Enterococcus faecium stimulates human neutrophils via the formyl-peptide receptor 2

Abstract

The human formyl-peptide receptor 2 (FPR2/ALX) senses phenol-soluble modulin (PSM) peptide toxins produced by pathogenic staphylococcal species and plays a crucial role in directing neutrophil influx during staphylococcal infection. However, it has remained unclear if FPR2 responds also to molecules from other bacterial pathogens. Here we analyzed a variety of gram-positive and gram-negative pathogens and found that apart from staphylococci only certain enterococcal strains have the capacity to stimulate FPR2/ALX. Most of the analyzed Enterococcus faecium but only sporadic Enterococcus faecalis strains released FPR2/ALX-stimulating molecules leading to neutrophil calcium ion fluxes, chemotaxis, and complement receptor upregulation. Among ten test strains vancomycin-resistant E. faecium had a significantly higher capacity to stimulate FPR2/ALX than vancomycin-susceptible strains, suggesting an association of strong FPR2/ALX activation with health-care associated strains. The enterococcal FPR2/ALX agonists were found to be peptides or proteins, which appear, however, to be unrelated to staphylococcal PSMs in sequence and physicochemical properties. Enterococci are among the most frequent invasive bacterial pathogens but the basis of enterococcal virulence and immune activation has remained incompletely understood. Our study indicates that previously unrecognized proteinaceous agonists contribute to Enterococcus-host interaction and underscores the importance of FPR2/ALX in host defense against major endogenous bacterial pathogens.

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.