Centro Ricerche Interdipartimentale Biotecnologie Innovative, University of Padova, Padova, Italy.
PLoS One. 2011;6(9):e25089. doi: 10.1371/journal.pone.0025089. Epub 2011 Sep 16.
The adhesin NadA favors cell adhesion/invasion by hypervirulent Neisseria meningitidis B (MenB). Its recombinant form NadA(Δ351-405,) devoid of the outer membrane domain, is an immunogenic candidate for an anti-MenB vaccine able to stimulate monocytes, macrophages and dendritic cells. In this study we investigated the molecular mechanism of NadA(Δ351-405) cellular effects in monocytes. We show that NadA(Δ351-405) (against which we obtained polyclonal antibodies in rabbits), binds to hsp90, but not to other extracellular homologous heat shock proteins grp94 and hsp70, in vitro and on the surface of monocytes, in a temperature dependent way. Pre-incubation of monocytes with the MenB soluble adhesin interfered with the binding of anti-hsp90 and anti-hsp70 antibodies to hsp90 and hsp70 at 37°C, a condition in which specific cell-binding occurs, but not at 0°C, a condition in which specific cell-binding is very diminished. Conversely, pre-incubation of monocytes with anti-hsp90 and anti-hsp70 antibodies did not affected NadA(Δ351-405) cell binding in any temperature condition, indicating that it associates to another receptor on their plasma membrane and then laterally diffuses to encounter hsp90. Consistently, polymixin B interfered with NadA(Δ351-405) /hsp90 association, abrogated the decrease of anti-hsp90 antibodies binding to the cell surface due to NadA(Δ351-405) and inhibited adhesin-induced cytokine/chemokine secretion without affecting monocyte-adhesin binding. Co-stimulation of monocytes with anti-hsp90 antibodies and NadA(Δ351-405) determined a stronger but polymixin B insensitive cell activation. This indicated that the formation of a recombinant NadA/hsp90/hsp70 complex, although essential for full monocyte stimulation, can be replaced by anti-hsp90 antibody/hsp90 binding. Finally, the activation of monocytes by NadA(Δ351-405) alone or in the presence of anti-hsp90 antibodies were both inhibited by neutralizing anti-TLR4 antibodies, but not by anti-TLR2 antibodies. We propose that hsp90-dependent recruitment into an hsp90/hsp70/TLR4 transducing signal complex is necessary for the immune-stimulating activity of NadA(Δ351-405) anti-MenB vaccine candidate.
粘附素 NadA 有利于高毒力脑膜炎奈瑟菌 B 型（MenB）的细胞粘附/入侵。其重组形式 NadA(Δ351-405)，缺失外膜结构域，是一种能够刺激单核细胞、巨噬细胞和树突状细胞的抗 MenB 疫苗的免疫原候选物。在这项研究中，我们研究了 NadA(Δ351-405)在单核细胞中的细胞作用的分子机制。我们表明，NadA(Δ351-405)（我们在兔子中获得了针对它的多克隆抗体），以依赖温度的方式在体外和单核细胞表面上与 hsp90 结合，但不与其他细胞外同源热休克蛋白 grp94 和 hsp70 结合。用 MenB 可溶性粘附素预孵育单核细胞，在 37°C 时干扰针对 hsp90 和 hsp70 的抗 hsp90 和抗 hsp70 抗体的结合，在该条件下会发生特异性细胞结合，但在 0°C 时不会发生特异性细胞结合，在该条件下特异性细胞结合大大减少。相反，用抗 hsp90 和抗 hsp70 抗体预孵育单核细胞不会影响 NadA(Δ351-405)在任何温度条件下的细胞结合，表明它与质膜上的另一个受体结合，然后侧向扩散以遇到 hsp90。一致地，多粘菌素 B 干扰 NadA(Δ351-405)/hsp90 结合，取消了由于 NadA(Δ351-405)导致的抗 hsp90 抗体与细胞表面结合的减少，并抑制粘附素诱导的细胞因子/趋化因子分泌，而不影响单核细胞-粘附素结合。用抗 hsp90 抗体和 NadA(Δ351-405)共同刺激单核细胞会导致更强但不受多粘菌素 B 影响的细胞激活。这表明尽管形成重组 NadA/hsp90/hsp70 复合物对于完全刺激单核细胞是必需的，但它可以被抗 hsp90 抗体/hsp90 结合所取代。最后，NadA(Δ351-405)单独或在抗 hsp90 抗体存在下激活单核细胞均被中和抗 TLR4 抗体抑制，但不受抗 TLR2 抗体抑制。我们提出 hsp90 依赖性募集到 hsp90/hsp70/TLR4 转导信号复合物是 NadA(Δ351-405)抗 MenB 疫苗候选物免疫刺激活性所必需的。
