Characterization of Listeria monocytogenes expressing anthrolysin O and phosphatidylinositol-specific phospholipase C from Bacillus anthracis

Abstract

Two virulence factors of Listeria monocytogenes, listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC), mediate escape of this pathogen from the phagocytic vacuole of macrophages, thereby allowing the bacterium access to the host cell cytosol for growth and spread to neighboring cells. We characterized their orthologs from Bacillus anthracis by expressing them in L. monocytogenes and characterizing their contribution to bacterial intracellular growth and cell-to-cell spread. We generated a series of L. monocytogenes strains expressing B. anthracis anthrolysin O (ALO) and PI-PLC in place of LLO and L. monocytogenes PI-PLC, respectively. We found that ALO was active at both acidic and neutral pH and could functionally replace LLO in mediating escape from a primary vacuole; however, ALO exerted a toxic effect on the host cell by damaging the plasma membrane. B. anthracis PI-PLC, unlike the L. monocytogenes ortholog, had high activity on glycosylphosphatidylinositol-anchored proteins. L. monocytogenes expressing B. anthracis PI-PLC showed significantly decreased efficiencies of escape from a phagosome and in cell-to-cell spread. We further compared the level of cytotoxicity to host cells by using mutant strains expressing ALO in combination either with L. monocytogenes PI-PLC or with B. anthracis PI-PLC. The results demonstrated that the mutant strain expressing the combination of ALO and B. anthracis PI-PLC caused less damage to host cells than the strain expressing ALO and L. monocytogenes PI-PLC. The present study indicates that LLO and L. monocytogenes PI-PLC has adapted for L. monocytogenes intracellular growth and virulence and suggests that ALO and B. anthracis PI-PLC may have a role in B. anthracis pathogenesis.

FIG. 1.

Diagram of the L. monocytogenes PrfA regulon and the mutants constructed in the present study. The star indicates a nonfunctional H86A mutation in L. monocytogenes PI-PLC (1), the black arrows indicate the replacement of L. monocytogenes PI-PLC with B. anthracis PI-PLC, and the gray arrows indicate the replacement of LLO with ALO. BaPI-PLC, B. anthracis PI-PLC.

FIG. 2.

Specific hemolytic activities of recombinant cholesterol-dependent cytolysins (CDC) on sheep red blood cells at the indicated pH. Values represent the mean ± the SD of three independent experiments.

FIG. 3.

Detection of PI-PLC activities. (A) Activity of indicated strains on PI using ALOA Listeria agar plates; (B) supernatant activity on PI using a quantitative [³H]inositol-PI method (13). For L. monocytogenes PI-PLC (LmPI-PLC) and B. anthracis PI-PLC (BaPI-PLC), the P value was >0.1 as determined by unpaired t test of three experiments. There is no activity detected when an inactive mutant form of L. monocytogenes PI-PLC, H86A, was used. (C) Activity on the GPI-anchored protein CD90 as determined by FACS. Left, pure B. thuringiensis PI-PLC (BtPI-PLC; 6 μg/ml); middle, supernatant of strain HG-L2002 (B. anthracis PI-PLC [BaPI-PLC]); right, supernatant of strain Lmdd (L. monocytogenes PI-PLC [LmPI-PLC]). In several separate experiments the negative control of untreated cells overlapped with the Lmdd curve (not shown).

FIG. 4.

Intracellular growth in J774 murine macrophage-like cells. The data shown are representative of at least three experiments. (A) Growth of Lmdd strains. (B) Light micrograph of J774 cells 6 h postinfection with Lmdd or Lmdd expressing ALO. For panels A and B, gentamicin at 50 μg/ml was added at 1 h postinfection for the duration of the experiment. (C and D) Growth of wild-type L. monocytogenes expressing LLO (10403S), PFO (DP-L4055), or ALO (DP-L4450). Gentamicin at 50 μg/ml was added at 1 h postinfection for the duration of the experiment (C) or for a short pulse from 1 to 1.5 h postinfection (D). L. monocytogenes with the LLO gene deleted (Δhly, DP-L2161) controls for killing of extracellular bacteria during the gentamicin pulse. (E and F) Light micrographs of J774 cells 6 h postinfection with WT strain 10403S or derivatives expressing either PFO or ALO. Gentamicin at 50 μg/ml was added at 1 h postinfection for the duration of the experiment (E) or for a short pulse from 1 to 1.5 h postinfection (F). LmPI-PLC, L. monocytogenes PI-PLC; BaPI-PLC, B. anthracis PI-PLC.

FIG. 5.

Evaluation of the integrity of the host cell plasma membrane after L. monocytogenes infections. (A) LDH release by infected J774 cells. The release of LDH after infection of J774 cells by each strain was compared to total LDH determined after lysis with detergent. Values represent the mean ± the SD of three independent experiments. ✽, P < 0.05; ✽✽✽, P < 0.001 (as determined by unpaired t test compared to strain HG-L2003). (B) Cellular DNA staining by propidium iodide using mouse bone marrow-derived macrophages. The gray histogram represents the uninfected cells as negative control. The percentage of cells that were stained with propidium iodide is indicated. These data are representative of three experiments.