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

Mucosal homeostasis and restitution of injury depends on epithelial proliferation and migration. The commensal microbiota of the gut lumen is essential for normal intestinal development, renewal and restitution. N‐formyl peptide receptors (FPRs) are seven membrane‐pass G protein coupled receptors that represent a family of mammalian pattern recognition receptors. FPR ligands include an array of peptides, lipids and small molecules derived from bacteria and the host. FPRs modulate phagocyte chemotaxis and oxidative burst. Intestinal epithelial cells (IECs) also express functional FPRs on the apical surface that can stimulate migration of cultured IECs in response to bacterial formyl peptides and endogenous ligands. Herein we report that a specific member of the gut microbiota requires FPR1 to stimulate reactive oxygen species (ROS) generation in IECs adjoining wounds resulting in phosphorylation of focal adhesion kinase (FAK) and ERK MAPK signaling in an intestinal epithelial NADPH oxidase Nox1 dependent manner. Moreover, we show that enteric microbiota requires FPR1 and Nox1 to stimulate redox dependent proliferation and migration of IECs, and to enhance recovery of mechanical or chemically induced mucosal injury in mouse distal colon. Taken together, these findings demonstrate a novel role for FPR1 in perceiving enteric microbiota to regulate homeostasis and restitution of the intestinal mucosa.

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.

Commensal bacteria and/or their products engender beneficial effects to the mammalian gut, including stimulating homeostatic cellular turnover and enhancing wound healing, without activating innate immune pathways. In the present study we observed commensal bacterial mediated activation of the pro‐proliferative ERK MAPK signaling pathway in gut epithelial cells and describe a mechanism for commensal‐host crosstalk. A range of commensal bacteria tested induced ERK phosphorylation without JNK or NF‐κB signaling. ERK‐specific phosphorylation was recapitulated using a purified small peptide, N‐formyl‐Met‐Leu‐Phe (fMLF), a bacterial product known to stimulate signaling in mammalian phagocytes. The fMLF tripeptide is recognized via the recently characterized epithelial formyl peptide receptors (FPR) located on the apical surface. Both commensal bacteria and fMLF application to apical surfaces of polarized gut epithleilal cells resulted in FPR activation. Also, pretreatment of model epithelia and the murine gastrointestinal tract with Boc2 (specific fMLF peptide antagonist) or pertussis toxin (Gi‐protein inhibitor) abolished commensal‐mediated ERK phosphorylation. Together, these data show commensal bacteria specifically activate the pro‐proliferative ERK pathway in an FPR‐dependent manner, and suggest a mechanism by which commensal bacteria contribute to gut epithelia homeostasis.

Many bacterial pathogens can influence eukaryotic immune signaling pathways by translocating preformed effector proteins into host cells. The Salmonella protein AvrA is known to have immunosuppressive effects in mammalian cells. We have studied the effects of AvrA in Drosophila, a model system with striking conservation between its immune signaling pathways and those of mammals. We created transgenic Drosophila harboring avrA under the transcriptional control of the yeast UAS promoter, allowing regulated expression of AvrA. Flies expressing AvrA were immuno‐compromised and failed to mount an anti‐microbial response specifically against gram negative bacteria. Drosophila expressing a catalytically inactive form of AvrA had a normal immune response. Further studies showed AvrA inhibited translocation of the NF‐kB homolog Relish, and unexpectedly, potently inhibited phosphorylation of the fly homologue of JNK. Studies in human cell culture recapitulated specific AvrA mediated inhibition of inducible JNK phosphorylation and NF‐kB inhibition. Furthermore, in cell culture transfected AvrA co‐immunoprecipitated with endogenous JNK showing that AvrA has direct interaction with JNK. These studies reveal Salmonella has evolved a protein able to inhibit the pro‐apoptotic JNK pathway while suppressing innate immunity. Research funding provided by the CCFA and Burroughs Welcome fund.