The SecA2 auxiliary secretion system of Gram(+) bacteria promotes the export of virulence proteins essential to colonize the host both in the case of Mycobacterium tuberculosis and Listeria monocytogenes, two intracellular bacteria causing diseases in humans. We and others demonstrated that this secretion system is also linked to the onset of long-term CD8(+) T cell-mediated protective immunity in mice. In the case of L. monocytogenes, expression of SecA2 inside the cytosol of infected cells correlates with the generation of CCL3-secreting memory CD8(+) T cells that are required for protection against secondary challenge with wild type (wt) L. monocytogenes. Since the SecA2 ATPase is well-conserved amongst Gram(+) pathogenic bacteria, we hypothesized that SecA2 itself bears evolutionary conserved motifs recognized by cytosolic pattern recognition receptors, leading to signaling events promoting the CCL3(+) memory CD8(+) T cells differentiation. To test this possibility, we generated a stable chromosomic L. monocytogenes mutant that expressed a SecA2 ATPase bearing mutated Nucleotide Binding Sites (NBS). Similarly to the deleted mutant of SecA2, the NBS mutant exhibited rough colonies and bacteria chaining phenotype, impaired protein secretion profile and in vivo virulence in comparison to wt L. monocytogenes. Importantly, mice immunized with the NBS-SecA2 mutant were not protected against secondary infection with wt L. monocytogenes and did not develop CCL3(+) memory CD8 T cells. NBS- and wt- SecA2 proteins were expressed to comparable extent by bacteria, suggesting that SecA2 itself is unlikely to promote the induction of these cells. Rather, one or several of the SecA2 substrate protein(s) released inside the cytosol of infected cells may be involved.