Priming of protective anti-Listeria monocytogenes memory CD8+ T cells requires a functional SecA2 secretion system

Abstract

The SecA2 auxiliary secretion system of Gram-positive bacteria promotes the export of virulence proteins essential for colonization of the host in the case of both Mycobacterium tuberculosis and Listeria monocytogenes, two intracellular bacteria causing diseases in humans. We and others have 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 among Gram-positive pathogenic bacteria, we hypothesized that SecA2 itself bears evolutionarily conserved motifs recognized by cytosolic pattern recognition receptors, leading to signaling events promoting the differentiation of CCL3(+) memory CD8(+) T cells. To test this possibility, we generated a stable L. monocytogenes chromosomal mutant that expressed a SecA2 ATPase bearing a mutated nucleotide binding site (NBS). Similarly to a SecA2 deletion mutant, the NBS mutant exhibited rough colonies, a bacterial chaining phenotype, an impaired protein secretion profile, and in vivo virulence in comparison to wt L. monocytogenes. Importantly, mice immunized with the SecA2 NBS mutant were not protected against secondary infection with wt L. monocytogenes and did not develop CCL3(+) memory CD8(+) T cells. NBS mutant and wt SecA2 proteins were expressed to comparable extents by bacteria, suggesting that SecA2 itself is unlikely to promote the induction of these cells. Rather, one or several of the SecA2 substrate proteins released inside the cytosol of infected cells may be involved.

Fig. 1.

Schematic representation of the mutated positions in the nucleotide binding sites (NBS) of SecA2. The main secA2 gene coding regions are represented by NBS-I and -II (which bind ATP), PPB (which defines a putative binding region of SecA2-dependent proteins), and CD (which is a conserved domain of SecA proteins). The nucleotides modified by site-directed mutagenesis and the corresponding amino acids changes are indicated on the detailed NBS-I and -II sequences.

Fig. 2.

Phenotypic and functional characterization of the NBS-SecA2 mutant of L. monocytogenes. (A) Morphology of wt, Del-SecA2, and NBS-SecA2 L. monocytogenes colonies grown on BHI agar plates at 37°C O/N as seen by light microscopy (magnification, ×10). wt bacteria exhibit smooth edges, whereas both SecA2 mutants (Del-SecA2 and NBS-SecA2) have craggy/rough edges. (B) Electron micrographs of wt, Del-SecA2, and NBS-SecA2 L. monocytogenes cultured at log phase. Left and right panels show low- and high-magnification views, respectively, of mutants and wt bacteria. Both mutants exhibit long filaments formed by multiple bacterial units, in contrast to individualized bacteria formed by wt L. monocytogenes. (C) SDS-PAGE analysis of TCA-concentrated stationary-phase culture supernatants from O/N-grown NBS-SecA2, Del-SecA2, and wt L. monocytogenes. Proteins were visualized by immunoblotting using a Listeria-specific antiserum.

Fig. 3.

The wt and NBS-deficient SecA2 proteins are expressed to equivalent levels in L. monocytogenes. (A) Summary of independently obtained recombinant L. monocytogenes clones that express chromosome-encoded wt (+) or NBS-mutated (−) SecA2-FLAG proteins. While wt SecA2-FLAG bacteria formed smooth (S) colonies, NBS-SecA2-FLAG bacteria formed rough (R) colonies on BHI plates. (B) Western blot analysis of SecA2 expression in three distinct wt or NBS-mutated bacterial clones encoding or not encoding (wt control) a C-terminal SecA2-FLAG protein. The cell pellet fraction of ∼3.3 × 10⁸ log-phase bacteria was separated by SDS-PAGE, blotted, and revealed using a FLAG-specific MAb. The results shown are representative of two independent experiments that gave comparable results.

Fig. 4.

Mice immunized with NBS-SecA2 L. monocytogenes are not protected against secondary infection with wt bacteria. Mice (four or five per group) were immunized with 0.1× the LD₅₀ of the indicated bacteria (wt, 2 × 10³; Del-SecA2, NBS-SecA2, and NBS-SecA2-FLAG, 10⁶) or injected with PBS. One month later, mice were given a secondary challenge with 3 × 10⁵ wt bacteria and killed 48 h after the challenge infection. Data show the number of bacteria in the spleen and the liver for each individual mouse and represent pooled results from two experiments. P values were calculated between groups of mice immunized with wt versus NBS-SecA2 and NBS-SecA2-FLAG L. monocytogenes and PBS versus Del-SecA2 and NBS-SecA2 L. monocytogenes in the experiment shown. P values close to 0.05 have low statistical significance.

Fig. 5.

Effector memory but not CCL3-secreting CD8⁺ T cells are present during secondary infection in mice given a primary immunization with NBS-SecA2 L. monocytogenes. BALB/c mice (10 to 12 mice per group) were immunized with PBS or 0.1× the LD₅₀ of the indicated bacteria and challenged 30 days later with 10× the LD₅₀ of wt bacteria. At 6 h after secondary challenge, spleen cells from individual mice were stained for expression of the cell surface markers CD8, CD3, CD62L, and H2-K^d/LLO_91-99 tetramers and/or restimulated in vitro with LLO_91-99 peptide for intracellular CCL3 staining and further processed for FACS analysis. Dot plots are representative of the staining obtained for CD8⁺ T cells expressing H2-K^d/LLO_91-99 tetramers. The right upper panel shows the mean frequency (± standard error of the mean [SEM]) of H2-K^d/LLO_91-99 tetramer-positive cells among the total CD8⁺ T cells. The left lower panel shows the mean frequency of H2-K^d/LLO_91-99 tetramer-specific CD62L^low (effector memory) CD8⁺ T cells among the total tetramer⁺ CD8⁺ T cells. The right lower panel represents the mean frequency (± SEM) of CCL3-secreting cells among the total CD3⁺ CD8⁺ T cells. Data are pooled results from three independent experiments with n = 15 mice. P values were calculated between groups of mice immunized with wt versus Del-SecA2 and NBS-SecA2 L. monocytogenes or injected with PBS. NS, not significant.