Sing Andreas, Reithmeier-Rost Dagmar, Granfors Kaisa, Hill Jim, Roggenkamp Andreas, Heesemann Jürgen
Lehrstuhl Bakteriologie, Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Pettenkoferstrasse 9a, 80336 Munich, Germany.
Proc Natl Acad Sci U S A. 2005 Nov 1;102(44):16049-54. doi: 10.1073/pnas.0504728102. Epub 2005 Oct 20.
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.
小肠结肠炎耶尔森菌O:8的毒力抗原LcrV通过Toll样受体2（TLR2）在巨噬细胞中诱导白细胞介素-10（IL-10）。LcrV的TLR2活性区域位于其N端氨基酸（aa）31 - 57内。对三种致病性耶尔森菌属的59个菌株的lcrV基因的25 - 92密码子进行测序，发现在aa 40 - 61内有一个高变热点。根据这些序列差异，鉴定出七个LcrV组，鼠疫耶尔森菌和假结核耶尔森菌在第一组中，其他六个分布在小肠结肠炎耶尔森菌中。通过在CD14/TLR2转染的人胚肾293细胞中测试所有七个LcrV组的LcrV序列衍生的合成寡肽，我们发现来自仅包含小肠结肠炎耶尔森菌O:8菌株的第四组的肽具有最高的TLR2活性。通过使用代表来自不同耶尔森菌属的aa 1 - 130的重组LcrV截短体，在小鼠腹腔巨噬细胞中验证了这些发现。通过在合成寡肽中用谷氨酰胺系统地取代带电荷的aa残基，我们表明K42Q取代导致两种体外细胞系统中TLR2活性的丧失。将这种K42Q取代引入小肠结肠炎耶尔森菌O:8 WA-C（pYV）的lcrV基因中，产生WA-C（pYVLcrV（K42Q）），结果证明其对C57BL/6小鼠的毒力低于亲本菌株。在TLR2（-/-）或IL-10（-/-）小鼠中未观察到这种毒力差异，证明LcrV通过TLR2介导的IL-10诱导作用对毒力有贡献。LcrV是一种确定的细菌毒力因子，显示其靶向TLR系统以逃避宿主的免疫反应。
