Modulation of enzymatic activity and biological function of Listeria monocytogenes broad-range phospholipase C by amino acid substitutions and by replacement with the Bacillus cereus ortholog

Abstract

The secreted broad-range phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC) of Listeria monocytogenes plays a role in the bacterium's ability to escape from phagosomes and spread from cell to cell. Based on comparisons with two orthologs, Clostridium perfringens alpha-toxin and Bacillus cereus PLC (PLCBc), we generated PC-PLC mutants with altered enzymatic activities and substrate specificities and analyzed them for biological function in tissue culture and mouse models of infection. Two of the conserved active-site zinc-coordinating histidines were confirmed by single amino acid substitutions H69G and H118G, which resulted in proteins inactive in broth culture and unstable intracellularly. Substitutions D4E and H56Y remodeled the PC-PLC active site to more closely resemble the PLCBc active site, while a gene replacement resulted in L. monocytogenes secreting PLCBc. All of these mutants yielded similar amounts of active enzyme as wild-type PC-PLC both in broth culture and intracellularly. D4E increased activity on and specificity for PC, while H56Y and D4E H56Y showed higher activity on both PC and sphingomyelin, with reduced specificity for PC. As expected, PLCBc expressed by L. monocytogenes was highly specific for PC. During early intracellular growth in human epithelial cells, the D4E mutant and the PLCBc-expressing strain performed significantly better than the wild type, while the H56Y and D4E H56Y mutants showed a significant defect. In assays for cell-to-cell spread, the H56Y and D4E mutants had close to wild-type characteristics, while the spreading efficiency of PLCBc was significantly lower. These studies emphasize the species-specific features of PC-PLC important for growth in mammalian cells.

FIG. 1

Detection of PC-PLC and PLC_Bc in the supernatant of L. monocytogenes broth cultures. For this experiment, strain DP-L1553 and isogenic mutants were used. The same volume of concentrated bacterial secreted proteins was loaded per lane. Secreted phospholipase phenotypes are indicated above the lanes. The position of the 31-kDa molecular weight (m.w.) marker is shown at the left. Pro and mature forms of PC-PLC and PLC_Bc (hybrid pro-PLC_Bc; see text) were identified by Western blotting with affinity-purified antibodies and are indicated to the right. (a) Coomassie blue staining of secreted proteins resolved by SDS-PAGE. Recombinant purified mature form PLC_Bc (rPLC_Bc) was run as a size and activity control. (b) Egg yolk overlay assay. An empty gel lane between D4E H56Y and PLC_Bc, originally introduced to prevent confluence of zones of opacity, was removed so that panels a and b could be aligned without changing the vertical position.

FIG. 2

Detection of PC-PLC and PLC_Bc from lysates of infected cells. J774 cells were infected with strain 10403S and isogenic mutants. At 4 h after infection, cells were pulse-labeled for 30 min with [³⁵S]methionine. Immediately after the pulse, the cells were lysed and PC-PLC or PLC_Bc was immunoprecipitated with the respective affinity-purified antibodies, followed by fractionation by SDS-PAGE and detection of radiolabeled proteins by fluorography. (a) Expressed phospholipase phenotypes are indicated above the lanes; positions of the 31-kDa protein size marker and pro and mature forms of the enzymes are marked on the sides. (b) Affinity-purified antibodies used for immunoprecipitation and expressed phospholipase phenotypes are shown at the top and bottom, respectively. Where indicated, prokaryotic (chloramphenicol [Cm]) and eukaryotic (anisomycin/cycloheximide [Am/Ch]) protein synthesis inhibitors were present during labeling.

FIG. 3

Intracellular growth of L. monocytogenes in Henle 407 human epithelial cells. Henle 407 cells were infected with DP-L2161 (LLO⁻) and isogenic mutant strains. The data shown are from representative experiments (see also Table 4). Phospholipase phenotypes are indicated. (a) Active-site mutants; (b) B. cereus plc gene replacement. Each datum point and error bar represents the means ± standard deviations of viable bacteria recovered from three coverslips.

FIG. 4

Plaque formation in infected L2 mouse fibroblasts. L2 cells were infected with strain 10403S (wild type) or DP-L1552 (ΔplcA PI-PLC⁻) and isogenic mutants thereof. Cell monolayers were stained with neutral red at 4 days after infection, highlighting clear areas of dead cells resulting from L. monocytogenes intracellular growth and cell-to-cell spread. The mean plaque diameters were calculated from several individual experiments (Table 4) and are indicated below each well. Inserts are magnified sections of the shown wells, showing the altered plaque morphology obtained with strains expressing B. cereus PLC_Bc.

FIG. 5

Influence of cytochalasin D on growth of PLC_Bc-expressing L. monocytogenes in Henle 407 cells. Henle 407 cells were infected with 10403S (wild type) and isogenic strains. The data shown are from one of three experiments. Phospholipase phenotypes are indicated. +cytD indicates cytochalasin D addition. Each datum point and error bar represents the means ± standard deviations of viable bacteria recovered from three coverslips.