Listeria monocytogenes mutants that fail to compartmentalize listerolysin O activity are cytotoxic, avirulent, and unable to evade host extracellular defenses

Abstract

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. Escape of the bacterium from the phagosome to the cytosol is mediated by the bacterial pore-forming protein listeriolysin O (LLO). LLO has multiple mechanisms that optimize activity in the phagosome and minimize activity in the host cytosol. Mutants that fail to compartmentalize LLO activity are cytotoxic and have reduced virulence. We sought to determine why cytotoxic bacteria have attenuated virulence in the mouse model of listeriosis. In this study, we constructed a series of strains with mutations in LLO and with various degrees of cytotoxicity. We found that the more cytotoxic the strain in cell culture, the less virulent it was in mice. Induction of neutropenia increased the relative virulence of the cytotoxic strains 100-fold in the spleen and 10-fold in the liver. The virulence defect was partially restored in neutropenic mice by adding gentamicin, an antibiotic that kills extracellular bacteria. Additionally, L. monocytogenes grew more slowly in extracellular fluid (mouse serum) than within tissue culture cells. We concluded that L. monocytogenes controls the cytolytic activity of LLO to maintain its nutritionally rich intracellular niche and avoid extracellular defenses of the host.

FIG. 1.

Growth of the cytotoxic mutants in J774 macrophage-like cells. (A) Numbers of CFU in monolayers of J774 cells on 12-mm glass coverslips at different times in the presence of the extracellular antibiotic gentamicin added 1 h postinfection. The data are means derived from three coverslips. (B) Numbers of CFU in monolayers of J774 cells on 12-mm glass coverslips at different times after gentamicin (Gent.) treatment from 1 to 2 h postinfection (indicated by the arrow) followed by three washes in PBS. The data are means derived from three coverslips.

FIG. 2.

LLO mutants permeabilize the plasma membrane. C57BL/6 bone marrow-derived macrophages were infected for 4 h without gentamicin and then stained with the membrane-impermeant dye propidium iodide (PI), which increases fluorescence when it passes through the membrane and interacts with host DNA. A total of 2.5 × 10⁴ cells were examined by flow cytometry; the results for half of the cells are shown. The gray histogram represents uninfected cells. The fluorescence range for cells considered permeabilized, as indicated by the marker M1, was defined by adding 10⁶ hemolytic units of purified LLO L461T to the macrophages, as shown in Fig. 1A. The infecting strain and the percentage of cells falling within the marker M1 range are indicated.

FIG. 3.

The greater the cytotoxicity, the less the cytotoxic bacteria grow in mice. A total of 1 × 10⁵ CFU of each strain were injected into the tail veins of C57BL/6 mice. After 24 h the liver and spleen of each mouse were removed, homogenized, and plated to determine the number of CFU in each organ. The error bars indicate standard deviations for five mice.

FIG. 4.

The greater the cytotoxicity, the greater the virulence defect. A competitive index was established by injecting both wild-type bacteria (wt) and erythromycin resistance-marked mutants into C57BL/6 mouse tail veins. Competitive indices were determined for the spleen and liver by performing competition assays with wild-type and LLO L461T Erm strains (A), wild-type and LLO S44A Erm strains (B), and wild-type and LLO S44A L461T Erm strains (C). The y axis indicates the ratio of the number of mutant CFU to the number of CFU of wild-type bacteria isolated from the spleen or liver of mice at different times on a log scale. Therefore, the nearer the bottom of the graph, the fewer mutant bacteria were retrieved from a mouse compared to the number of wild-type bacteria. The ratio for each mouse is represented by a single marker for the spleen and a single marker for the liver. The +RB6 mice received an injection of the neutrophil-depleting monoclonal antibody RB6-8C5 6 h before infection with L. monocytogenes (9). The thick horizontal lines indicate a competitive index of 1.

FIG. 5.

Cytotoxic mutants are more sensitive to gentamicin. (A) A total of 1 × 10⁵ CFU wild-type bacteria were injected into the tail veins of C57BL/6 mice. Then 1 mg of gentamicin was injected subcutaneously, and at different times the livers and spleens were removed, homogenized, and plated to determine the number of CFU in each organ. The error bars indicate standard deviations for a minimum of seven mice. (B) Competitive indices were established at 48 h as described in the legend to Fig. 4. The +RB6 gent mice received an injection of the RB6-8C5 monoclonal antibody 6 h before infection, as well as a 1-mg gentamicin sulfate injection subcutaneously 6 h before organs were harvested. SALT, LLO S44A L461T strain; Gent, gentamicin; wt, wild type.

FIG. 6.

Virulence defects are not due to defects in cell-to-cell spread. Competitive indices were determined as described in the legend to Fig. 4. For the ΔActA data sets, all strains, including the reference strain secreting wild-type LLO, had an in-frame deletion in actA that eliminated actin-based motility. (A) 24 hr and 48 hr indicate the times of organ harvest. The data points are the ratios of the number of CFU of erythromycin-resistant LLO L461T bacteria to the number of CFU of wild-type bacteria in the spleen and liver. The 24 hr ΔActA and 48 hr ΔActA data points are the ratios of the number of erythromycin-resistant ΔActA LLO L461T bacteria to the number of wild-type LLO-secreting bacteria without the ActA gene recovered from the spleen and liver at 24 and 48 h, respectively. (B) Competitive indices established like those described for panel A, except that LLO S44A erythromycin-resistant mutants were used. wt, wild type.

FIG. 7.

Model for the virulence defect associated with cytotoxic L. monocytogenes. When L. monocytogenes is injected into a mouse through the tail vein, the majority of the bacteria are found in the spleen and liver. In these organs the bacteria invade cells. Bacteria lacking LLO activity (ΔLLO) are unable to escape from the phagosome and thus are unable to multiply, which terminates the infection. Both wild-type and cytotoxic bacteria are able to escape from the phagosome and initiate growth in the cytosol. The wild-type bacteria are able to efficiently grow and spread to neighboring cells before the initial cell's integrity is compromised. This allows the wild-type bacteria to spread through the tissues and continue the infection. In contrast, the cytotoxic mutant damages the host cell soon after it enters the cytosol and exposes the bacteria to the extracellular milieu, which results in a reduction in the net bacterial growth and therefore a reduction in virulence.