Salmonellae are bacterial pathogens that have evolved sophisticated strategies to evade host immune defenses. These strategies include the secretion of effector proteins into mammalian cells so as to subvert innate immune and apoptotic signaling pathways, thereby allowing Salmonella to avoid elimination. Here, we show that the secreted Salmonella typhimurium effector protein AvrA possesses acetyltransferase activity toward specific mitogen-activated protein kinase kinases (MAPKKs) and potently inhibits c-Jun N-terminal kinase (JNK) and NF-kappaB signaling pathways in both transgenic Drosophila and murine models. Furthermore, we show that AvrA dampens the proapoptotic innate immune response to Salmonella at the mouse intestinal mucosa. This activity is consistent with the natural history of Salmonella in mammalian hosts, where the bacteria elicit transient inflammation but do not destroy epithelial cells. Our findings suggest that targeting JNK signaling to dampen apoptosis may be a conserved strategy for intracellular pathogens.

The mammalian gut microbiota is essential for normal intestinal development, renewal and repair. Injury to the intestinal mucosa can occur with infection, surgical trauma, and in idiopathic inflammatory bowel disease. Repair of mucosal injury, termed restitution, as well as restoration of intestinal homeostasis involves induced and coordinated proliferation and migration of intestinal epithelial cells. N-formyl peptide receptors (FPRs) are widely expressed pattern recognition receptors that can specifically bind and induce responses to host derived and bacterial peptides and small molecules. Here we report that specific members of the gut microbiota stimulate FPR1 on intestinal epithelial cells to generate reactive oxygen species via enterocyte NADPH oxidase NOX1, causing rapid phosphorylation of Focal Adhesion Kinase (FAK) and ERK MAPK. These events stimulate migration and proliferation of enterocytes adjacent to colonic wounds. Together, these findings identify a novel role of FPR1 as pattern recognition receptors for perceiving the enteric microbiota that promotes repair of mucosal wounds via generation of ROS from the enterocyte NOX1.

The normal microbial occupants of the mammalian intestine are crucial for maintaining gut homeostasis, yet the mechanisms by which intestinal cells perceive and respond to the microbiota are largely unknown. Intestinal epithelial contact with commensal bacteria and/or their products has been shown to activate noninflammatory signaling pathways, such as extracellular signal-related kinase (ERK), thus influencing homeostatic processes. We previously demonstrated that commensal bacteria stimulate ERK pathway activity via interaction with formyl peptide receptors (FPRs). In the current study, we expand on these findings and show that commensal bacteria initiate ERK signaling through rapid FPR-dependent reactive oxygen species (ROS) generation and subsequent modulation of MAP kinase phosphatase redox status. ROS generation induced by the commensal bacteria Lactobacillus rhamnosus GG and the FPR peptide ligand, N-formyl-Met-Leu-Phe, was abolished in the presence of selective inhibitors for G protein-coupled signaling and FPR ligand interaction. In addition, pretreatment of cells with inhibitors of ROS generation attenuated commensal bacteriainduced ERK signaling, indicating that ROS generation is required for ERK pathway activation. Bacterial colonization also led to oxidative inactivation of the redox-sensitive and ERKspecific phosphatase, DUSP3/VHR, and consequent stimulation of ERK pathway signaling. Together, these data demonstrate that commensal bacteria and their products activate ROS signaling in an FPR-dependent manner and define a mechanism by which cellular ROS influences the ERK pathway through a redox-sensitive regulatory circuit.

Commensal bacteria and/or their products engender beneficial effects to the mammalian gut, including stimulating physiological cellular turnover and enhancing wound healing, without activating overt inflammation. In the present study, we observed commensal bacteriamediated activation of the noninflammatory extracellular signal-regulated kinase[ERK]/mitogen-activated protein kinase and Akt signaling pathways in gut epithelial cells and delineated a mechanism for this bacterially activated signaling. All tested strains of commensal bacteria induced ERK phosphorylation without stimulating pro-inflammatory phospho-IB or pro-apoptotic phospho-c-Jun NH 2 -terminal kinase, with Lactobacillus species being most potent. This pattern of signaling activation was recapitulated using the peptide N-formyl-MetLeu-Phe, a bacterial product known to stimulate signaling events in mammalian phagocytes. Sensing of N-formyl-Met-Leu-Phe by gut epithelial cells occurs via recently characterized formyl peptide receptors located in the plasma membrane. Both commensal bacteria and N-formyl-Met-Leu-Phe application to the apical surface of polarized gut epithelial cells resulted in specific formyl peptide receptor activation. In addition, pretreatment of model epithelia and murine colon with Boc2 (a specific peptide antagonist) or pertussis toxin (a G i -protein inhibitor) abolished commensalmediated ERK phosphorylation. Taken together, these data show that commensal bacteria specifically activate the ERK/mitogen-activated protein kinase pathway in an formyl peptide receptor-dependent manner, delineating a mechanism by which commensal bacteria contribute to cellular signaling in gut epithelia. (Am J

N‐formyl peptide receptors (FPRs), which in humans include FPR1, FPR2 and FPR3, are an important family of pattern recognition receptors that bind an array of bacterial as well as host derived peptide and nonpeptide ligands. FPRs are G protein coupled surface receptors, which are well described in mediating phagocyte functions. We have demonstrated FPRs on the apical surface of intestinal epithelial cells (IEC) and have shown that in IECs FPR induces ROS generation in response to bacterial fMLF (a formylated tripeptide), viable Lactobacillus rhamnosus GG, and endogenous annexin A1 mimetic (Ac 2‐26). Furthermore, we showed that FPR‐elicited ROS activates the noninflammatory ERK MAPK pathway and enhances migration of IECs. We hypothesize that FPR1, the high‐affinity receptor, is important for motility of intestinal epithelia. Mice deficient in FPR1 demonstrates normal intestinal tissue architecture; however, intestinal crypts of the FPR1 null mouse contain more proliferating cells, and show slower migration along the crypt‐villus axis. These data suggest that FPR1 is important in maintaining homeostasis of the intestinal epithelia.