Davidson Anthony C, Humphreys Daniel, Brooks Andrew B E, Hume Peter J, Koronakis Vassilis
Department of Pathology, Cambridge University, Cambridge, United Kingdom.
Department of Pathology, Cambridge University, Cambridge, United Kingdom
mBio. 2015 Feb 10;6(1):e02253-14. doi: 10.1128/mBio.02253-14.
To establish intracellular infections, Salmonella bacteria trigger host cell membrane ruffling and invasion by subverting cellular Arf guanine nucleotide exchange factors (GEFs) that activate Arf1 and Arf6 GTPases by promoting GTP binding. A family of cellular Arf GTPase-activating proteins (GAPs) can downregulate Arf signaling by stimulating GTP hydrolysis, but whether they do this during infection is unknown. Here, we uncovered a remarkable role for distinct Arf GAP family members in Salmonella invasion. The Arf6 GAPs ACAP1 and ADAP1 and the Arf1 GAP ASAP1 localized at Salmonella-induced ruffles, which was not the case for the plasma membrane-localized Arf6 GAPs ARAP3 and GIT1 or the Golgi-associated Arf1 GAP1. Surprisingly, we found that loss of ACAP1, ADAP1, or ASAP1 impaired Salmonella invasion, revealing that GAPs cannot be considered mere terminators of cytoskeleton remodeling. Salmonella invasion was restored in Arf GAP-depleted cells by expressing fast-cycling Arf derivatives, demonstrating that Arf GTP/GDP cycles facilitate Salmonella invasion. Consistent with this view, both constitutively active and dominant-negative Arf derivatives that cannot undergo GTP/GDP cycles inhibited invasion. Furthermore, we demonstrated that Arf GEFs and GAPs colocalize at invading Salmonella and collaborate to drive Arf1-dependent pathogen invasion. This study revealed that Salmonella bacteria exploit a remarkable interplay between Arf GEFs and GAPs to direct cycles of Arf GTPase activation and inactivation. These cycles drive Salmonella cytoskeleton remodeling and enable intracellular infections.
To initiate infections, the Salmonella bacterial pathogen remodels the mammalian actin cytoskeleton and invades host cells by subverting host Arf GEFs that activate Arf1 and Arf6 GTPases. Cellular Arf GAPs deactivate Arf GTPases and negatively regulate cell processes, but whether they target Arfs during infection is unknown. Here, we uncovered an important role for the Arf GAP family in Salmonella invasion. Surprisingly, we found that Arf1 and Arf6 GAPs cooperate with their Arf GEF counterparts to facilitate cycles of Arf GTPase activation and inactivation, which direct pathogen invasion. This report illustrates that GAP proteins promote actin-dependent processes and are not necessarily restricted to negatively regulating cellular signaling. It uncovers a remarkable interplay between Arf GEFs and GAPs that is exploited by Salmonella to establish infection and expands our understanding of Arf GTPase-regulated cytoskeleton remodeling.
为了建立细胞内感染，沙门氏菌通过破坏细胞内的Arf鸟嘌呤核苷酸交换因子（GEF）来触发宿主细胞膜的褶皱和入侵，这些因子通过促进GTP结合来激活Arf1和Arf6 GTP酶。一类细胞内的Arf GTP酶激活蛋白（GAP）可以通过刺激GTP水解来下调Arf信号，但它们在感染过程中是否发挥作用尚不清楚。在这里，我们发现了不同的Arf GAP家族成员在沙门氏菌入侵中的显著作用。Arf6 GAPs ACAP1和ADAP1以及Arf1 GAP ASAP1定位于沙门氏菌诱导的褶皱处，而质膜定位的Arf6 GAPs ARAP3和GIT1或高尔基体相关的Arf1 GAP1则不然。令人惊讶的是，我们发现ACAP1、ADAP1或ASAP丢失会损害沙门氏菌的入侵，这表明GAP不能仅仅被视为细胞骨架重塑的终止者。通过表达快速循环的Arf衍生物，在Arf GAP缺失的细胞中恢复了沙门氏菌的入侵，这表明Arf GTP/GDP循环促进了沙门氏菌的入侵。与此观点一致，不能进行GTP/GDP循环的组成型激活和显性负性Arf衍生物均抑制入侵。此外，我们证明了Arf GEFs和GAPs在入侵的沙门氏菌处共定位，并协同驱动Arf1依赖性病原体入侵。这项研究表明，沙门氏菌利用Arf GEFs和GAPs之间的显著相互作用来指导Arf GTP酶的激活和失活循环。这些循环驱动沙门氏菌细胞骨架重塑并实现细胞内感染。
为了引发感染，沙门氏菌病原体通过破坏激活Arf1和Arf6 GTP酶的宿主Arf GEFs来重塑哺乳动物肌动蛋白细胞骨架并入侵宿主细胞。细胞内的Arf GAPs使Arf GTP酶失活并对细胞过程进行负调控，但它们在感染过程中是否靶向Arfs尚不清楚。在这里，我们发现了Arf GAP家族在沙门氏菌入侵中的重要作用。令人惊讶的是，我们发现Arf1和Arf6 GAPs与其Arf GEF对应物协同作用，促进Arf GTP酶的激活和失活循环，从而指导病原体入侵。本报告表明，GAP蛋白促进肌动蛋白依赖性过程，不一定局限于对细胞信号进行负调控。它揭示了Arf GEFs和GAPs之间的显著相互作用，沙门氏菌利用这种相互作用来建立感染，并扩展了我们对Arf GTP酶调节的细胞骨架重塑的理解。
