L. monocytogenes strains were grown to stationary phase and used to infect tissue culture cells grown on glass coverslips. At 30 minutes (J774 cells) or 60 minutes (PtK2 cells) post-infection, monolayers were washed, gentamicin was added, and at the indicated time points cell monolayers were lysed and the number of bacteria per coverslip was determined. The results are expressed as the mean number of bacterial CFU + the standard deviation for three coverslips per time point. The data for one of three experiments with similar results is shown. (A) Bacterial intracellular growth in J774 macrophage-like tissue culture cells. (B) Bacterial intracellular growth in PtK2 epithelial cells. ◆, parent strain NF-L1124; , prfA Y154C (NF-L1213). (C) Intracellular growth and cell-to-cell spread of L. monocytogenes in murine L2 fibroblasts, as indicated by plaque formation. The prfA Y154C strain failed to form visible plaques after three days of incubation, whereas multiple plaques were visible in monolayers infected with wild type bacteria. Data shown is representative of three experiments.

(A) Cytotoxicity was measured by lactate dehydrogenase (LDH) release from bacteria-infected PtK2 epithelial cells at 24 hours post-infection with the indicated strains. Maximum release (max) was determined from complete host cell lysis and set to 100%; all values are expressed as a percentage of max ± standard deviation. The data shown is representative of three independent experiments. (B–C) Cytotoxicity visualized by microscopy of PtK2 cells infected with the parent strain (NF- L1124) (B) or prfA Y154C (NF-L1213) (C) at 24 hours post-infection. PtK2 cells were infected with bacteria at an MOI of ~5:1. At one hour post-infection the monolayers were washed and gentamicin was added to kill any extracellular bacteria. At 24 hr post-infection the monolayers were fixed and stained with Diff Quik stain. Data shown is representative of three independent experiments. (D) Intracellular expression of LLO by L. monocytogenes in PtK2 cells. PtK2 cells were infected with L. monocytogenes: at 5 hr post-infection the infected cells were starved for methionine and cysteine and host protein synthesis was blocked as described in Materials and Methods. Infected cells were incubated with [³⁵S]-methionine for 1 hr prior to host cell lysis and immunoprecipitation with monoclonal anti-LLO antibody B3–19 [41]. The amount of sample loaded per lane was normalized for the number of bacteria. An autoradiograph representative of two independent experiments is shown, which was exposed for 100 hours using the Storm Phosphorimager and analyzed with ImageQuant software. The arrow indicates the position of the LLO band, which is approximately 58 kD.

(A) Bacterial actA expression levels as measured by monitoring GUS activity. Levels of actA expression are indicated by symbols denoting GUS activity as measured at the indicated time points. GUS activity was measured for bacterial cultures grown in BHI broth and the units of GUS were normalized for optical density as described by Shetron-Rama et al., [38]. Each time point was measured in triplicate, and the data shown is representative of three experiments ± SEM. ◆, parent strain NF-L1124; , prfA* mutant strain NF-L943 (prfA G155S); , original plasmid integration prfA Y154C mutant (NF-L1215); , isogenic chromosomal prfA Y154C (NF-L1213). Large symbols indicate GUS activity, the smaller symbols reflect bacterial growth as measured by optical density. (B) Detailed view of NF-L1124, NF-L1213, and NF-L1215 from panel A. (C) Measurement of phospholipase activity. Bacteria were grown on media containing egg-yolk; the white precipitate surrounding bacterial growth is indicative of PC-PLC activity. Both NF- L1213 and NF-L1215 prfA Y154C strains exhibit enhanced PC-PLC activity in comparison to wild type (NF-L1124) or ΔprfA (NF-L1123) strains, but have lower levels than the prfA* mutant strain NF-L943 (prfA G155S). Data shown is representative of two experiments. (D) Secreted hemolytic activity as measured by erythrocyte (RBC) lysis. Serial dilutions of bacterial supernatants were incubated with RBCs and cell lysis was determined by measuring absorbance at 450 nm (OD₄₅₀). Data shown is representative of three experiments. Symbols: ◆, parent strain NF-L1124; , isogenic chromosomal prfA Y154C (NF-L1213); ● ΔprfA (NF-L1123); , prfA G155S (NF- L943).

(A–F) Visualization by fluorescence microscopy of polymerized actin in PtK2 epithelial cells infected with L. monocytogenes. PtK2 cells were infected with either NF-L1124 (wild type prfA) (A, C, E) or NF-L1213 (prfA Y154C) (B, D, F) bacteria at an MOI of ~5:1. At one hour post-infection the monolayers were washed and gentamicin was added to kill any extracellular bacteria. At 3 hr (A, B), 5 hr (C, D), and 7 hr (E, F) post-infection the monolayers were fixed, permeabilized, and stained with NBD-phallicidin for filamentous actin (green) and an anti-Listeria antibody and tetramethylrhodamine goat anti-rabbit conjugated secondary antibody to detect bacteria (red) as described in Materials and Methods. Results shown are representative of two independent experiments. (G) Quantitation of actin association for wild type NF-L1124 and prfA Y154C strain NF-L1213. Actin association in the form of clouds (polymerized actin surrounding bacteria) or tails was scored for approximately 50 bacteria in infected cells from at least 10 independent microscopic fields for each time-point listed. Numbers listed represent the mean ± standard error derived from two independent experiments. (H) Bacterial intracellular actA expression as measured by GUS activity. Tissue culture dishes containing monolayers of PtK2 cells were infected with the indicated L. monocytogenes strains for 8 h as described in Materials and Methods. GUS activity was determined following lysis of the monolayers. The number of CFU per dish was determined by lysing infected PtK2 cells grown on coverslips in duplicate dishes and plating a portion of the lysates. Units of GUS are as described by Shetron-Rama et al. [38]. Each assay was done in triplicate and the data represent the mean ± standard deviation for at least three individual experiments.

Left panel: BALB/c mice were injected intravenously with wild type (■, 1900 CFU), ΔactA (●, 1.55 × 10⁶ CFU), or prfA Y154C (▲, 4.4 × 10⁴ CFU). On the days indicated the spleens from 2–3 mice were homogenized individually and numbers of CFU/spleen determined by dilution plating. Right panel. BALB/c mice were immunized with either wildtype L. monocytogenes or prfA Y154C (NF-L1213) at the indicated doses. Seven days later, spleens were removed, a single cell suspensions prepared and the frequency of LLO_91–99 (grey bars) or p60_217–225 (white bars) specific IFN-γ producing CD8+ cells by determined by intracellular cytokine staining.