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

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.

L-fucose stimulates utilization of D-ribose by Escherichia coli MG1655 DeltafucAO and E. coli Nissle 1917 DeltafucAO mutants in the mouse intestine and in M9 minimal medium.

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 DeltafucAO mutant and an E. coli MG1655 DeltarbsK mutant were fed separately to mice along with wild-type E. coli MG1655. The E. coli MG1655 DeltafucAO mutant colonized the intestine at a level 2 orders of magnitude lower than that of the wild type, but the E. coli MG1655 DeltarbsK mutant and the wild type colonized at nearly identical levels. Surprisingly, an E. coli MG1655 DeltafucAO DeltarbsK mutant was eliminated from the intestine by either wild-type E. coli MG1655 or E. coli MG1655 DeltafucAO, suggesting that the DeltafucAO 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 DeltafucAO mutant but not by an E. coli MG1655 DeltafucK mutant. Since the DeltafucK mutant cannot convert L-fuculose to L-fuculose-1-phosphate, whereas the DeltafucAO 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 DeltafucAO mutant, derived from a human probiotic commensal strain, acted in a manner identical to that of E. coli MG1655 DeltafucAO 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.

Role of motility and the flhDC Operon in Escherichia coli MG1655 colonization of the mouse intestine.

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, FlhD(4)C(2). 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 FlhD(4)C(2) 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.

Role of gluconeogenesis and the tricarboxylic acid cycle in the virulence of Salmonella enterica serovar Typhimurium in BALB/c mice.

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 Deltacra 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 DeltasdhCDA mutant, which blocks the conversion of succinate to fumarate, were both attenuated, whereas both an SR-11 DeltaaspA mutant and an SR-11 DeltafrdABC 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 DeltafadD mutant and an SR-11 DeltaaceA mutant were both fully virulent.

Glycolytic and gluconeogenic growth of Escherichia coli O157:H7 (EDL933) and E. coli K-12 (MG1655) in the mouse intestine.

Escherichia coli EDL933, an O157:H7 strain, is known to colonize the streptomycin-treated CD-1 mouse intestine by growing in intestinal mucus (E. A. Wadolkowski, J. A. Burris, and A. D. O'Brien, Infect. Immun. 58:2438-2445, 1990), but what nutrients and metabolic pathways are employed during colonization has not been determined. In this study, when the wild-type EDL933 strain was fed to mice along with an EDL933 DeltappsA DeltapckA mutant, which is unable to utilize tricarboxylic acid cycle intermediates and gluconeogenic substrates for growth, both strains colonized the mouse intestine equally well. Therefore, EDL933 utilizes a glycolytic substrate(s) for both initial growth and maintenance when it is the only E. coli strain fed to the mice. However, in the presence of large numbers of MG1655, a K-12 strain, it is shown that EDL933 utilizes a glycolytic substrate(s) for initial growth in the mouse intestine but appears to utilize both glycolytic and gluconeogenic substrates in an attempt to maintain colonization. It is further shown that MG1655 predominantly utilizes glycolytic substrates for growth in the mouse intestine whether growing in the presence or absence of large numbers of EDL933. Data are presented showing that although small numbers of EDL933 grow to large numbers in the intestine in the presence of large numbers of MG1655 when both strains are fed to mice simultaneously, precolonization with MG1655 affords protection against subsequent colonization by EDL933. Moreover, in mice that are precolonized with EDL933, small numbers of MG1655 are able to grow rapidly in the intestine and EDL933 is eliminated. In situ hybridization experiments using E. coli-specific rRNA probes showed that while MG1655 is found only in mucus, EDL933 is found both in mucus and closely associated with intestinal epithelial cells. The data are discussed with respect to competition for nutrients and to the protection that some intestinal commensal E. coli strains might afford against infection by O157:H7 strains.

Carbon nutrition of Escherichia coli in the mouse intestine.

Whole-genome expression profiling revealed Escherichia coli MG1655 genes induced by growth on mucus, conditions designed to mimic nutrient availability in the mammalian intestine. Most were nutritional genes corresponding to catabolic pathways for nutrients found in mucus. We knocked out several pathways and tested the relative fitness of the mutants for colonization of the mouse intestine in competition with their wild-type parent. We found that only mutations in sugar pathways affected colonization, not phospholipid and amino acid catabolism, not gluconeogenesis, not the tricarboxylic acid cycle, and not the pentose phosphate pathway. Gluconate appeared to be a major carbon source used by E. coli MG1655 to colonize, having an impact on both the initiation and maintenance stages. N-acetylglucosamine and N-acetylneuraminic acid appeared to be involved in initiation, but not maintenance. Glucuronate, mannose, fucose, and ribose appeared to be involved in maintenance, but not initiation. The in vitro order of preference for these seven sugars paralleled the relative impact of the corresponding metabolic lesions on colonization: gluconate > N-acetylglucosamine > N-acetylneuraminic acid = glucuronate > mannose > fucose > ribose. The results of this systematic analysis of nutrients used by E. coli MG1655 to colonize the mouse intestine are intriguing in light of the nutrient-niche hypothesis, which states that the ecological niches within the intestine are defined by nutrient availability. Because humans are presumably colonized with different commensal strains, differences in nutrient availability may provide an open niche for infecting E. coli pathogens in some individuals and a barrier to infection in others.

Lysophosphatidic acid inhibition of the accumulation of Pseudomonas aeruginosa PAO1 alginate, pyoverdin, elastase and LasA.

The pathogenesis of Pseudomonas aeruginosa is at least partially attributable to its ability to synthesize and secrete the siderophore pyoverdin and the two zinc metalloproteases elastase and LasA, and its ability to form biofilms in which bacterial cells are embedded in an alginate matrix. In the present study, a lysophospholipid, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate [also called monopalmitoylphosphatidic acid (MPPA)], which accumulates in inflammatory exudates, was shown to inhibit the extracellular accumulation of P. aeruginosa PAO1 alginate, elastase, LasA protease and the siderophore pyoverdin. MPPA also inhibited biofilm formation. The inhibitory effects of MPPA occur independently of rpoS expression and without affecting the accumulation of the autoinducers N-(3-oxododecanoyl) homoserine lactone and N-butyryl-L-homoserine lactone, and may be due, at least in part, to the ability of MPPA to bind divalent cations.