Cell invasion by Trypanosoma cruzi extracellular amastigotes (EAs) relies significantly upon the host cell actin cytoskeleton. In past decades EAs have been established as a reliable model for phagocytosis inducer in non-phagocytic cells. Our current hypothesis is that EAs engage a phagocytosis-like mechanism in non-professional phagocytic cells; however, the molecular mechanisms in professional phagocytes still remain unexplored. In this work, we evaluated the involvement of Rac1 and Cdc42 in the actin-dependent internalization of EAs in RAW 264.7 macrophages. Kinetic assays showed similar internalization of EAs in unstimulated RAW and non-phagocytic HeLa cells but increased in LPS/IFN-γ stimulated RAW cells. However, depletion of Rac1, Cdc42 or RhoA inhibited EA internalization similarly in both unstimulated and stimulated RAW cells. Overexpression of active, but not the dominant-negative, construct of Rac1 increased EA internalization. Remarkably, for Cdc42, both the active and the inactive mutants decreased EA internalization when compared to wild type groups. Despite that, both Rac1 and Cdc42 activation mutants were similarly recruited to and colocalized with actin at the EA-macrophage contact sites when compared to their native isoforms. Altogether, these results corroborate that EAs engage phagocytic processes to invade both professional and non-professional phagocytic cells providing evidences of converging actin mediated mechanisms induced by intracellular pathogens in both cell types.

克氏锥虫对细胞的侵袭细胞外无鞭毛体 (EA) 显着依赖于宿主细胞肌动蛋白细胞骨架。在过去的几十年中，EA 已被确立为非吞噬细胞中吞噬诱导剂的可靠模型。我们目前的假设是 EA 在非专业吞噬细胞中具有类似吞噬作用的机制。然而，专业吞噬细胞的分子机制仍未探索。在这项工作中，我们评估了 Rac1 和 Cdc42 在 RAW 264.7 巨噬细胞中 EA 的肌动蛋白依赖性内化中的参与。动力学分析显示 EA 在未刺激的 RAW 和非吞噬性 HeLa 细胞中的内化相似，但在 LPS/IFN-γ 刺激的 RAW 细胞中增加。然而，Rac1、Cdc42 或 RhoA 的消耗在未刺激和刺激的 RAW 细胞中类似地抑制了 EA 内化。活性过度表达，但不是显性负性，Rac1 的构造增加了 EA 内化。值得注意的是，对于 Cdc42，与野生型组相比，活性和非活性突变体均降低了 EA 内化。尽管如此，与它们的天然亚型相比，Rac1 和 Cdc42 激活突变体在 EA 巨噬细胞接触位点同样被招募并与肌动蛋白共定位。总而言之，这些结果证实 EA 参与吞噬过程以侵入专业和非专业吞噬细胞，提供了两种细胞类型中由细胞内病原体诱导的趋同肌动蛋白介导机制的证据。与它们的天然亚型相比，Rac1 和 Cdc42 激活突变体在 EA 巨噬细胞接触位点被类似地招募并与肌动蛋白共定位。总而言之，这些结果证实 EA 参与吞噬过程以侵入专业和非专业吞噬细胞，提供了两种细胞类型中由细胞内病原体诱导的趋同肌动蛋白介导机制的证据。与它们的天然亚型相比，Rac1 和 Cdc42 激活突变体在 EA 巨噬细胞接触位点被类似地招募并与肌动蛋白共定位。总而言之，这些结果证实 EA 参与吞噬过程以侵入专业和非专业吞噬细胞，提供了两种细胞类型中由细胞内病原体诱导的趋同肌动蛋白介导机制的证据。