Enterococcus faecalis capsular polysaccharide serotypes C and D and their contributions to host innate immune evasion - PubMed (original) (raw)

Enterococcus faecalis capsular polysaccharide serotypes C and D and their contributions to host innate immune evasion

Lance R Thurlow et al. Infect Immun. 2009 Dec.

Abstract

It has become increasingly difficult to treat infections caused by Enterococcus faecalis due to its high levels of intrinsic and acquired antibiotic resistance. However, few studies have explored the mechanisms that E. faecalis employs to circumvent the host innate immune response and establish infection. Capsular polysaccharides are important virulence factors that are associated with innate immune evasion. We demonstrate, using cultured macrophages (RAW 264.7), that capsule-producing E. faecalis strains of either serotype C or D are more resistant to complement-mediated opsonophagocytosis than unencapsulated strains. We show that differences in opsonophagocytosis are not due to variations in C3 deposition but are due to the ability of capsule to mask bound C3 from detection on the surface of E. faecalis. Similarly, E. faecalis capsule masks lipoteichoic acid from detection, which correlates with decreased tumor necrosis factor alpha production by cultured macrophages in the presence of encapsulated strains compared to that in the presence of unencapsulated strains. Our studies confirm the important role of the capsule as a virulence factor of E. faecalis and provide several mechanisms by which the presence of the capsule influences evasion of the innate immune response and suggest that the capsule could be a potential target for developing alternative therapies to treat E. faecalis infections.

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Figures

FIG. 1.

FIG. 1.

Capsule serotypes C and D are resistant to opsonophagocytosis in the presence of complement. (A) Representative micrographs depicting LT12 (V583 expressing GFP), LT13 (Δ_cpsF_ expressing GFP), and LT14 (Δ_cpsC_ expressing GFP) incubated with RAW 264.7 macrophage-like cells. (B) Quantification of the phagocytic index expressed as a percentage of that of the unencapsulated LT14 strain (see Materials and Methods for calculation of the phagocytic index). The light-gray bar (LT12; serotype C) and the dark-gray bar (LT13; serotype D) both show a significant reduction in the phagocytic index compared to LT14 (black bar). The error bars represent the standard errors of three replicates.

FIG. 2.

FIG. 2.

Amounts of C3 deposition do not differ between strains. Western blot analysis was employed to examine the amounts of C3 deposited on the cell surfaces of serotype C (FA2-2), serotype D (LT01), and unencapsulated (LT05 and OG1RF) strains. The blot shows the 75-kDa β chain of C3 for FA2-2 (A), LT02 (B), LT05 (C), OG1RF (D), and the negative control, FA2-2 (E), incubated with heat-inactivated serum. The additional bands present on the blot are unprocessed C3, as well as C3 and C3b breakdown products recognized by the polyclonal antibodies to C3.

FIG. 3.

FIG. 3.

Complement C3 is masked from detection by capsule. Flow cytometry was used, in conjunction with anti-C3 antibodies and FITC-conjugated secondary antibodies, to evaluate the availability of C3 to detection. (A) Representative histograms depicting (from left to right) flow cytometry results for serotype C (V583), serotype D (LT02), and unencapsulated (LT06) E. faecalis strains. The isotype controls are light gray, and the C3 antibody-treated cells are dark gray. (B) Quantification of the C3-positive cells. Using one-way ANOVA, in conjunction with a Newman-Keuls posttest, statistical analysis of three replicates showed statistically significant differences (P < 0.05) in the amounts of positively labeled bacteria when V583 (light-gray bar) and LT06 (black bar) were compared and when LT02 (dark-gray bar) and LT06 were compared. Statistical analysis also revealed a significant difference in C3 detection between V583 and LT02 (P < 0.05). The error bars represent standard errors for three replicates. Approximately 50,000 bacteria were analyzed for each replicate.

FIG. 4.

FIG. 4.

The presence of capsule masks LTA from detection by antibodies. Flow cytometry was used, in conjunction with LTA antiserum and FITC-conjugated secondary antibodies, to evaluate the levels of LTA accessibility. (A) Representative histograms depicting (from left to right) flow cytometry results for serotype C (V583), seroypte D (LT02), and unencapsulated (LT06) E. faecalis strains. The isotype controls are light gray, and the anti-LTA antibody-treated cells are dark gray. (B) Quantification of LTA detection by flow cytometry. Statistical analysis of three replicates using one-way ANOVA, in conjunction with a Newman-Keuls posttest, showed significant differences (P < 0.05) between the amounts of LTA detected in V583 (light-gray bar) and LT06 (black bar) and between LT02 (dark-gray bar) and LT06, with P values of less than 0.05. However, there was no statistical difference in LTA detection when LT02 was compared V583. The error bars represent standard errors for three replicates. Approximately 50,000 bacteria were analyzed for each replicate.

FIG. 5.

FIG. 5.

E. faecalis capsule reduces TNF-α production by RAW 264.7 cells. Macrophage-like RAW 264.7 cells were incubated with serotype C (V583), serotype D (T-5 and LT02), and unencapsulated (LT06, 12030, and OG1RF) E. faecalis strains. The supernatants were collected and analyzed by ELISA for TNF-α content. The results show TNF-α production by RAW 264.7 cells in the presence of each strain. Statistical analysis of three replicates using one-way ANOVA and a Newman-Kuels post hoc test showed significant differences between the amounts of TNF-α produced in response to T-5, V583, and LT02 and those produced in response to LT06, 12030, and OG1RF. Interestingly, there was no statistically significant difference between the amounts of TNF-α produced by uninduced RAW cells and the three encapsulated strains. The error bars represent standard errors for three replicate experiments.

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