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CD8+cytotoxic T lymphocytes play a major role in defense against intracellular pathogens, and their functions are specified by antigen recognition and innate cytokines. IL-12 and IFN-α/β are potent “signal 3” cytokines that are involved in both effector and memory cell development. Although the majority of effector cells are eliminated as inflammation resolves, some survive within the pool of memory cells and retain immediate effector function. In this study, we demonstrate that IL-12 instructs a unique program of effector cell differentiation that is distinct from IFN-α/β. Moreover, effector memory (TEM) cells within peripheral blood display many common attributes of cells differentiated in vitro in response to IL-12, including proinflammatory cytokine secretion and lytic activity. A pattern of IL-12–induced genes was identified that demarcate TEMfrom central memory cells, and the ontologies of these genes correlated precisely with their effector functions. Further, we uncovered a unique program of gene expression that was acutely regulated by IL-12 and reflected in stable gene expression patterns within TEM, but not T central memory cells in vivo. Thus, this study directly links a selective set of IL-12–induced genes to the programming of effector functions within the stable population of human CD8+TEMcells in vivo.

ABSTRACTThe intestine is inhabited by a large microbial community consisting primarily of anaerobes and, to a lesser extent, facultative anaerobes, such as Escherichia coli, which we have shown requires aerobic respiration to compete successfully in the mouse intestine (S. A. Jones et al., Infect. Immun. 75:4891-4899, 2007). If facultative anaerobes efficiently lower oxygen availability in the intestine, then their sustained growth must also depend on anaerobic metabolism. In support of this idea, mutants lacking nitrate reductase or fumarate reductase have extreme colonization defects. Here, we further explore the role of anaerobic respiration in colonization using the streptomycin-treated mouse model. We found that respiratory electron flow is primarily via the naphthoquinones, which pass electrons to cytochrome bdoxidase and the anaerobic terminal reductases. We found that E. coliuses nitrate and fumarate in the intestine, but not nitrite, dimethyl sulfoxide, or trimethylamine N-oxide. Competitive colonizations revealed that cytochrome bdoxidase is more advantageous than nitrate reductase or fumarate reductase. Strains lacking nitrate reductase outcompeted fumarate reductase mutants once the nitrate concentration in cecal mucus reached submillimolar levels, indicating that fumarate is the more important anaerobic electron acceptor in the intestine because nitrate is limiting. Since nitrate is highest in the absence of E. coli, we conclude that E. coliis the only bacterium in the streptomycin-treated mouse large intestine that respires nitrate. Lastly, we demonstrated that a mutant lacking the NarXL regulator (activator of the NarG system), but not a mutant lacking the NarP-NarQ regulator, has a colonization defect, consistent with the advantage provided by NarG. The emerging picture is one in which gene regulation is tuned to balance expression of the terminal reductases that E. coliuses to maximize its competitiveness and achieve the highest possible population in the intestine.

ABSTRACTMutant screens and transcriptome studies led us to consider whether the metabolism of glucose polymers, i.e., maltose, maltodextrin, and glycogen, is important for Escherichia colicolonization of the intestine. By using the streptomycin-treated mouse model, we found that catabolism of the disaccharide maltose provides a competitive advantage in vivo to pathogenic E. coliO157:H7 and commensal E. coliK-12, whereas degradation of exogenous forms of the more complex glucose polymer, maltodextrin, does not. The endogenous glucose polymer, glycogen, appears to play an important role in colonization, since mutants that are unable to synthesize or degrade glycogen have significant colonization defects. In support of the hypothesis that E. colirelies on internal carbon stores to maintain colonization during periods of famine, we found that by providing a constant supply of a readily metabolized sugar, i.e., gluconate, in the animal's drinking water, the competitive disadvantage of E. coliglycogen metabolism mutants is rescued. The results suggest that glycogen storage may be widespread in enteric bacteria because it is necessary for maintaining rapid growth in the intestine, where there is intense competition for resources and occasional famine. An important implication of this study is that the sugars used by E. coliare present in limited quantities in the intestine, making endogenous carbon stores valuable. Thus, there may be merit to combating enteric infections by using probiotics or prebiotics to manipulate the intestinal microbiota in such a way as to limit the availability of sugars preferred by E. coliO157:H7 and perhaps other pathogens.

ABSTRACTThe carbon sources that support the growth of pathogenic Escherichia coliO157:H7 in the mammalian intestine have not previously been investigated. In vivo, the pathogenic E. coliEDL933 grows primarily as single cells dispersed within the mucus layer that overlies the mouse cecal epithelium. We therefore compared the pathogenic strain and the commensal E. colistrain MG1655 modes of metabolism in vitro, using a mixture of the sugars known to be present in cecal mucus, and found that the two strains used the 13 sugars in a similar order and cometabolized as many as 9 sugars at a time. We conducted systematic mutation analyses of E. coliEDL933 and E. coliMG1655 by using lesions in the pathways used for catabolism of 13 mucus-derived sugars and five other compounds for which the corresponding bacterial gene system was induced in the transcriptome of cells grown on cecal mucus. Each of 18 catabolic mutants in both bacterial genetic backgrounds was fed to streptomycin-treated mice, together with the respective wild-type parent strain, and their colonization was monitored by fecal plate counts. None of the mutations corresponding to the five compounds not found in mucosal polysaccharides resulted in colonization defects. Based on the mutations that caused colonization defects, we determined that both E. coliEDL933 and E. coliMG1655 used arabinose, fucose, and N-acetylglucosamine in the intestine. In addition, E. coliEDL933 used galactose, hexuronates, mannose, and ribose, whereas E. coliMG1655 used gluconate and N-acetylneuraminic acid. The colonization defects of six catabolic lesions were found to be additive with E. coliEDL933 but not with E. coliMG1655. The data indicate that pathogenic E. coliEDL933 uses sugars that are not used by commensal E. coliMG1655 to colonize the mouse intestine. The results suggest a strategy whereby invading pathogens gain advantage by simultaneously consuming several sugars that may be available because they are not consumed by the commensal intestinal microbiota.

ABSTRACTMammals are aerobes that harbor an intestinal ecosystem dominated by large numbers of anaerobic microorganisms. However, the role of oxygen in the intestinal ecosystem is largely unexplored. We used systematic mutational analysis to determine the role of respiratory metabolism in the streptomycin-treated mouse model of intestinal colonization. Here we provide evidence that aerobic respiration is required for commensal and pathogenic Escherichia colito colonize mice. Our results showed that mutants lacking ATP synthase, which is required for all respiratory energy-conserving metabolism, were eliminated by competition with respiratory-competent wild-type strains. Mutants lacking the high-affinity cytochrome bdoxidase, which is used when oxygen tensions are low, also failed to colonize. However, the low-affinity cytochrome bo3oxidase, which is used when oxygen tension is high, was found not to be necessary for colonization. Mutants lacking either nitrate reductase or fumarate reductase also had major colonization defects. The results showed that the entire E. colipopulation was dependent on both microaerobic and anaerobic respiration, consistent with the hypothesis that the E. coliniche is alternately microaerobic and anaerobic, rather than static. The results indicate that success of the facultative anaerobes in the intestine depends on their respiratory flexibility. Despite competition for relatively scarce carbon sources, the energy efficiency provided by respiration may contribute to the widespread distribution (i.e., success) of E. colistrains as commensal inhabitants of the mammalian intestine.