Table 1.
Bacterial strains.
Figure 1.
Induction of calcium ion flux in FPR1-transfected (A) or FPR2/ALX-transfected (B) HL60 cells.
Data represent means ± SEM of 3 independent experiments with 3 different culture filtrates.
Figure 2.
Calcium influx stimulated by enterococcal culture filtrates of various clinical blood culture or stool isolates.
(A) Activation of FPR2/ALX-transfected cells by 3% E. faecium or E. faecalis culture filtrates. (B) Significantly stronger FPR2/ALX activation through 3% culture filtrates from vancomycin-resistant E. faecium (VRE) compared to vancomycin-sensitive E. faecium (VSE). Of each, VRE and VSE, five strains were analyzed. Data represent means ± SEM of 3 independent experiments and at least 3 different culture filtrates. P-value for Figure 2B was determined by the two-tailed Student's paired t-test. **** p<0.0001.
Figure 3.
Enterococcal supernatants induce calcium influx and chemotaxis in human neutrophils.
(A) Culture supernatants of E. faecalis and E. faecium, both vancomycin-resistant and vancomycin-sensitive, activate human leukocytes at different levels. (B) Activation of human leukocytes by E. faecium culture filtrates is stronger inhibited by the FPR2/ALX-specific inhibitor FLIPr than by E. faecalis culture filtrates. (C) Chemotaxis induced by E. faecium culture supernatants is inhibited by the FPR2/ALX-specific inhibitor FLIPr. (D) CD11b upregulation by E. faecium and E. faecium VRE 3% culture filtrates can be inhibited by FLIPr. Data represent means ± SEM of 3 independent experiments with 3 different culture filtrates. P-values were determined by one-way ANOVA with Bonferroni's post test. * p<0.05, ** p<0.001, *** p<0.0005, **** p<0.0001; ns, non-significant.
Figure 4.
No detectable PSM-like peptides in E. faecium culture filtrates (A) and inactivation of FPR2/ALX-dependent activation by proteolytic treatment of culture filtrates (B).
(A) 100 µl of 24-h culture filtrates of E. faecium, E. faecium VRE, and S. aureus USA300 were analyzed for the presence of PSMs using RP-HPLC/ESI-MS. The graph shows total ion chromatograms recorded in the characteristic PSM elution range (between dotted vertical lines). (B) Activation of FPR2/ALX-transfected cells is abolished by proteolytic digestion of E. faecium culture filtrates. Bacterial culture filtrate concentration was 1.5%. MMK1 concentration was 50 nM. Proteinase K was used at 1 unit ml−1. Data represent means ± SEM of 3 independent experiments with 3 different culture filtrates. P-values were determined by one-way ANOVA with Bonferroni's post test. * p<0.05, ** p<0.001, *** p<0.0005, **** p<0.0001; ns, non-significant.