These findings support the conclusion that escape from the double-membrane vacuole during secondary infection of cells is necessary for complete NLRP6 activation by Listeria. It is however important to note that ActA and PC-PLC deficiency did not reduce NLRP6 activation to the level elicited by the Listeriolysin O-deficient mutant, which indicates that while escape from the secondary vacuole is the main driver of NLRP6 activation, another event, for example, primary vacuolar escape, could still contribute to NLRP6 activation (Fig. 5H). This evidence concerns the gene ACTA1 and infection.