A hypervariable N-terminal region of Yersinia LcrV determines Toll-like receptor 2-mediated IL-10 induction and mouse virulence

Abstract

The virulence antigen LcrV of Yersinia enterocolitica O:8 induces IL-10 in macrophages via Toll-like receptor 2 (TLR2). The TLR2-active region of LcrV is localized within its N-terminal amino acids (aa) 31-57. Sequencing of codons 25-92 of the lcrV gene from 59 strains of the three pathogenic Yersinia species revealed a hypervariable hotspot within aa 40-61. According to these sequence differences, seven LcrV groups were identified, with Y. pestis and Y. pseudotuberculosis represented in group I and the other six distributed within Y. enterocolitica. By testing LcrV sequence-derived synthetic oligopeptides of all seven LcrV groups in CD14/TLR2-transfected human embryonic kidney 293 cells, we found the highest TLR2 activity with a peptide derived from group IV comprising exclusively Y. enterocolitica O:8 strains. These findings were verified in murine peritoneal macrophages by using recombinant LcrV truncates representing aa 1-130 from different Yersinia spp. By systematically replacing charged aa residues by glutamine in synthetic oligopeptides, we show that the K42Q substitution leads to abrogation of TLR2 activity in both in vitro cell systems. This K42Q substitution was introduced in the lcrV gene from Y. enterocolitica O:8 WA-C(pYV), resulting in WA-C(pYVLcrV(K42Q)), which turned out to be less virulent for C57BL/6 mice than the parental strain. This difference in virulence was not observed in TLR2(-/-) or IL-10(-/-) mice, proving that LcrV contributes to virulence by TLR2-mediated IL-10 induction. LcrV is a defined bacterial virulence factor shown to target the TLR system for evasion of the host's immune response.

Fig. 1.

Variable TLR2-activity in the N-terminal LcrV of different Yersinia strains. (a) NF-κB-dependent luciferase reporter activity of 36-meric synthetic peptides (20 μg/ml; 5 μM) representing the N-terminal aa 31-66 of LcrV, derived from different Yersinia strains in CD14/TLR2-transfected HEK 293 cells. I-VII indicate the respective LcrV groups listed in Table 1, c (control) indicates unstimulated cells. (b) NF-κB-dependent luciferase reporter activity in CD14/TLR2-transfected HEK 293 cells after treatment with recombinant proteins comprising the 130 N-terminal aa from Y. enterocolitica O:8, O:3, and Y. pseudotuberculosis (pstb) (1.9 μg/ml; 135 nM).

Fig. 2.

A single point mutation in the N terminus of LcrV abolishes its TLR2-activity. (a) NF-κB-dependent luciferase reporter activity of 20-meric synthetic peptides (10 μg/ml; 5 μM) representing the N-terminal aa 31-49 of Y. enterocolitica O:8 strain WA-314 LcrV without or with single aa mutations. (b and c) NF-κB-dependent luciferase reporter activity in CD14/TLR2-transfected HEK 293 cells (b) and IL-10-production by C57BL/6 proteose peptone (c) elicited peritoneal macrophages after treatment with recombinant proteins (5 μg/ml; 135 nM), representing wild-type LcrV or LcrV derivates bearing a single point mutation. **, P < 0.01 compared with negative controls (PBS).

Fig. 3.

The K42Q mutation impairs the TLR2-dependent pathogenicity of Y. enterocolitica. Survival curves of C57BL/6 mice infected i.p. (5 × 10³ and 10⁴ CFU; n = 11 to 12 mice) (a) or orally (10⁷ CFU; n = 16 mice for lcrV_WT, n = 11 mice for lcrV_K42Q)(b) with lcrV_WT or lcrV_K42Q, respectively. (c) Survival curves of C57BL/6 (n = 12 per group), TLR2^-/- (n = 10 per group), and IL-10^-/- (n = 12 per group) mice infected i.p. (10⁵ CFU) with lcrV_WT or lcrV_K42Q, respectively. (d) Bacterial load in spleen and Peyer's patches (PP) of C57BL/6(n = 21 per group), TLR2^-/- (n = 11 per group), and IL-10^-/- (n = 10 per group) mice 7 days after peroral infection (5 × 10⁸ CFU) with lcrV_WT or lcrV_K42Q, respectively. *, P < 0.05 when comparing lcrV_WT-vs. lcrV_K42Q-infected mice of the indicated strain. (e) Splenic IL-10 production in C57BL/6(n = 12 per group) and TLR2^-/- (n = 10 per group) mice 7 days after peroral infection (5 × 10⁸ CFU) with lcrV_WT or lcrV_K42Q, respectively. **, P < 0.01 when comparing lcrV_WT-vs. lcrV_K42Q-infected mice of the indicated strain.

Fig. 4.

3D structure and sequence of Y. pestis LcrV. (a) The image shows a 3D model of Y. pestis LcrV as reported in ref. . The α1-helix (green), the β1-strand (turquoise), the site for the K42Q mutation (red), and the hotspots at aa positions 40, 43, and 61 (yellow) are highlighted. The hotspot at aa 53 cannot be shown, because this region revealed no interpretable crystallographic structure. The ribbon diagram was generated with deepview pdb.(b) Sequence alignment of the N terminus from Y. pestis LcrV and P. aeruginosa PcrV. The α1-helix and the β1-strand of LcrV according to ref. are colored in green and turquoise, respectively. The sequence alignment was performed with dnaman.