Institute for Medical Microbiology, Virology, and Hygiene, University Medical Center Eppendorf, Hamburg, Germany.
Institute for Medical Microbiology, Virology, and Hygiene, University Medical Center Eppendorf, Hamburg, Germany
FASEB J. 2014 Jul;28(7):3075-89. doi: 10.1096/fj.13-247049. Epub 2014 Apr 2.
Borrelia burgdorferi is the causative agent of Lyme disease, an infectious disease that primarily affects the skin, nervous system, and joints. Uptake of borreliae by immune cells is decisive for the course of the infection, and remodelling of the host actin cytoskeleton is crucial in this process. In this study, we showed that the actin-regulatory formin Daam1 is important in Borrelia phagocytosis by primary human macrophages. Uptake of borreliae proceeds preferentially through capture by filopodia and formation of coiling pseudopods that enwrap the spirochetes. Using immunofluorescence, we localized endogenous and overexpressed Daam1 to filopodia and to F-actin-rich uptake structures. Live-cell imaging further showed that Daam1 is enriched at coiling pseudopods that arise from the macrophage surface. This filopodia-independent step was corroborated by control experiments of phagocytic cup formation with latex beads. Moreover, siRNA-mediated knockdown of Daam1 led to a 65% reduction of borreliae-induced filopodia, and, as shown by the outside-inside staining technique, to a 50% decrease in phagocytic uptake of borreliae, as well as a 37% reduction in coiling pseudopod formation. Collectively, we showed that Daam1 plays a dual role in the phagocytic uptake of borreliae: first, as a regulator of filopodia, which are used for capturing spirochetes, and second, in the formation of the coiling pseudopod that enwraps the bacterial cell. These data identify Daam1 as a novel regulator of B. burgdorferi phagocytosis. At the same time, this is the first demonstration of a role for Daam1 in phagocytic processes in general.-Hoffmann, A.-K., Naj, X., Linder, S. Daam1 is a regulator of filopodia formation and phagocytic uptake of Borrelia burgdorferi by primary human macrophages.
伯氏疏螺旋体是莱姆病的病原体，莱姆病是一种主要影响皮肤、神经系统和关节的传染病。免疫细胞摄取伯氏疏螺旋体对感染过程至关重要，宿主肌动蛋白细胞骨架的重塑在此过程中起着关键作用。在这项研究中，我们表明，肌动蛋白调节因子 Daam1 在伯氏疏螺旋体被原代人巨噬细胞吞噬的过程中很重要。伯氏疏螺旋体的摄取优先通过被丝状伪足捕获和形成螺旋伪足来进行，这些伪足包裹着螺旋体。通过免疫荧光，我们将内源性和过表达的 Daam1 定位在丝状伪足和富含 F-肌动蛋白的摄取结构上。活细胞成像进一步显示，Daam1 在从巨噬细胞质膜上产生的螺旋伪足中富集。这个丝状伪足不依赖的步骤得到了用乳胶珠进行吞噬杯形成的对照实验的证实。此外，Daam1 的 siRNA 介导敲低导致诱导的丝状伪足减少 65%，并且如通过内外染色技术所示，对伯氏疏螺旋体的吞噬摄取减少 50%，以及螺旋伪足形成减少 37%。总的来说，我们表明 Daam1 在伯氏疏螺旋体的吞噬摄取中起双重作用：首先，作为丝状伪足的调节剂，丝状伪足用于捕获螺旋体，其次，在包裹细菌细胞的螺旋伪足形成中起作用。这些数据将 Daam1 确定为伯氏疏螺旋体吞噬作用的新调节剂。同时，这是 Daam1 在吞噬过程中起作用的第一个例证。
