Question

explain e coli o157 h7 metabolic pathways

Answer 1

From ingestion to colonization

When E. coli is eliminated by a host animal it is not growing because it cannot grow in the luminal contents of the intestine. E. coli persists in the environment until its next host consumes viable bacteria in contaminated water or adulterated food. Following ingestion, a stressor faced by E. coli is acidity in the stomach, which it survives because stationary phase bacteria induce protective acid resistance systems . Extreme acid tolerance makes E. coli transmissible by as few as ten bacterial cells . Upon reaching the colon, E. coli must find the nutrients it needs to exit lag phase and grow from low to high numbers. Failure to transition from lag phase to logarithmic phase will lead to elimination of the invading E. coli bacteria. Successful colonization of the colon by E. coli depends upon competition for nutrients with a dense and diverse microbiota , penetration of the mucus layer , but not motility , and the abilities to avoid host defenses, and grow rapidly, E. coli resides in mucus until being sloughed into the lumen of the intestine , from whence some cells are eliminated in the host feces and the cycle begins again. This circle of colonization and extra-intestinal survival is the reality for commensal and pathogenic E. coli alike. Basic principles of colonization Colonization is defined as the indefinite persistence of a particular bacterial population without reintroduction of that bacterium. We agree with Rolf Freter, a true pioneer in the field of intestinal colonization, who concluded that although several factors could theoretically contribute to an organism’s ability to colonize, competition for nutrients is paramount for success in the intestinal ecosystem (26). According to Freter’s nutrient-niche hypothesis, the mammalian intestine is analogous to a chemostat in which several hundreds of species of bacteria are in equilibrium. To co-colonize, each species must use at least one limiting nutrient better than all the other species (18, 27, 28). The nutrient-niche hypothesis further predicts that invading species will have difficulty colonizing a stable ecosystem, such as the healthy intestine. The ability of the microbiota to resist invasion is termed colonization resistance (29), an example of which being that when human volunteers were fed E. coli strains isolated from their own feces those E. coli failed to colonize (30). Yet, despite colonization resistance, humans are colonized on average with five different E. coli strains and there is a continuous succession of strains in individuals (30). This suggests that diversity exists among commensal E. coli strains and that different strains may possess different strategies for utilizing growth-limiting nutrients. If diversity amongst E. coli commensal strains plays a role in colonization resistance, then mice pre-colonized with a human E. coli commensal strain would resist colonization by the same strain (isogenic challenge strain) because bacteria that consume the nutrients it needs to colonize already occupy its preferred niche. However, if mice pre-colonized with one human E. coli commensal strain were subsequently fed a different E. coli strain (nonisogenic challenge strain) then if the second strain could occupy a distinct niche in the intestine it would co-colonize with the first strain. The results of such experiments showed that each of several pre-colonized E. coli strains nearly eliminated its isogenic challenge strain from the intestine, confirming that colonization resistance can be modeled in mice, but non-isogenic challenge E. coli strains grew to higher numbers in the presence of different pre-colonized strains, suggesting that the newly introduced non-isogenic challenge strain either grows faster than the pre-colonized strain on one or more nutrients or uses nutrient(s) not being used by the pre-colonized strain. How might an invading enteric pathogen subvert colonization resistance? According to the nutrient-niche hypothesis, upon reaching the intestine the pathogen would first have to outcompete the resident microbiota for at least one nutrient, allowing it initially to colonize the intestine. However, colonization would not in itself result in pathogenesis if the pathogen must reach the epithelium and either bind to epithelial cells or invade the epithelium. In such instances, the pathogen must presumably penetrate the mucus layer. In a series of groundbreaking studies, Stecher, Hardt, and colleagues showed that when Salmonella enterica serovar Typhimurium induces inflammation in a mouse colitis model. The serovar Typhimurium is attracted by chemotaxis to galactose-containing nutrients on the mucosal surface (e.g. galactose-containing glycoconjugates and mucin) and, as expected, flagella and motility were required (32). Thus, to quote the authors , “Triggering the host’s immune defense can shift the balance between the protective microbiota and the pathogen in favor of the pathogen.” In streptomycin-treated mice, nutrient consumption by colonized E. coli strains can prevent invading E. coli strains from colonizing . By examining the sugars used by various human commensal E. coli strains to colonize, we identified a pair of strains(E. coli HS and E. coli Nissle 1917) that together use the five sugars previously found to be most important for colonization by the enterohemorrhagic Escherichia coli (EHEC) strain EDL933 (O157:H7). When mice were pre-colonized with E. coli HS and E. coli Nissle 1917, invading E. coli EDL933 was eliminated from the intestine . Clearly, one therapeutic strategy to prevent pathogenesis would be to outcompete the pathogen for nutrients normally present in the intestine and eliminate it before it can colonize and subsequently cause inflammation. Implicit in the nutrient-niche hypothesis is the idea that different species compete for preferred nutrients from a mixture that is equally available to all species. However, there is growing evidence that at least under some circumstances E. coli receives the nutrients it needs through direct interactions with neighboring microbes in the intestinal community. Thus, we take a renewed look at the metabolism of and nutrient flow between members of the intestinal microbiota. Central metabolism and intestinal colonization E. coli is a Gram negative, prototrophic, facultative anaerobe with the ability to respire oxygen, use alternative anaerobic electron acceptors, or ferment, depending on electron acceptor availability. Central metabolism in E. coli consists of the Embden-MeyerhofParnas glycolytic pathway (EMP), the pentose phosphate pathway (PP), the EntnerDoudoroff pathway (ED), the TCA cycle, and diverse fermentation pathways. E. coli grows best on sugars, including a wide range of mono- and disaccharides, but it cannot grow on complex polysaccharides because it lacks the necessary hydrolase enzymes. E. coli also can grow on amino acids and dicarboxylates that feed into the TCA cycle; the metabolism of these nutrients requires gluconeogenesis, the biosynthesis of glucose phosphate to be used as precursors of macromolecules such as LPS and peptidoglycan. Central metabolic pathways in E. coli are highly conserved, constituting a significant part of the core E. coli genome (38). The role of central metabolism during intestinal colonization has been studied in E. coli. The results of these experiments are summarized below (Table 1). Mutants blocked in glycolysis or the ED pathway, but not the PP pathway, have major colonization defects in competition with their wild type parents (39). Given its role in hexose metabolism, it is expected that glycolysis is important for colonization. Indeed, a pgi mutant lacking the key enzyme, phosphoglucose isomerase, of the EMP glycolytic pathway Conway and Cohen Page 3 Microbiol Spectr. Author manuscript; available in PMC 2015 July 22. Author Manuscript Author Manuscript Author Manuscript Author Manuscript has a substantial colonization defect when competed against its wild type E. coli K-12 parent (Table 1). The role of the EMP pathway goes beyond colonization by E. coli. For example, glucose catabolism and glycolysis are known to play a role in intracellular growth of serovar Typhimurium within macrophage vacuoles and proper regulation of glucose catabolism and glycolysis are coupled to virulence factor expression in EHEC . A recent study of Shigella flexneri revealed similar usage of these central metabolic pathways to support replication within host cells . We conclude that glycolysis is important for E. coli colonization and other aspects of enteric pathogenesis. Gluconate was the first nutrient that was shown to be used by E. coli to colonize the streptomycin-treated mouse intestine . Since gluconate and other sugar acids are primarily catabolized via the ED pathway, it is reasonable to expect that mutants lacking the pathway will be defective in colonization . The ED pathway is encoded by the edd-eda operon . The promoter-proximal edd gene encodes 6-phosphogluconate dehydratase, which converts 6-phosphogluconate to 2-keto-3-deoxy-6-phosphogluconate. The eda gene encodes 2-keto-3-deoxy-6-phosphogluconate aldolase, which converts 2-keto-3-deoxy-6- phosphogluconate to glyceraldehye-3-phosphate and pyruvate. E. coli edd mutants lacking the ED pathway, but retaining the pentose phosphate (PP) pathway, are poor colonizers of the mouse intestine, suggesting that E. coli utilizes the ED pathway for growth in the intestine (43). Other enteric bacteria require the ED pathway. For example, intracellular serovar Typhimurium induces genes of the ED pathway and gluconate catabolism during growth in macrophages (46). Moreover, the ED pathway is induced by Vibrio cholerae in vivo and an edd mutant failed to colonize the mouse intestine (47). In contrast to the importance of the ED pathway, an E. coli gnd mutant, missing 6- phosphogluconate dehydrogenase and therefore deficient in the oxidative branch of the PP pathway, was as good a mouse intestine colonizer as the wild type . It should be noted that gnd mutants retain the non-oxidative PP pathway; therefore they retain the ability to make essential precursor metabolites (e.g. ribose-5-phosphate) . We conclude that E. coli has alternative mechanisms for generating reducing power (NADPH) other than the oxidative PP pathway, but that the ED pathway for sugar acid catabolism is required to colonize efficiently . The role of the TCA cycle in commensal E. coli colonization of the intestine and in E. coli pathogenesis is poorly studied. It has been reported that an sdhB mutant lacking succinate dehydrogenase colonized as well as its wild type parent . However, E. coli has a second isoform of succinate dehydrogenase: fumarate reductase, which provides redundant enzyme function under some circumstances . Indeed, an E. coli sdhAB frdA double mutant has a significant colonization defect . The role of the TCA cycle in colonization and pathogenesis by other Enterobacteriaceae is better understood, as described immediately below. A fully functional TCA cycle is required for virulence of Salmonella enterica serovar Typhimurium via oral infection of BALB/c mice, i.e., a sucCD mutant, which prevents the conversion of succinyl coenzyme A to succinate, was attenuated. Also, an sdhCDA mutant, which blocks the conversion of succinate to fumarate, was attenuated, whereas both an aspA Conway and Cohen Page 4 Microbiol Spectr. Author manuscript; available in PMC 2015 July 22. Author Manuscript Author Manuscript Author Manuscript Author Manuscript mutant and an frdABC mutant, deficient in the ability to run the reductive branch of the TCA cycle, were fully virulent (50). Moreover, although it appears that serovar Typhimurium replenishes TCA cycle intermediates from substrates present in mouse tissues, fatty acid degradation and the glyoxylate bypass are not required, since a fadD, fadF, and aceA mutants were all fully virulent during acute infection (50–52). Interestingly, it appears that the TCA cycle is required for virulence of Edwardsiella ictaluri in catfish fingerlings (53) and that the glyoxylate bypass is required for serovar Typhimurium persistent infection of mice (51). The fact that E. coli depends on the TCA cycle for colonization implies that gluconeogenesis also is important. Using mutants that are unable to synthesize glucose from fatty acids, acetate, and TCA cycle intermediates because they are blocked in converting pyruvate to phosphoenolpyruvate (ppsA pckA), a critical step in gluconeogenesis, it was shown that neither the commensal E. coli K-12 strain MG1655 nor EHEC use gluconeogenesis for growth in the streptomycin-treated mouse intestine when each is the only E. coli strain fed to mice . However, E. coli Nissle 1917, the probiotic strain does use gluconeogenesis to colonize (Schinner, et al, unpublished). In addition, while E. coli EDL933 did not use gluconeogenic nutrients when it was the only E. coli strain in the mouse intestine, it used metabolic flexibility to switch to gluconeogenic nutrients when in competition in the intestine with either E. coli MG1655 (54) or E. coli Nissle 1917 (Schinner, et al, unpublished). These findings are of extreme interest in view of a recent report showing that E. coli EDL933 activates expression of virulence factor genes only under gluconeogenic conditions (41). Catabolic pathway diversity in E. coli The substrate range of E. coli is limited to mono-saccharides, disaccharides, a small number of larger sugars, some polyols, and sugar acids (55). Amino acids and carboxylates also are consumed (55, 56). The corresponding catabolic pathways feed these substrates into central metabolism. While the genes encoding central metabolism in E. coli fall within the highly conserved core genome (38), there is predicted to be some variation between strains with respect to the catabolic pathways that feed various substrates into central metabolism, as indicated by genome based metabolic modeling . For example, pathogenic E. coli strains are predicted to grow on sucrose while commensals are not. In contrast, commensals are predicted to grow on galactonate while pathogens are not. However, most of the substrates predicted by modeling to be used differentially by different E. coli strains are not known to be present in the intestine . E. coli EDL933, the prototypical EHEC strain, is able to grow on sucrose, whereas most commensal strains do not because they lack the sac genes, and some strains are missing genes within the N-acetylgalactosamine operon and are thus unable to grow on this substrate . Despite the modest differences between strains regarding their substrate range, in laboratory cultures containing a mixture of thirteen different sugars known to be present in mucus polysaccharides, E. coli EDL933 and E. coli MG1655 each use the sugars in the same order. However, although E. coli strains have nearly identical catabolic potential, they vary significantly in the sugars that support their colonization