Secondary cell wall polymers of Enterococcus faecalis are critical for resistance to complement activation via mannose-binding lectin

Abstract

The complement system is part of our first line of defense against invading pathogens. The strategies used by Enterococcus faecalis to evade recognition by human complement are incompletely understood. In this study, we identified an insertional mutant of the wall teichoic acid (WTA) synthesis gene tagB in E. faecalis V583 that exhibited an increased susceptibility to complement-mediated killing by neutrophils. Further analysis revealed that increased killing of the mutant was due to a higher rate of phagocytosis by neutrophils, which correlated with higher C3b deposition on the bacterial surface. Our studies indicated that complement activation via the lectin pathway was much stronger on the tagB mutant compared with wild type. In concordance, we found an increased binding of the key lectin pathway components mannose-binding lectin and mannose-binding lectin-associated serine protease-2 (MASP-2) on the mutant. To understand the mechanism of lectin pathway inhibition by E. faecalis, we purified and characterized cell wall carbohydrates of E. faecalis wild type and V583ΔtagB. NMR analysis revealed that the mutant strain lacked two WTAs with a repeating unit of →6)[α-l-Rhap-(1→3)]β-D-GalpNAc-(1→5)-Rbo-1-P and →6) β-D-Glcp-(1→3) [α-D-Glcp-(1→4)]-β-D-GalpNAc-(1→5)-Rbo-1-P→, respectively (Rbo, ribitol). In addition, compositional changes in the enterococcal rhamnopolysaccharide were noticed. Our study indicates that in E. faecalis, modification of peptidoglycan by secondary cell wall polymers is critical to evade recognition by the complement system.

FIGURE 1.

Opsonophagocytosis and complement deposition of E. faecalis mutants in WTA biosynthesis. A, opsonophagocytic killing of E. faecalis V583 wild type and WTA biosynthesis mutants in the presence of 1.7% absorbed baby rabbit serum in combination with human WBCs, or combined with WBC plus specific rabbit Ab. Percentage of killing was determined as a relative to colony-forming units from control tubes containing bacteria and neutrophils only. B, phagocytosis of FITC-labeled bacteria after 15 min of incubation with absorbed human serum. Error bars represent the mean ± S.E. of three separate experiments using different donors. *, p < 0.05 for mutant versus wild type (two-tailed Student's t test). ***, p < 0.001 (one-way analysis of variance with Dunnett's multiple comparison test for comparison with E. faecalis V583 wild type). MFI, mean fluorescence intensity.

FIGURE 2.

Increased C3b deposition on E. faecalis V583ΔtagB. C3b deposition on enterococcal bacterial cells measured by flow cytometry after incubation with human absorbed serum. A, left: dot plot showing bacterial cells and how they were gated for fluorescence analyses. Right: representative histogram of C3b deposition analysis (2.5% serum). B, C3b deposition on E. faecalis V583 wild type and ΔtagB in absorbed serum. Error bars represent the mean ± S.E. of three separate experiments. *, p < 0.05; **, p < 0.005 for mutant versus wild type (two-tailed Student's t test). MFI, mean fluorescence intensity.

FIGURE 3.

Increased C3b deposition on E. faecalis V583ΔtagB is mediated via the lectin pathway. A, C3b deposition on E. faecalis in human absorbed serum via the alternative pathway. B, C4b deposition on E. faecalis in human absorbed serum. C, C3b deposition on E. faecalis cells in C1q-depleted human serum. Error bars represent the mean ± S.E. of three separate experiments. *, p < 0.05; **, p < 0.005 for mutant versus wild type (two-tailed Student's t test). MFI, mean fluorescence intensity.

FIGURE 4.

E. faecalis V583ΔtagB cells bind higher amounts of MBL. E. faecalis was incubated with human absorbed serum and binding of MASP-2 (A), L-ficolin (B), and MBL (C) was detected. D, binding of purified MBL to E. faecalis cells. Error bars represent the mean ± S.E. of three separate experiments. *, p < 0.05; **, p < 0.005 for mutant versus wild type (two-tailed Student's t test). MFI, mean fluorescence intensity.

FIGURE 5.

SDS-PAGE of enzymatically digested cell wall extracts. A, staining with periodic acid-Schiff's reagent (PAS). B, staining with Stains All. Lane 1, molecular mass standard; lane 2, cell wall extracts of E. faecalis V583 wild type; lane 3, cell wall extracts of E. faecalis V583ΔtagB.

FIGURE 6.

Chemical structures of teichoic acids from E. faecalis V583 wild type. A, structure of OS I and OS II. B, structure of WTA I and WTA II. A, α-l-Rha; B, β-d-GalNAc; K, Rbo; D, α-d-Glc; E, β-d-GalNAc; F, β-d-Glc; G, Rbo. For corresponding ¹H and ¹³C NMR chemical shifts, see supplemental Tables 1 and 3. The numbering of atoms in Rbo residue is reversed in oligosaccharides compared with the polymers according to the International Union of Pure and Applied Chemistry rules.