Fig 1.
Inhibition of S. aureus internalization into bMEC by L. casei.
Internalization rates of S. aureus N305 after 2 h of interaction with bMEC and co-incubation with wt, fbpA (panel A) and sortase mutant strains (panel B) of L. casei BL23 at an MOI of 2,000:1. S. aureus was used at an MOI of 100:1. The internalization assay of S. aureus alone was used as a reference (control). Internalization rates were then defined as the internalized S. aureus population in the presence of the different L. casei strains relative to the internalized S. aureus population of the reference experiment. Data are presented as means ± standard deviations. Each experiment was done in triplicate, and differences between groups were compared using one-way ANOVA with Bonferroni's Multiple Comparison Test. *: P < 0.05.
Fig 2.
Microscopic observation of internalized S. aureus.
Internalization of S. aureus N305 as observed by transmission electron microscopy. S. aureus N305 (at an MOI of 100:1) was incubated for 2 h with bMEC either alone (A, B) or in the presence of L. casei BL23 wt (C, D) or srtA2 mutant (E, F) strains, at an MOI of 2,000:1.
Fig 3.
Internalization of wild type and mutant strains of L. casei BL23 into bMEC.
L. casei populations internalized into bMEC were determined after 2 h of interaction at an MOI 2,000:1. The internalization assay of the L. casei BL23 wild type (wt) strain was used as a reference. Internalization rates were defined as the internalized population of mutant strains relative to the internalized L. casei BL23 wt strain population. Data are presented as mean ± standard deviations. Each experiment was done in triplicate and differences between groups were compared using Student’s t-test. *: P < 0.05.
Fig 4.
Internalization of L. casei BL23 wt (A) and srtA2 mutant (B) strains as observed by transmission electron microscopy. Degradation vesicles (white arrows) were observed in a greater proportion in cells containing mutant srtA2.
Fig 5.
Resistance of L. casei BL23 wt and srtA2 strains to H2O2.
Resistance of L. casei BL23 wt (●, ○) and srtA2 (■, □) strains to H2O2 was evaluated in the stationary phase of growth of L. casei (24h—MRS). The residual population was evaluated at 0, 10, 20 and 30 min after exposure to 0.25% (●, ■) and 0.5% H2O2 (○, □). Data are presented as means ± standard deviations. Each experiment was done in triplicate, and differences between groups were compared using Student’s t-test. *: P < 0.05.
Table 1.
Cell surface proteome of L. casei BL23 wt and srtA2 mutant as determined by enzymatic shaving.
Fig 6.
L. casei BL23 srtA2 displayed a lower cell wall thickness compared to the wt strain.
L. casei wt (A) and srtA2 mutant (B) strains were grown in UF milk medium for 48 h and observed by transmission electron microscopy.
Fig 7.
Auto-aggregation capacities of L. casei BL23 wt and srtA2 strains.
Strains were grown in UF milk medium for 48 h at 37°C. Auto-aggregation was evaluated by spectrophotometry (600 nm) and expressed as the auto-aggregation percentage. Cell suspension OD after growth (48 h) and homogenization was used as a reference (100%). Data are presented as means ± standard deviations. Each experiment was done in triplicate, and differences between groups were compared using Student’s t-test. *: P < 0.05.
Fig 8.
Impact of L. casei BL380 (BL23 bnaG) on S. aureus internalization into bMEC.
Internalization rates of S. aureus N305 after 2 h of interaction with bMEC and co-incubation with L. casei BL23 and L. casei BL380 (bnaG) at an MOI of 2,000:1. S. aureus was used at an MOI of 100:1. The internalization assay of S. aureus alone was used as a reference. Internalization rates were then defined as the internalized S. aureus population in the presence of the different L. casei strains relative to the internalized S. aureus population of the reference experiment. Data are presented as means ± standard deviations. Each experiment was done in triplicate, and differences between groups were compared using one-way ANOVA with Bonferroni's Multiple Comparison Test. *: P < 0.05.