Summary An optimal gut microbiota influences many beneficial processes in the metazoan host. However, the molecular mechanisms that mediate and function in symbiont-induced host responses have not yet been fully characterized. Here, we report that cellular ROS enzymatically generated in response to contact with lactobacilli in both mice and Drosophila has salutary effects against exogenous insults to the intestinal epithelium via the activation of Nrf2 responsive cytoprotective genes. These data show that the xenobiotic inducible Nrf2 pathway participates as a signaling conduit between the prokaryotic symbiont and the eukaryotic host. Indeed, our data imply that the capacity of lactobacilli to induce redox signaling in epithelial cells is a highly conserved hormetic adaptation to impel cellular conditioning to exogenous biotic stimuli. These data also highlight the role the microbiota plays in eukaryotic cytoprotective pathways, and may have significant implications in the characterization of a eubiotic microbiota.

A high sugar and high‐fat diet Western‐style diet can result in significant dyslipidemia, often leading to non‐alcoholic fatty liver disease (NAFLD), with females particularly affected by this diet irrespective of total caloric intake. Dietary supplementation with beneficial bacteria has been advocated as therapeutic intervention to modulate liver adiposity resulting from a Western‐style diet. Here, we assess the activity of beneficial bacteria on modulating the impact of a Western‐style diet in female mice. Of those tested, we show that a previously uncharacterized beneficial bacterium, namely Lactococcus lactis sp. cremoris significantly protected against Western style diet‐induced hepatic steatosis, elevated cholesterol levels, glucose intolerance, increased body mass index (BMI), and adiposity. Due to these effects, we propose the use of L. lactis sp. cremoris as a therapeutic modality to promote metabolic health in individuals suffering adverse health events resulting from a Western style diet. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PIX proteins are guanine nucleotide exchange factors (GEFs) that activate Rac and Cdc42, and are known to have numerous functions in various cell types. Here, we show that a PIX protein has an important function in muscle. From a genetic screen in C. elegans, we found that pix-1 is required for the assembly of integrin adhesion complexes (IACs) at borders between muscle cells, and is required for locomotion of the animal. A pix-1 null mutant has a reduced level of activated Rac in muscle. PIX-1 localizes to IACs at muscle cell boundaries, M-lines and dense bodies. Mutations in genes encoding proteins at known steps of the PIX signaling pathway show defects at muscle cell boundaries. A missense mutation in a highly conserved residue in the RacGEF domain results in normal levels of PIX-1 protein, but a reduced level of activated Rac in muscle, and abnormal IACs at muscle cell boundaries.

Mutations in the unc-82 locus of Caenorhabditis elegans were previously identified by screening for disrupted muscle cytoskeleton in otherwise apparently normal mutagenized animals. Here we demonstrate that the locus encodes a serine/threonine kinase orthologous to human ARK5/SNARK (NUAK1/NUAK2) and related to the PAR-1 and SNF1/AMP-Activated kinase (AMPK) families. The predicted 1600-amino-acid polypeptide contains an N-terminal catalytic domain and noncomplex repetitive sequence in the remainder of the molecule. Phenotypic analyses indicate that unc-82 is required for maintaining the organization of myosin filaments and internal components of the M-line during cellshape changes. Mutants exhibit normal patterning of cytoskeletal elements during early embryogenesis. Defects in localization of thick filament and M-line components arise during embryonic elongation and become progressively more severe as development proceeds. The phenotype is independent of contractile activity, consistent with unc-82 mutations preventing proper cytoskeletal reorganization during growth, rather than undermining structural integrity of the M-line. This is the first report establishing a role for the UNC-82/ARK5/SNARK kinases in normal development. We propose that activation of UNC-82 kinase during cell elongation regulates thick filament attachment or growth, perhaps through phosphorylation of myosin and paramyosin. We speculate that regulation of myosin is an ancestral characteristic of kinases in this region of the kinome.

A distinct taxon of the Drosophila microbiota, Lactobacillus plantarum, is capable of stimulating the generation of reactive oxygen species (ROS) within cells, and inducing epithelial cell proliferation. Here, we show that microbial-induced ROS generation within Drosophila larval stem cell compartments exhibits a distinct spatial distribution. Lactobacilli-induced ROS is strictly excluded from defined midgut compartments that harbor adult midgut progenitor (AMP) cells, forming a functional 'ROS sheltered zone' (RSZ). The RSZ is undiscernible in germ-free larvae, but forms following monocolonization with L. plantarum. L. plantarum is a strong activator of the ROS-sensitive CncC/Nrf2 signaling pathway within enterocytes. Enterocyte-specific activation of CncC stimulated the proliferation of AMPs, which demonstrates that pro-proliferative signals are transduced from enterocytes to AMPs. Mechanistically, we show that the cytokine Upd2 is expressed in the gut following L. plantarum colonization in a CncC-dependent fashion, and may function in lactobacilli-induced AMP proliferation and intestinal tissue growth and development.

Intestinal homeostasis is regulated in-part by reactive oxygen species (ROS) that are generated in the colonic mucosa following contact with certain lactobacilli. Mechanistically, ROS can modulate protein function through the oxidation of cysteine residues within proteins. Recent advances in cysteine labeling by the Isotope Coded Affinity Tags (ICATs) technique has facilitated the identification of cysteine thiol modifications in response to stimuli. Here, we used ICATs to map the redox protein network oxidized upon initial contact of the colonic mucosa with Lactobacillus rhamnosus GG (LGG). We detected significant LGG-specific redox changes in over 450 proteins, many of which are implicated to function in cellular processes such as endosomal trafficking, epithelial cell junctions, barrier integrity, and cytoskeleton maintenance and formation. We particularly noted the LGG-specific oxidation of Rac1, which is a pleiotropic regulator of many cellular processes. Together, these data reveal new insights into lactobacilli-induced and redox-dependent networks involved in intestinal homeostasis.

Recent evidence has demonstrated that reactive oxygen (eg, hydrogen peroxide) can activate host cell signaling pathways that function in repair. We show that mice deficient in their capacity to generate reactive oxygen by the NADPH oxidase 2 holoenzyme, an enzyme complex highly expressed in neutrophils and macrophages, have disrupted capacity to orchestrate signaling events that function in mucosal repair. Similar observations were made for mice after neutrophil depletion, pinpointing this cell type as the source of the reactive oxygen driving oxidation-reduction protein signaling in the epithelium. To simulate epithelial exposure to high levels of reactive oxygen produced by neutrophils and gain new insight into this oxidation-reduction signaling, epithelial cells were treated with hydrogen peroxide, biochemical experiments were conducted, and a proteome-wide screen was performed using isotope-coded affinity tags to detect proteins oxidized after exposure. This analysis implicated signaling pathways regulating focal adhesions, cell junctions, and maintenance of the cytoskeleton. These pathways are also known to act via coordinated phosphorylation events within proteins that constitute the focal adhesion complex, including focal adhesion kinase and Crk-associated substrate. We identified the Rho family small GTPebinding protein Ras-related C3 botulinum toxin substrate 1 and p21 activated kinases 2 as operational in these signaling and localization pathways. These data support the hypothesis that reactive oxygen species from neutrophils can orchestrate epithelial cellesignaling events functioning in intestinal repair.

The conservation of intestinal stem cell crypt dynamics between Drosophila melanogaster and mammals allows for the genetically tractable fly model to be used for analyses of intestinal development, homeostasis, and renewal in relation to microbiota. The invertebrate fly model is advantageous for genetic research due to its anatomical and genetic simplicity and short lifespan. Accordingly, experimental resources such as large numbers of mutant and genetically modified flies have been developed. We have developed techniques to generate germ-free Drosophila, monoassociate them with candidate bacteria, and assess ensuing physiological responses within the gut tissue that include the generation of reactive oxygen species and cell proliferation.