A prl mutation in SecY suppresses secretion and virulence defects of Listeria monocytogenes secA2 mutants

Abstract

The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogen Listeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence in L. monocytogenes; however, the mechanism of export via this pathway is poorly understood. L. monocytogenes secA2 mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility to L. monocytogenes secA2 mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by the lmo2769 operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation in secY was identified that conferred smooth colony morphology to secA2 mutants, restored wild-type plaque formation, and increased virulence in mice. This secY mutation resembled a prl suppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ΔsecA2prlA1 mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although the prl mutation largely suppressed almost all of the measurable SecA2-dependent traits, the ΔsecA2prlA1 mutant was still less virulent in vivo than the wild-type strain, suggesting that SecA2 function was still required for pathogenesis.

FIG 1

Swarming motility of suppressor mutants. Motility assessed on semisolid LB agar following incubation at 30°C after 48 h. Motility is expressed as a percentage of the swarming area of the WT strain seeded on the same plate. Error bars represent standard deviations of the mean zone ratios from three independent experiments. Images show motility zones for the suppressor mutants indicated.

FIG 2

Plaque size of secA2 suppressor mutants. L2 fibroblasts were infected with secA2 suppressor mutants, and mean plaque size was measured 3 days postinfection. Plaque area of each mutant is shown as a percentage compared to that of the WT strain. Error bars represent standard deviations of the mean zone ratios from three independent experiments. Images show plaques for the suppressor mutants indicated.

FIG 3

Characterization of the R57 revertant. (A) Fluorescence microscopy of the ΔsecA2 and R57 strains. Mid-exponential-phase cells were stained with SYTO9 green. (B) Disk diffusion susceptibility to 1 mg of lysozyme expressed as a ratio of the WT, where a ratio of >1 indicates increased susceptibility to lysozyme. Error bars represent standard deviations of the mean zone ratios. Student's t test was used to analyze statistical significance: ***, P < 0.0001; ns, P ≥ 0.05. Bacterial burdens in spleens (C) and livers (D) 48 h postinfection with 1 × 10⁵ CFU in CD1 mice. The dashed line represents the limit of detection. Results show CFU from three independent experiments. Statistical significance was evaluated using a Mann-Whitney test. ***, P < 0.0001; ns, P ≥ 0.05.

FIG 4

Characterization of an lmo2769 mutant in a ΔsecA2 background. (A) Swarming motility of mutants and strains complemented with the lmo2767 operon was assessed on semisolid LB agar following incubation at 30°C after 48 h. Motility is expressed as a percentage of the swarming area of the WT strain and set at 100% (represented by a red dotted line). Error bars represent standard deviations of the mean zone ratios from three independent experiments. (B) Disk diffusion susceptibility to 1 mg of lysozyme expressed as a ratio of the WT. Error bars represent standard deviations of the mean zone ratios. (C and D) Plaque area of each mutant is shown as a percentage of that of the WT strain, set at 100% (represented by a red dotted line). Error bars represent standard deviations of the mean plaque size ratios from three independent experiments. Student's t test was used to analyze statistical significance: ***, P < 0.0001; **, P < 0.01; *, P < 0.05; and ns, P ≥ 0.05.

FIG 5

Characterization of the prlA1 mutation. (A) Swarming motility of mutants and strains complemented with the lmo2767 operon was assessed on semisolid LB agar following incubation at 30°C after 48 h. Motility is expressed as a percentage of the swarming area of the WT strain and set at 100% (represented by a red dotted line). Error bars represent standard deviations of the mean zone ratios from three independent experiments. (B) Plaque area of each mutant is shown as a percentage compared to that of the WT strain, set at 100% (represented by a dotted line). Error bars represent standard deviations of the mean plaque size ratios from three independent experiments. Student's t test was used to analyze statistical significance: ***, P < 0.0001; **, P < 0.01; *, P < 0.05; and ns, P ≥ 0.05. (C) Secreted levels of P60 in supernatants from mid-log (5-h) cultured cells in LB broth expressed as a percentage of protein secreted by the WT strain. (D) Secreted levels of P60 and LLO in supernatants from 1-h, 2-h, and 3-h cultures in LB broth. Bacterial burdens in spleens (E) and livers (F) 48 h post-intravenous infection of 1 × 10⁵ CFU in mice, showing data from three independent experiments. The dashed line represents the limit of detection. Statistical significance was evaluated using a Mann-Whitney test. ***, P < 0.0001; *, P < 0.05; ns, P ≥ 0.05.