Department of Chemical and Materials Engineering, University of Kentucky, Lexington, Kentucky 40506-0054, USA.
Bioconjug Chem. 2012 Feb 15;23(2):184-95. doi: 10.1021/bc200436x. Epub 2012 Jan 19.
Past work has shown that Treponema pallidum, the causative agent of syphilis, binds host fibronectin (FN). FN and other host proteins are believed to bind to rare outer membrane proteins (OMPs) of T. pallidum, and it is postulated that this interaction may facilitate cell attachment and mask antigenic targets on the surface. This research seeks to prepare a surface capable of mimicking the FN binding ability of T. pallidum in order to investigate the impact of FN binding with adsorbed Tp0483 on the host response to the surface. By understanding this interaction, it may be possible to develop more effective treatments for infection and possibly mimic the stealth properties of the bacteria. Functionalized self-assembled monolayers (SAMs) on gold were used to investigate rTp0483 and FN adsorption. Using a quartz crystal microbalance (QCM), rTp0483 adsorption and subsequent FN adsorption onto rTp0483 were determined to be higher on negatively charged carboxylate-terminated self-assembled monolayers (-COO(-) SAMs) compared to the other surfaces analyzed. Kinetic analysis of rTp0483 adsorption using surface plasmon resonance (SPR) supported this finding. Kinetic analysis of FN adsorption using SPR revealed a multistep event, where the concentration of immobilized rTp0483 plays a role in FN binding. An examination of relative QCM dissipation energy compared to the shift in frequency showed a correlation between the physical properties of adsorbed rTp0483 and SAM surface chemistry. In addition, AFM images of rTp0483 on selected SAMs illustrated a preference of rTp0483 to bind as aggregates. Adsorption on -COO(-) SAMs was more uniform across the surface, which may help further explain why FN bound more strongly. rTp0483 antibody studies suggested the involvement of amino acids 274-289 and 316-333 in binding between rTp0483 to FN, while a peptide blocking study only showed inhibition of binding with amino acids 316-333. Finally, surface adsorbed rTp0483 with FN bound significantly less anti-RGD and gelatin compared to FN adsorbed directly to -COO(-) SAMs, indicating that one or both binding regions may play a role in binding between rTp0483 and FN.
过去的研究表明，梅毒螺旋体（导致梅毒的病原体）结合宿主纤维连接蛋白（FN）。FN 和其他宿主蛋白被认为与梅毒螺旋体罕见的外膜蛋白（OMPs）结合，据推测这种相互作用可能有助于细胞附着并掩盖表面的抗原靶标。这项研究旨在制备一种能够模拟梅毒螺旋体结合 FN 能力的表面，以研究 FN 与吸附的 Tp0483 结合对宿主对表面反应的影响。通过了解这种相互作用，有可能开发出更有效的感染治疗方法，并可能模拟细菌的隐身特性。金的功能化自组装单层（SAM）用于研究 rTp0483 和 FN 的吸附。使用石英晶体微天平（QCM），确定 rTp0483 的吸附以及随后 rTp0483 上 FN 的吸附在带负电荷的羧酸盐端自组装单层（-COO（-）SAM）上比分析的其他表面更高。使用表面等离子体共振（SPR）对 rTp0483 吸附的动力学分析支持了这一发现。使用 SPR 对 FN 吸附的动力学分析显示出多步事件，其中固定化 rTp0483 的浓度在 FN 结合中起作用。相对 QCM 耗散能量与频率变化的比较表明，吸附 rTp0483 的物理性质与 SAM 表面化学之间存在相关性。此外，选定 SAM 上 rTp0483 的 AFM 图像说明了 rTp0483 作为聚集体结合的偏好。-COO（-）SAM 上的吸附在整个表面上更均匀，这可能有助于进一步解释为什么 FN 结合得更强。rTp0483 抗体研究表明，rTp0483 与 FN 之间的结合涉及氨基酸 274-289 和 316-333，而肽阻断研究仅显示与氨基酸 316-333 的结合抑制。最后，与 FN 直接吸附到-COO（-）SAM 相比，表面吸附的带 FN 的 rTp0483 与抗 RGD 和明胶的结合显著减少，表明一个或两个结合区域可能在 rTp0483 与 FN 之间的结合中起作用。
