Cytosolic expression of SecA2 is a prerequisite for long-term protective immunity

Abstract

SummaryInduction of efficient adaptive T cell-mediated immunity against the intracellular bacterium Listeria monocytogenes requires its successful invasion of host cell cytosol. However, it is not clear whether its cytosolic escape and growth are sufficient to induce T cell-mediated clearance and protection upon secondary infection. To investigate this issue, we have searched for mutants that do not induce long-term protective immunity yet invade the cytosol of infected cells. We found that mice immunized with L. monocytogenes lacking the SecA2 ATPase, an auxiliary protein secretion system present in several Gram-positive pathogenic bacteria, mounted a robust cytolytic IFN-g-secreting CD8+ T cell response but were not protected against a secondary challenge with wild-type (wt) bacteria. Furthermore, CD8 + T cells from mice immunized with secA2 -bacteria failed to transfer protection when injected into recipient mice demonstrating that they were unable to confer protection. Also, secA2 -and wt L. monocytogenes spread to the same myeloid-derived cell types in vivo and SecA2 deficiency does not interfere with intracytosolic bacteria multiplication. Therefore, cytosol invasion is not sufficient for inducing secondary protective responses and induction of memory CD8 + T cells mediating long-term antibacterial protective immunity is dependent upon SecA2 expression inside the cytosol of host cells in vivo.

Priming of Protective Anti-Listeria monocytogenes Memory CD8+T Cells Requires a Functional SecA2 Secretion System

“…While some L. monocytogenes-derived proteins exhibited equal intensities in the supernatants from all bacteria, many other proteins detected in the culture supernatant of wt bacteria were reduced in NBS-and in Del-SecA2 bacteria to comparable extents and as previously described (24). Next, we tested whether the NBS-SecA2 mutant of L. monocytogenes was also impaired in its virulence in vivo, as described for the Del-SecA2 mutant (24,31). We found that the mutants exhibit comparable LD 50 s in mice (10 7 bacteria) (data not shown), a result in agreement with previously published reports on Del-SecA2 L. monocytogenes by us and others.…”

“…Six-to 8-week-old BALB/cByJ female mice (Janvier Laboratories) were used in all experiments. This study was carried out in strict accordance with the recommendations in the Guide for the Care , respectively, in BALB/c mice (24,31). All wt bacteria were prepared from clones grown from organs of infected mice.…”

“…However, these cells were suboptimally primed and did not differentiate into protective (CCL3-secreting) memory cells, which are required to confer protection against a secondary infection with wt L. monocytogenes (31,32). Interestingly, data from Porcelli's laboratory have also linked the presence of SecA2 from the intracellular bacterium M. tuberculosis to the onset of CD8 ϩ T cell-mediated immunity (16,32).…”

“…Recently, using L. monocytogenes deficient in SecA2, we further found that mice immunized with this mutant formed memory T cell populations equivalent to those of mice that received wt bacteria (31,32). However, these cells were suboptimally primed and did not differentiate into protective (CCL3-secreting) memory cells, which are required to confer protection against a secondary infection with wt L. monocytogenes (31,32).…”

“…In the case of M. tuberculosis, SecA2 deficiency affects the ability of this bacterium to secrete superoxide dismutase A (SodA), a critical enzyme involved in M. tuberculosis escape and survival inside infected host cells by eluding the oxidative attack of the macrophages (21). In the case of L. monocytogenes, at least 19 proteins are secreted through this auxiliary secretory pathway (2,10,24,25); among them, the L. monocytogenes murein hydrolases p60 and NamA are the two major SecA2 substrate proteins and account for the highly attenuated virulence in vivo of secA2 deletion mutant L. monocytogenes compared to the wild-type (wt) bacteria (ϳ500-fold) (24)(25)(26)31). Both hydrolases can degrade L. monocytogenes PGN during growth, and the observed attenuated virulence most likely results from the impaired release of NLR ligands inside the cytosol of infected host cells, which subverts host pattern recognition.…”