Critical Role for the Host GTPase-Activating Protein ARAP2 in InlB-Mediated Entry of Listeria monocytogenes

The bacterial pathogen Listeria monocytogenes causes food-borne illnesses culminating in gastroenteritis, meningitis, or abortion. Listeria induces its internalization into some mammalian cells through binding of the bacterial surface protein InlB to the host receptor tyrosine kinase Met. Interaction of InlB with the Met receptor elicits host downstream signaling pathways that promote F-actin cytoskeletal changes responsible for pathogen engulfment. Here we show that the mammalian signaling protein ARAP2 plays a critical role in cytoskeletal remodeling and internalization of Listeria. Depletion of ARAP2 through RNA interference (RNAi) caused a marked inhibition of InlB-mediated F-actin rearrangements and bacterial entry. ARAP2 contains multiple functional domains, including a GTPase-activating protein (GAP) domain that antagonizes the GTPase Arf6 and a domain capable of binding the GTPase RhoA. Genetic data indicated roles for both the Arf GAP and RhoA binding domains in Listeria entry. Experiments involving Arf6 RNAi or a constitutively activated allele of Arf6 demonstrated that one of the ways in which ARAP2 promotes bacterial uptake is by restraining the activity of Arf6. Conversely, Rho activity was dispensable for Listeria internalization, suggesting that the RhoA binding domain in ARAP2 acts by engaging a host ligand other than Rho proteins. Collectively, our findings indicate that ARAP2 promotes InlB-mediated entry of Listeria, in part, by antagonizing the host GTPase Arf6.Listeria monocytogenes is a Gram-positive, food-borne bacterial pathogen capable of causing gastroenteritis, meningitis, or abortions (39, 32). Listeria induces its own internalization (entry) into nonphagocytic mammalian cells, a process that likely plays an important role in traversal of the intestinal, placental, and blood-brain barriers (7, 14, 16, 23). One of the pathways of Listeria entry is mediated by interaction of the bacterial surface protein InlB with its host receptor, the Met receptor tyrosine kinase (16, 37).InlB-Met interaction triggers activation (tyrosine phosphorylation) of the Met receptor and subsequent rearrangements in the F-actin cytoskeleton of the mammalian cell (16, 29). These cytoskeletal changes remodel the host cell surface, resulting in engulfment of adherent Listeria. One of the host mammalian proteins that acts downstream of Met to promote F-actin rearrangements and bacterial entry is type IA phosphoinositide (PI) 3-kinase (8, 17, 18). This PI 3-kinase is a heterodimeric enzyme comprised of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit (4, 11). p85-p110 generates phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂)] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃], which are lipid second messengers that regulate a variety of biological processes, including growth, survival, and motility of mammalian cells. A plethora of downstream “target” proteins that bind PI(4,5)P₂ and/or PI(3,4,5)P₃ and mediate the biological effects of p85-p110 have been identified (4, 24). However, mammalian proteins that act downstream of type IA PI 3-kinase to control Listeria uptake have yet to found.In this work, we demonstrate that the human GTPase-activating protein (GAP) ARAP2 is required for InlB-mediated cytoskeletal changes and entry of Listeria. ARAP2 is known to bind PI(3,4,5)P₃, resulting in upregulation of a GAP domain that inactivates the mammalian GTPase Arf6 (42). We provide genetic evidence indicating that the ArfGAP domain of ARAP2 stimulates Listeria entry by antagonizing Arf6. ARAP2 has several functional domains in addition to its ArfGAP domain. One of these domains in ARAP2 interacts with the mammalian GTPase RhoA (42). Our genetic data demonstrate that this “RhoA binding” (RB) domain also plays a critical role in bacterial entry. Surprisingly, pharmacological experiments indicate that the RB domain controls Listeria uptake through an unknown mechanism that does not involve Rho proteins. Our work indicates a key role for host ARAP2 in InlB-mediated entry of Listeria. One of the likely ways that type IA PI 3-kinase controls entry of Listeria is through regulation of ARAP2.