Mary P. Leatham

Comparison of carbon nutrition for pathogenic and commensal Escherichia coli strains in the mouse intestine.

ABSTRACT The carbon sources that support the growth of pathogenic Escherichia coli O157:H7 in the mammalian intestine have not previously been investigated. In vivo, the pathogenic E. coli EDL933 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. coli strain 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. coli EDL933 and E. coli MG1655 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. coli EDL933 and E. coli MG1655 used arabinose, fucose, and N-acetylglucosamine in the intestine. In addition, E. coli EDL933 used galactose, hexuronates, mannose, and ribose, whereas E. coli MG1655 used gluconate and N-acetylneuraminic acid. The colonization defects of six catabolic lesions were found to be additive with E. coli EDL933 but not with E. coli MG1655. The data indicate that pathogenic E. coli EDL933 uses sugars that are not used by commensal E. coli MG1655 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.

Respiration of Escherichia coli in the Mouse Intestine

ABSTRACT Mammals 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 coli to 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 bd oxidase, which is used when oxygen tensions are low, also failed to colonize. However, the low-affinity cytochrome bo3 oxidase, 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. coli population was dependent on both microaerobic and anaerobic respiration, consistent with the hypothesis that the E. coli niche 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. coli strains as commensal inhabitants of the mammalian intestine.

Precolonized Human Commensal Escherichia coli Strains Serve as a Barrier to E. coli O157:H7 Growth in the Streptomycin-Treated Mouse Intestine

ABSTRACT Different Escherichia coli strains generally have the same metabolic capacity for growth on sugars in vitro, but they appear to use different sugars in the streptomycin-treated mouse intestine (Fabich et al., Infect. Immun. 76:1143-1152, 2008). Here, mice were precolonized with any of three human commensal strains (E. coli MG1655, E. coli HS, or E. coli Nissle 1917) and 10 days later were fed 105 CFU of the same strains. While each precolonized strain nearly eliminated its isogenic strain, confirming that colonization resistance can be modeled in mice, each allowed growth of the other commensal strains to higher numbers, consistent with different commensal E. coli strains using different nutrients in the intestine. Mice were also precolonized with any of five commensal E. coli strains for 10 days and then were fed 105 CFU of E. coli EDL933, an O157:H7 pathogen. E. coli Nissle 1917 and E. coli EFC1 limited growth of E. coli EDL933 in the intestine (103 to 104 CFU/gram of feces), whereas E. coli MG1655, E. coli HS, and E. coli EFC2 allowed growth to higher numbers (106 to 107 CFU/gram of feces). Importantly, when E. coli EDL933 was fed to mice previously co-colonized with three E. coli strains (MG1655, HS, and Nissle 1917), it was eliminated from the intestine (<10 CFU/gram of feces). These results confirm that commensal E. coli strains can provide a barrier to infection and suggest that it may be possible to construct E. coli probiotic strains that prevent growth of pathogenic E. coli strains in the intestine.

Role of Gluconeogenesis and the Tricarboxylic Acid Cycle in the Virulence of Salmonella enterica Serovar Typhimurium in BALB/c Mice

ABSTRACT In Salmonella enterica serovar Typhimurium, the Cra protein (catabolite repressor/activator) regulates utilization of gluconeogenic carbon sources by activating transcription of genes in the gluconeogenic pathway, the glyoxylate bypass, the tricarboxylic acid (TCA) cycle, and electron transport and repressing genes encoding glycolytic enzymes. A serovar Typhimurium SR-11 Δcra mutant was recently reported to be avirulent in BALB/c mice via the peroral route, suggesting that gluconeogenesis may be required for virulence. In the present study, specific SR-11 genes in the gluconeogenic pathway were deleted (fbp, glpX, ppsA, and pckA), and the mutants were tested for virulence in BALB/c mice. The data show that SR-11 does not require gluconeogenesis to retain full virulence and suggest that as yet unidentified sugars are utilized by SR-11 for growth during infection of BALB/c mice. The data also suggest that the TCA cycle operates as a full cycle, i.e., a sucCD mutant, which prevents the conversion of succinyl coenzyme A to succinate, and an ΔsdhCDA mutant, which blocks the conversion of succinate to fumarate, were both attenuated, whereas both an SR-11 ΔaspA mutant and an SR-11 ΔfrdABC mutant, deficient in the ability to run the reductive branch of the TCA cycle, were fully virulent. Moreover, although it appears that SR-11 replenishes TCA cycle intermediates from substrates present in mouse tissues, fatty acid degradation and the glyoxylate bypass are not required, since an SR-11 ΔfadD mutant and an SR-11 ΔaceA mutant were both fully virulent.

Glycogen and Maltose Utilization by Escherichia coli O157:H7 in the Mouse Intestine

ABSTRACT Mutant screens and transcriptome studies led us to consider whether the metabolism of glucose polymers, i.e., maltose, maltodextrin, and glycogen, is important for Escherichia coli colonization 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. coli O157:H7 and commensal E. coli K-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. coli relies 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 animals drinking water, the competitive disadvantage of E. coli glycogen 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. coli are 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. coli O157:H7 and perhaps other pathogens.

An Escherichia coli MG1655 Lipopolysaccharide Deep-Rough Core Mutant Grows and Survives in Mouse Cecal Mucus but Fails To Colonize the Mouse Large Intestine

ABSTRACT The ability of E. coli strains to colonize the mouse large intestine has been correlated with their ability to grow in cecal and colonic mucus. In the present study, an E. coli MG1655 strain was mutagenized with a mini-Tn5 Km (kanamycin) transposon, and mutants were tested for the ability to grow on agar plates with mouse cecal mucus as the sole source of carbon and nitrogen. One mutant, designated MD42 (for mucus defective), grew poorly on cecal-mucus agar plates but grew well on Luria agar plates and on glucose minimal-agar plates. Sequencing revealed that the insertion in MD42 was in the waaQ gene, which is involved in lipopolysaccharide (LPS) core biosynthesis. Like “deep-rough” E. coli mutants, MD42 was hypersensitive to sodium dodecyl sulfate (SDS), bile salts, and the hydrophobic antibiotic novobiocin. Furthermore, its LPS core oligosaccharide was truncated, like that of a deep-rough mutant. MD42 initially grew in the large intestines of streptomycin-treated mice but then failed to colonize (<102 CFU per g of feces), whereas its parent colonized at levels between 107 and 108 CFU per g of feces. When mouse cecal mucosal sections were hybridized with an E. coli-specific rRNA probe, MD42 was observed in cecal mucus as clumps 24 h postfeeding, whereas its parent was present almost exclusively as single cells, suggesting that clumping may play a role in preventing MD42 colonization. Surprisingly, MD42 grew nearly as well as its parent during growth in undiluted, highly viscous cecal mucus isolated directly from the mouse cecum and, like its parent, survived well after reaching stationary phase, suggesting that there are no antimicrobials in mucus that prevent MD42 colonization. After mini-mariner transposon mutagenesis, an SDS-resistant suppressor mutant of MD42 was isolated. The mini-mariner insertion was shown to be in the bipA gene, a known regulator of E. coli surface components. When grown in Luria broth, the LPS core of the suppressor mutant remained truncated; however, the LPS core was not truncated when the suppressor mutant was grown in the presence of SDS. Moreover, when the suppressor mutant was grown in the presence of SDS and fed to mice, it colonized the mouse large intestine. Collectively, the data presented here suggest that BipA may play a role in E. coli MG1655 LPS core biosynthesis and that because MD42 forms clumps in intestinal mucus, it is unable to colonize the mouse large intestine.

Mouse Intestine Selects Nonmotile flhDC Mutants of Escherichia coli MG1655 with Increased Colonizing Ability and Better Utilization of Carbon Sources

ABSTRACT d-Gluconate which is primarily catabolized via the Entner-Doudoroff (ED) pathway, has been implicated as being important for colonization of the streptomycin-treated mouse large intestine by Escherichia coli MG1655, a human commensal strain. In the present study, we report that an MG1655 Δedd mutant defective in the ED pathway grows poorly not only on gluconate as a sole carbon source but on a number of other sugars previously implicated as being important for colonization, including l-fucose, d-gluconate, d-glucuronate, N-acetyl-d-glucosamine, d-mannose, and d-ribose. Furthermore, we show that the mouse intestine selects mutants of MG1655 Δedd and wild-type MG1655 that have improved mouse intestine-colonizing ability and grow 15 to 30% faster on the aforementioned sugars. The mutants of MG1655 Δedd and wild-type MG1655 selected by the intestine are shown to be nonmotile and to have deletions in the flhDC operon, which encodes the master regulator of flagellar biosynthesis. Finally, we show that ΔflhDC mutants of wild-type MG1655 and MG1655 Δedd constructed in the laboratory act identically to those selected by the intestine; i.e., they grow better than their respective parents on sugars as sole carbon sources and are better colonizers of the mouse intestine.

Role of Motility and the flhDC Operon in Escherichia coli MG1655 Colonization of the Mouse Intestine

ABSTRACT Previously, we reported that the mouse intestine selected mutants of Escherichia coli MG1655 that have improved colonizing ability (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). These mutants grew 10 to 20% faster than their parent in mouse cecal mucus in vitro and 15 to 30% faster on several sugars found in the mouse intestine. The mutants were nonmotile and had deletions of various lengths beginning immediately downstream of an IS1 element located within the regulatory region of the flhDC operon, which encodes the master regulator of flagellum biosynthesis, FlhD4C2. Here we show that during intestinal colonization by wild-type E. coli strain MG1655, 45 to 50% of the cells became nonmotile by day 3 after feeding of the strain to mice and between 80 and 90% of the cells were nonmotile by day 15 after feeding. Ten nonmotile mutants isolated from mice were sequenced, and all were found to have flhDC deletions of various lengths. Despite this strong selection, 10 to 20% of the E. coli MG1655 cells remained motile over a 15-day period, suggesting that there is an as-yet-undefined intestinal niche in which motility is an advantage. The deletions appear to be selected in the intestine for two reasons. First, genes unrelated to motility that are normally either directly or indirectly repressed by FlhD4C2 but can contribute to maximum colonizing ability are released from repression. Second, energy normally used to synthesize flagella and turn the flagellar motor is redirected to growth.

l-Fucose Stimulates Utilization of d-Ribose by Escherichia coli MG1655 ΔfucAO and E. coli Nissle 1917 ΔfucAO Mutants in the Mouse Intestine and in M9 Minimal Medium

ABSTRACT Escherichia coli MG1655 uses several sugars for growth in the mouse intestine. To determine the roles of l-fucose and d-ribose, an E. coli MG1655 ΔfucAO mutant and an E. coli MG1655 ΔrbsK mutant were fed separately to mice along with wild-type E. coli MG1655. The E. coli MG1655 ΔfucAO mutant colonized the intestine at a level 2 orders of magnitude lower than that of the wild type, but the E. coli MG1655 ΔrbsK mutant and the wild type colonized at nearly identical levels. Surprisingly, an E. coli MG1655 ΔfucAO ΔrbsK mutant was eliminated from the intestine by either wild-type E. coli MG1655 or E. coli MG1655 ΔfucAO, suggesting that the ΔfucAO mutant switches to ribose in vivo. Indeed, in vitro growth experiments showed that l-fucose stimulated utilization of d-ribose by the E. coli MG1655 ΔfucAO mutant but not by an E. coli MG1655 ΔfucK mutant. Since the ΔfucK mutant cannot convert l-fuculose to l-fuculose-1-phosphate, whereas the ΔfucAO mutant accumulates l-fuculose-1-phosphate, the data suggest that l-fuculose-1-phosphate stimulates growth on ribose both in the intestine and in vitro. An E. coli Nissle 1917 ΔfucAO mutant, derived from a human probiotic commensal strain, acted in a manner identical to that of E. coli MG1655 ΔfucAO in vivo and in vitro. Furthermore, l-fucose at a concentration too low to support growth stimulated the utilization of ribose by the wild-type E. coli strains in vitro. Collectively, the data suggest that l-fuculose-1-phosphate plays a role in the regulation of ribose usage as a carbon source by E. coli MG1655 and E. coli Nissle 1917 in the mouse intestine.

A Salmonella enterica Serovar Typhimurium Succinate Dehydrogenase/Fumarate Reductase Double Mutant Is Avirulent and Immunogenic in BALB/c Mice

ABSTRACT Previously we showed that the tricarboxylic acid (TCA) cycle operates as a full cycle during Salmonella enterica serovar Typhimurium SR-11 peroral infection of BALB/c mice (M. Tchawa Yimga et al., Infect. Immun. 74:1130-1140, 2006). The evidence was that a ΔsucCD mutant (succinyl coenzyme A [succinyl-CoA] synthetase), which prevents the conversion of succinyl-CoA to succinate, and a ΔsdhCDA mutant (succinate dehydrogenase), which blocks the conversion of succinate to fumarate, were both attenuated, whereas an SR-11 ΔaspA mutant (aspartase) and an SR-11 ΔfrdABCD mutant (fumarate reductase), deficient in the ability to run the reductive branch of the TCA cycle, were fully virulent. In the present study, evidence is presented that a serovar Typhimurium SR-11 ΔfrdABCD ΔsdhCDA double mutant is avirulent in BALB/c mice and protective against subsequent infection with the virulent serovar Typhimurium SR-11 wild-type strain via the peroral route and is highly attenuated via the intraperitoneal route. These results suggest that fumarate reductase, which normally runs in the reductive pathway in the opposite direction of succinate dehydrogenase, can replace it during infection by running in the same direction as succinate dehydrogenase in order to run a full TCA cycle in an SR-11 ΔsdhCDA mutant. The data also suggest that the conversion of succinate to fumarate plays a key role in serovar Typhimurium virulence. Moreover, the data raise the possibility that S. enterica ΔfrdABCD ΔsdhCDA double mutants and ΔfrdABCD ΔsdhCDA double mutants of other intracellular bacterial pathogens with complete TCA cycles may prove to be effective live vaccine strains for animals and humans.