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Listeria monocytogenes is a facultative intracellular gram-positive pathogen of humans and animals that causes a serious, food-borne illness in immunocompromised and pregnant individuals (9). Although the oral route is the natural route of L. monocytogenes infection, highly reproducible and useful intravenous and intraperitoneal murine models have been developed that mimic characteristics of disseminated disease, specifically growth of the organism in the spleen and liver (17). Immunity to L. monocytogenes in these models is entirely cell mediated (18). Macrophages and neutrophils are essential for the initial innate resistance to L. monocytogenes (25) while cytotoxic T lymphocytes (CTLs) are central to the adaptive immune response (12).

The intracellular life cycle of L. monocytogenes has been well described in tissue culture models of infection. Once inside a host cell vacuole or phagosome, L. monocytogenes is able to escape into the cytosol and initiate replication. During intracellular growth, actin polymerizes at one pole of the bacterium and propels the bacillus through the cytoplasm and into neighboring host cells, thereby facilitating cell-to-cell spread without exposure to the extracellular environment (19, 24). ActA is an L. monocytogenes surface protein that facilitates actin nucleation at the bacterial surface (8, 14). The central region of ActA is composed of proline-rich repeats (Table 1) which bind to the eukaryotic protein vasodilator-stimulated phosphoprotein (VASP). This interaction leads to an increase in the bacterial movement rate and cell-to-cell spread (16, 20, 22). A strain of L. monocytogenes that is completely incapable of actin-based motility (ActA) is 1,000-fold less virulent in the murine model of listeriosis (3). Furthermore, deletion of the proline-rich repeats of ActA leads to a decreased bacterial movement rate and lower cell-to-cell spread efficiency in tissue culture cells as well as a 40-fold increase in 50% lethal dose (LD₅₀) (22). However, glycine substitution of critical proline residues within the ActA central region ablates VASP recruitment to the surface of L. monocytogenes and results in slower movement within tissue culture cells. Nevertheless, this loss of VASP binding does not translate into any virulence attenuation detectable by the LD₅₀ assay (22). As LD₅₀ experiments were restricted to primary infections, it is possible that they may not have revealed all relevant aspects of L. monocytogenes pathogenesis.

We have designed a competitive index assay which allows us to compare different bacterial strains in the context of both primary and secondary listeriosis (infections of naive and immunized mice, respectively). By using this sensitive assay we show that loss of VASP binding indeed results in decreased virulence. Furthermore, we have found that strains of L. monocytogenes deficient in recruiting VASP are even less pathogenic during a secondary immune response than during a primary one.

Bacterial and mouse strains. All L. monocytogenes strains used in this study were derived from 10403S (2). Bacteria were grown in brain heart infusion (BHI) (Becton Dickinson, Sparks, Md.) to log phase, washed once with phosphate-buffered saline, and resuspended in fresh BHI. Aliquots were frozen and stored at 80°C. Four- to six-week-old female CD-1 mice were purchased from Charles River Laboratories (Wilmington, Mass.). Four- to six-week-old female BALB/c mice and five- to seven-week-old female C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, Maine).

Selection of a L. monocytogenes reference strain. In order to establish a reliable reference strain for a competitive index assay, it was essential to ensure that the selected strain display full virulence in a murine model of infection. Accordingly, we selected for a virulent transposon-containing strain in vivo. An L. monocytogenes 10403S::Tn917-LTV3 random insertion library (5) was grown to log phase at 37°C. The culture was diluted in phosphate-buffered saline, and 10⁴ bacteria were injected intravenously (i.v.) into a single BALB/c mouse. After 48 h, the animal was sacrificed and its liver was homogenized in 0.2% NP-40. The homogenate was pelleted, resuspended in BHI containing chloramphenicol (10 µg/ml) (to select for the transposon, which contains the erm and cat genes), and allowed to reach stationary phase. The resulting culture was diluted in BHI containing chloramphenicol (10 µg/ml) and grown to log phase at 37°C, and 10⁴ bacteria were injected i.v. into a second BALB/c mouse. After 48 h, the animal was sacrificed and its liver was homogenized. Dilutions of the organ homogenate were plated onto Luria-Bertani agar, and the plates were incubated at 37°C. Random colonies were screened for full virulence in a primary infection competitive index assay (described below). One clone (DP-L3903) was chosen as a reference strain for our assay after characterization in three different mouse strains during primary and secondary infection (Fig. 1). DP-L3903 competed equally with 10403S in inbred (BALB/c and C57BL/6) and outbred (CD-1) mice during primary listeriosis. In addition, DP-L3903 behaved indistinguishably from the wild type during secondary infection of BALB/c mice (Fig. 1).

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Competitive index analysis of the reference strain and wild-type 10403S L. monocytogenes. Groups of 3 to 10 naive or L. monocytogenes-immunized mice were infected with a 1:1 mixture of DP-L3903 and 10403S. CD-1 mice were sacrificed 48 h postinoculation, while BALB/c and C57BL/6 mice were euthanized after 60 h. A competitive index of 1 indicates that the two strains are proliferating equally in vivo. Abbreviations: S, spleen; L, liver. 1°, infection of naive mice; 2°, infection of L. monocytogenes-immunized mice; y axis, competitive index.

Competitive index analysis during primary listeriosis. To validate the competitive index assay as an accurate measure of virulence, we compared published LD₅₀s with our competitive indexes for several strains of L. monocytogenes. Briefly, frozen culture stocks were thawed, grown to log phase in fresh BHI for 2 h, and mixed in a 1:1 ratio. BALB/c and C57BL/6 mice received i.v. injections of 6 × 10⁴ to 8 × 10⁴ total bacteria (reference and test strains combined). A dose of 1 × 10⁵ to 7 × 10⁵ was used for CD-1 mice. Animals were sacrificed anywhere from 48 to 60 h later, and their spleens and livers were harvested. Spleens and livers were homogenized in 5 and 10 ml 0.2% NP-40, respectively, for 1 min in a tissue homogenizer (Ultra Turrax T-25 basic; IKA Works, Inc., Wilmington, N.C.), and homogenates were plated onto Luria-Bertani agar. At least 100 colonies per organ were replica plated or patched onto BHI agar containing erythromycin (2 µg/ml). Competitive indexes were calculated by dividing the number of test strain CFU (erythromycin sensitive) by the number of reference strain CFU (erythromycin resistant). The LD₅₀ of a strain containing a large in-frame deletion within actA was reported to be 1,000-fold greater than that of its wild-type parent in BALB/c mice (3). Similarly, the mean competitive index of a ActA strain during primary infection demonstrated a 1,000-fold defect in the liver and 500-fold defect in the spleen of BALB/c mice 48 h postinoculation (data not shown). Likewise, the LD₅₀ of a strain which lacks the repeat region of ActA (ActA6) (Table 1) was found to be 40-fold greater than that of the wild type in BALB/c mice (22). The mean competitive index of this strain in the liver during primary infection reflected approximately the same virulence attenuation 60 h postinoculation (Fig. 2B). Interestingly, the mean competitive index in the spleens of these mice was significantly higher than that in the livers (Fig. 2). We observed this difference with most L. monocytogenes actA mutant strains analyzed.

View larger version (27K): [in this window] [in a new window]   FIG. 2.   Competitive index analysis during primary infection (A and B) or secondary infection (C and D) in the spleen (A and C) or liver (B and D). Groups of four or five naive or immunized BALB/c mice were infected with a 1:1 mixture of the reference strain (DP-L3903) and ActA3, ActA3-GG, or ActA6. All mice were sacrificed 60 h postinoculation. Average fold defect was calculated by dividing the number of reference strain CFU by the number of test strain CFU for each organ. Primary infection competitive indexes of ActA6 and ActA3-GG were significantly lower in livers than spleens, with two-sided P values of 0.0004 for ActA6 and 0.0002 for ActA3-GG by the Mann-Whitney test as calculated by MiniTab (State College, Pa.) software. Liver competitive indexes of ActA6 and ActA3-GG were significantly lower during secondary infection than during primary infection, with two-sided P values of 0.037 for ActA6 and 0.0048 for ActA3-GG. Symbols: , outlying value as determined by KaleidaGraph (Synergy Software, Reading, Pa.); *, underestimated values (1 or 0 erythromycin-sensitive colonies out of approximately 200 screened). y axis, competitive index.

Competitive index analysis during secondary listeriosis. To test whether a given bacterial strain displayed similar virulence attenuation in immunized animals compared to that in naive animals, we performed competitive index analysis during secondary infection of the three actA mutants. Mice were immunized with 2 × 10³ L. monocytogenes 10403S bacteria by i.v. injection. Three weeks later, the immunized animals were challenged with 6 × 10⁵ to 10 × 10⁵ total bacteria (reference and test strains combined). Animals were sacrificed 60 h postinoculation and their organs were treated as described above. In order to determine the degree of resistance displayed by the immunized animals, BALB/c mice immunized as described above and naive mice of the same age were challenged with 6 × 10⁴ L. monocytogenes 10403S bacteria by i.v. injection. After 48 h, the numbers of CFU in the spleen and liver were enumerated and the difference in bacterial load between primary and secondary infection was determined (2). Under these conditions, immunized animals had 10⁴- to 10⁵-fold fewer bacteria in both organs than did naive mice.