the methyl green-DNA complex is degraded (Sinicropi et al. 1994). The SpnA knockout had significantly lower DNAse function than wildtype GAS whilst the non-functional complements showed similar activity to the knockouts and the functional complements showed an intermediate function in between that of the knockout and that of the wildtype (Fig. 5) All strains showed higher activity than buffer alone (Fig. 5).
Previous experiments have characterised the role of SpnA as a cell wall anchored DNAse important for immune evasion through the destruction of neutrophil extracellular traps (NETs) (Buchanan et al. 2006). While small scale in vivo experiments in mice have been conducted (Hasegawa et al. 2010), a Galleria mellonella infection model allows for a larger scale screening of mutants to further refine the role of SpnA in GAS virulence. This is due to the lower cost, easy maintenance, small size and suitability of the wax worm (Ramarao et al. 2012).
In the above experiments, the role of SpnA in GAS virulence was clearly demonstrated by the significant difference in killing and the health index of the caterpillars between wild type and ΔSpnA. They also revealed that complementation with cell wall bound SpnA on a plasmid