Sudden exposure of human populations to chemicals, pathogens, or radiation has the potential to result in substantial morbidity. A potential means of rapidly protecting such populations might be to activate innate host defense pathways, which can provide broad protection against a variety of insults. However, innate immune activators can, by themselves, result in severe inflammatory pathology, which in large part is driven by hemopoietic-derived cytokines such as TNF-α. We reasoned that, because it preferentially activates epithelial cells, the TLR5 agonist flagellin might not induce severe inflammatory pathology and yet be an ideal agent to provide such non-specific protection, particularly at the mucosal surfaces that serve as a front line of host defense. In accordance, we observed that systemic treatment of mice with purified flagellin did not induce the serologic, histopathologic, and clinical hallmarks of inflammation that are induced by LPS but yet protected mice against chemicals, pathogens, and ionizing radiation. Flagellin-elicited radioprotection required TLR5, the TLR signaling adaptor MyD88, and was effective if given between 2 h before to 4 h after exposure to irradiation. Flagellin-elicited radioprotection was, in part, mediated via effects on cells in bone marrow but yet rescued mortality without a pronounced rescue of radiation-induced anemia or leukopenia. Thus, systemic administration of flagellin may be a relatively safe means of providing temporary non-specific protection against a variety of challenges.

Flagellin, the primary structural component of bacterial flagella, is recognized by Toll-like receptor 5 (TLR5) present on the basolateral surface of intestinal epithelial cells. Utilizing biochemical assays of proinflammatory signaling pathways and mRNA expression profiling, we found that purified flagellin could recapitulate the human epithelial cell proinflammatory responses activated by flagellated pathogenic bacteria. Flagellin-induced proinflammatory activation showed similar kinetics and gene specificity as that induced by the classical endogenous proinflammatory cytokine TNF-α, although both responses were more rapid than that elicited by viable flagellated bacteria. Flagellin, like TNF-α, activated a number of antiapoptotic mediators, and pretreatment of epithelial cells with this bacterial protein could protect cells from subsequent bacterially mediated apoptotic challenge. However, when NF-κB-mediated or phosphatidylinositol 3-kinase/Akt proinflammatory signaling was blocked, flagellin could induce programmed cell death. Consistently, we demonstrate that flagellin and viable flagellate Salmonella induces both the extrinsic and intrinsic caspase activation pathways, with the extrinsic pathway (caspase 8) activated by purified flagellin in a TLR5-dependant fashion. We conclude that interaction of flagellin with epithelial cells induces caspase activation in parallel with proinflammatory responses. Such intertwining of proinflammatory and apoptotic signaling mediated by bacterial products suggests roles for host programmed cell death in the pathogenesis of enteric infections.

The human enteric flora plays a significant role in intestinal health and disease. Certain enteric bacteria can inhibit the NF-κB pathway by blockade of IκB-α ubiquitination. IκB-α ubiquitination is catalyzed by the E3-SCFβTrCP ubiquitin ligase, which is itself regulated via covalent modification of the cullin-1 subunit by the ubiquitin-like protein NEDD8. Neddylation is a biochemical event associated with diverse cellular processes related to cell signaling, however, physiological regulation of cullin neddylation has not been described in mammalian systems. We report that interaction of nonpathogenic bacteria with epithelial cells resulted in a rapid loss of neddylated Cul-1 and consequent repression of the NF-κB pathway. This observation may explain the ability of intestinal bacterial communities to influence diverse eukaryotic processes in general and inflammatory tolerance of the mammalian intestinal epithelia specifically.

Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis in all plants, animals, and bacteria. The Escherichia coli form is a multimeric protein complex consisting of three distinct and separate components: biotin carboxylase, carboxyltransferase, and the biotin carboxyl carrier protein. The biotin carboxylase component catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the carboxylate source and has a distinct architecture that is characteristic of the ATP-grasp superfamily of enzymes. Included in this superfamily are D-Ala D-Ala ligase, glutathione synthetase, carbamyl phosphate synthetase, N 5 -carboxyaminoimidazole ribonucleotide synthetase, and glycinamide ribonucleotide transformylase, all of which have known three-dimensional structures and contain a number of highly conserved residues between them. Four of these residues of biotin carboxylase, Lys-116, Lys-159, His-209, and Glu-276, were selected for sitedirected mutagenesis studies based on their structural homology with conserved residues of other ATP-grasp enzymes. These mutants were subjected to kinetic analysis to characterize their roles in substrate binding and catalysis. In all four mutants, the K m value for ATP was significantly increased, implicating these residues in the binding of ATP. This result is consistent with the crystal structures of several other ATP-grasp enzymes, which have shown specific interactions between the corresponding homologous residues and cocrystallized ADP or nucleotide analogs. In addition, the maximal velocity of the reaction was significantly reduced (between 30-and 260-fold) in the 4 mutants relative to wild type. The data suggest that the mutations have misaligned the reactants for optimal catalysis.Acetyl-CoA carboxylase catalyzes the first committed step in long chain fatty acid synthesis, namely the formation of malonyl-CoA from acetyl-CoA, MgATP, and bicarbonate. Found in all plants, animals, and bacteria, this enzyme is biotin-dependent, with the following two-step reaction mechanism (1).Enzyme-biotin-CO 2The Escherichia coli form of this enzyme consists of three separable components. The biotin carboxylase component catalyzes the first half-reaction, which involves the phosphorylation of bicarbonate to form a carboxyphosphate intermediate, followed by the transfer of the carboxyl group to the 1Ј nitrogen of biotin (2). The carboxyltransferase component catalyzes the second half-reaction. In vivo the biotin molecule is linked to the biotin carboxyl carrier protein through an amide bond to a specific lysine residue. Both biotin carboxylase and carboxyltransferase retain activity in the absence of the other two components and will also use free biotin as a substrate (3). The crystal structure of the biotin carboxylase component has been solved and is the only three-dimensional structure of a biotindependent carboxylase, making it the paradigm for structurefunction analysis of this class of enzymes (4). Two years after the solution of the crystal structure, Artymiu...

Flagellin is a promising adjuvant for suppressing ionising radiation-induced injury. MKP-7 activity exhibits cytoprotective effects, and is thus a candidate cellular molecule for limiting the damaging effect of radiotherapy on the gastreointestinal system.