CwpFM (EntFM) is a Bacillus cereus potential cell wall peptidase implicated in adhesion, biofilm formation, and virulence

Abstract

Bacillus cereus EntFM displays an NlpC/P60 domain, characteristic of cell wall peptidases. The protein is involved in bacterial shape, motility, adhesion to epithelial cells, biofilm formation, vacuolization of macrophages, and virulence. These data provide new information on this, so far, poorly studied toxin and suggest that this protein is a cell wall peptidase, which we propose to rename CwpFM.

FIG. 1.

(A) Domain organization of B. cereus CwpFM and B. subtilis LytF cell wall peptidases. Green boxes represent SH3 cell wall binding domains. Red boxes define the NlpC/P60 peptidase domain. (B) Bacterial morphology, as observed with phase-contrast microscopy. Bacterial morphology of the B. thuringiensis 407 and the cwpFM mutant strains was analyzed by phase-contrast light microscopy (Leica). Magnification, ×100. Cultures were sampled at different growth phases: the beginning of the exponential phase (OD₆₀₀, 0.3) (a) or the beginning (OD₆₀₀, 3) (b), middle (OD₆₀₀, 7) (c), or end (OD₆₀₀, 9) (d) of the stationary phase. Bar, 10 μm. (C) Motility assay. Bacteria were inoculated on 0.3% agar plates. Motility of the wild-type B. thuringiensis 407, the cwpFM mutant, and the complemented strain cwpFM/pHTΩcwpFM was assessed after 24 h of incubation at 37°C. Images are representative of two independent experiments with three replicates. (D) Quantitative analysis of bacterial adhesion to HeLa cells. HeLa cells were incubated with wild-type B. thuringiensis 407 and the cwpFM mutant strain, at a density of 10 bacteria per cell, for 20 min. Cells were washed to eliminate nonadherent bacteria, and the bacteria associated with cells were quantified by dilution plating on LB agar plates. Adhesion was calculated as the ratio of adherent bacteria to the total number of bacteria used for inoculation. Results are shown as means of two independent experiments, each including three replicates. (E) Biofilm formation by B. cereus at the air-liquid interface. The B. thuringiensis 407 strain, the cwpFM mutant, and the complemented strain cwpFM/pHTΩcwpFM were incubated in the wells of a PVC microtiter plate for 48 h at 30°C. Biofilm formation was quantified using crystal violet. Bars represent OD₅₉₅ values, and results are the mean of three independent experiments with 16 replicates.

FIG. 2.

(A and B) Evaluation of cytotoxicity using trypan blue dye. HeLa (A) or macrophage (B) cells (1 × 10⁵) were incubated with the culture supernatant of B. thuringiensis 407, the cwpFM mutant, or B. subtilis, or with purified GST-CwpFM (1 μg/ml). Control cells were either noninfected (NI) or incubated with purified GST (1 μg/ml). After 2 h of incubation, cell mortality was evaluated by trypan blue dye exclusion. (C) CwpFM induces vacuolization of macrophages. Macrophages (10⁶ cells/well) were incubated with 1 μg/ml of purified GST-CwpFM or GST. After treatment, cells were fixed with 4% paraformaldehyde for 25 min at 4°C. Cell morphology was observed under a phase-contrast microscope (100× objective; Leica) (top panels). Arrows indicate cell vacuoles. Alternatively, cells were stained using the DeadEnd fluorometric TUNEL kit and observed under a fluorescence microscope (bottom panels). All cells are labeled in red, and potential apoptotic cells are stained green. Images are representative of at least three independent experiments. (D) CwpFM virulence against G. mellonella larvae. Various concentrations of wild-type and cwpFM strains were inoculated into the hemocoel of three groups of 20 G. mellonella larvae. Mortality was recorded after 24 h at 37°C, and the LD₅₀ was determined using the probit method.