Search

Using interleukin 10-deficient (IL-10(-/-) ) and wild-type mice monoassociated with either the adherent-invasive Escherichia coli UNC or the probiotic E. coli Nissle, the effect of a mild intestinal inflammation on the bacterial proteome was studied. Within 8 weeks, IL-10(-/-) mice monoassociated with E. coli UNC exhibited an increased expression of several proinflammatory markers in caecal mucosa. Escherichia coli Nissle-associated IL-10(-/-) mice did not do so. As observed previously for E. coli from mice with acute colitis, glycolytic enzymes were downregulated in intestinal E. coli UNC from IL-10(-/-) mice. In addition, the inhibitor of vertebrate C-type lysozyme, Ivy, was upregulated on messenger RNA (mRNA) and protein level in E. coli Nissle from IL-10(-/-) mice compared with E. coli UNC from these mice. Higher expression of Ivy in E. coli Nissle correlated with an improved growth of this probiotic strain in the presence of lysozyme-ethylenediaminetetraacetic acid (EDTA). By overexpressing Ivy, we demonstrated that Ivy contributes to a higher lysozyme resistance of E. coli, supporting the role of Ivy as a potential fitness factor. However, deletion of Ivy did not alter the growth phenotype of E. coli Nissle in the presence of lysozyme-EDTA, suggesting the existence of additional lysozyme inhibitors that can take over the function of Ivy., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Using a gnotobiotic mouse model, we previously observed the upregulation of 2-deoxy-D-gluconate 3-dehydrogenase (KduD) in intestinal E. coli of mice fed a lactose-rich diet and the downregulation of this enzyme and of 5-keto 4-deoxyuronate isomerase (KduI) on a casein-rich diet. The present study aimed to define the role of the so far poorly characterized E. coli proteins KduD and KduI in vitro. Galacturonate and glucuronate induced kduD and kduI gene expression 3-fold and 7 to 11-fold, respectively, under aerobic conditions as well as 9 to 20-fold and 19 to 54-fold, respectively, under anaerobic conditions. KduI facilitated the breakdown of these hexuronates. In E. coli, galacturonate and glucuronate are normally degraded by UxaABC and UxuAB. However, osmotic stress represses the expression of the corresponding genes in an OxyR-dependent manner. When grown in the presence of galacturonate or glucuronate, kduID-deficient E. coli had a 30% to 80% lower maximal cell density and 1.5 to 2-fold longer doubling times under osmotic stress conditions than wild type E. coli. Growth on lactose promoted the intracellular formation of hexuronates, which possibly explain the induction of KduD on a lactose-rich diet. These results indicate a novel function of KduI and KduD in E. coli and demonstrate the crucial influence of osmotic stress on the gene expression of hexuronate degrading enzymes.

To identify Escherichia coli proteins involved in adaptation to intestinal inflammation, mice were monoassociated with the colitogenic E. coli strain UNC or with the probiotic E. coli strain Nissle. Intestinal inflammation was induced by treating the mice with 3.5% dextran sodium sulfate (DSS). Differentially expressed proteins in E. coli strains collected from cecal contents were identified by 2-dimensional difference gel electrophoresis. In both strains, acute inflammation led to the downregulation of pathways involved in carbohydrate breakdown and energy generation. Accordingly, DSS-treated mice had lower concentrations of bacterial fermentation products in their cecal contents than control mice. Differentially expressed proteins also included the Fe-S cluster repair protein NfuA, the tryptophanase TnaA, and the uncharacterized protein YggE. NfuA expression was 3-fold higher in E. coli strains from DSS-treated than from control mice. Reporter experiments confirmed the induction of nfuA in response to iron deprivation, mimicking Fe-S cluster destruction by inflammation. YggE expression, which has been reported to reduce the intracellular level of reactive oxygen species, was 4- to 8-fold higher in E. coli Nissle than in E. coli UNC. This was confirmed by in vitro reporter gene assays indicating that Nissle is better equipped to cope with oxidative stress than UNC. Nissle isolated from DSS-treated and control mice had TnaA levels 4- to 7-fold-higher than those of UNC. Levels of indole resulting from the TnaA reaction were higher in control animals associated with E. coli Nissle. Because of its anti-inflammatory effect, indole is hypothesized to be involved in the extension of the remission phase in ulcerative colitis described for E. coli Nissle.

To study the adaptation of an intestinal bacterium to its natural environment, germfree mice were associated with commensal Escherichia coli MG1655. Two-dimensional gel electrophoresis was used to identify proteins differentially expressed in E. coli MG1655 collected from either cecal contents or anaerobic in vitro cultures. Fourteen differentially expressed proteins (>3-fold; P < 0.05) were identified, nine of which were upregulated in cecal versus in vitro-grown E. coli. Four of these proteins were investigated further for their role in gut colonization. After deletion of the corresponding genes, the resulting E. coli mutants were tested for their ability to colonize the intestines of gnotobiotic mice in competition with the wild-type strain. A mutant devoid of ydjG, which encodes a putative NADH-dependent methylglyoxal reductase, reached a 1.2-log-lower cecal concentration than the wild type. Deletion of the nanA gene encoding N-acetylneuraminate lyase affected the colonization and persistence of E. coli in the intestines of the gnotobiotic mice only slightly. A mutant devoid of 5'-phosphoribosyl 4-(N-succinocarboxamide)-5-aminoimidazole synthase, a key enzyme of purine synthesis, displayed intestinal cell counts >4 logs lower than those of the wild type. Deletion of the gene encoding aspartate carbamoyltransferase, a key enzyme of pyrimidine synthesis, even resulted in the washout of the corresponding mutant from the mouse intestinal tract. These findings indicate that E. coli needs to synthesize purines and pyrimidines to successfully colonize the mouse intestine.

Commensal bacteria play a role in the aetiology of inflammatory bowel diseases (IBD). High intestinal numbers of Escherichia coli in IBD patients suggest a role of this organism in the initiation or progression of chronic gut inflammation. In addition, some E. coli genotypes are more frequently detected in IBD patients than others. We aimed to find out whether gut inflammation in an IBD mouse model is associated with a particular E. coli strain. Intestinal contents and tissue material were taken from 1-, 8-, 16- and 24-week-old interleukin 10-deficient (IL-10(-/-)) mice and the respective wild-type animals. Caecal and colonic inflammation was observed in IL-10(-/-) animals from the 8 weeks of life on accompanied by a lower intestinal microbial diversity than in the respective wild-type animals. Culture- based and molecular approaches revealed that animals with gut inflammation harboured significantly higher numbers of E. coli than healthy controls. Phylogenetic grouping according to the E. coli Reference Collection (ECOR) system and strain typing by random-amplified polymorphic DNA and pulsed-field gel electrophoresis revealed that all mice were colonized by one single E. coli strain. The strain was shown to have the O7:H7:K1 serotype and to belong to the virulence-associated phylogenetic group B2. In a co-association experiment with gnotobiotic mice, the strain outnumbered E. coli ECOR strains belonging to the phylogenetic group A and B2 respectively. A high number of virulence- and fitness-associated genes were detected in the strain's genome possibly involved in the bacterial adaptation to the murine intestine.

The gastrointestinal tract of mammals is inhabited by several hundred bacterial species. While the effects of the gut microbiota upon the host have been widely studied, the microbial response to host factors has only recently attracted attention. In order to investigate the influence of the host on the physiology of gastrointestinal bacteria, a simplified model of host-bacteria interaction was created by associating germfree mice with commensal Escherichia coli. Here we demonstrate the feasibility of analysing the bacterial response to the conditions in the digestive system by a proteomics-based approach. Two-dimensional gel electrophoresis (2D-GE) followed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was used to identify bacterial proteins from caecal and faecal samples. In a set of 60 arbitrarily chosen spots of stably and differentially expressed proteins, 50 different bacterial proteins were identified. Their ascribed functions suggest that the host-associated bacteria adapt their metabolism to the conditions in the intestine by utilizing arginine, asparagine and aspartate as well as glucose/galactose, ribose, maltose, glucuronate, galacturonate and gluconate as substrates. Thirteen proteins not previously detected on 2D-gels and 10 proteins with unknown or poorly characterized physiological function were identified, while the existence of three proteins had so far only been inferred from predictions or by homology.

Using a gnotobiotic mouse model, we previously observed the upregulation of 2-deoxy-D-gluconate 3-dehydrogenase (KduD) in intestinal E. coli of mice fed a lactose-rich diet and the downregulation of this enzyme and of 5-keto 4-deoxyuronate isomerase (KduI) on a casein-rich diet. The present study aimed to define the role of the so far poorly characterized E. coli proteins KduD and KduI in vitro. Galacturonate and glucuronate induced kduD and kduI gene expression 3-fold and 7 to 11-fold, respectively, under aerobic conditions as well as 9 to 20-fold and 19 to 54-fold, respectively, under anaerobic conditions. KduI facilitated the breakdown of these hexuronates. In E. coli, galacturonate and glucuronate are normally degraded by UxaABC and UxuAB. However, osmotic stress represses the expression of the corresponding genes in an OxyR-dependent manner. When grown in the presence of galacturonate or glucuronate, kduID-deficient E. coli had a 30% to 80% lower maximal cell density and 1.5 to 2-fold longer doubling times under osmotic stress conditions than wild type E. coli. Growth on lactose promoted the intracellular formation of hexuronates, which possibly explain the induction of KduD on a lactose-rich diet. These results indicate a novel function of KduI and KduD in E. coli and demonstrate the crucial influence of osmotic stress on the gene expression of hexuronate degrading enzymes. [ABSTRACT FROM AUTHOR]

To study the impact of nutritional factors on protein expression of intestinal bacteria, gnotobiotic mice monoassociated with Escherichia coli K-12 were fed three different diets: a diet rich in starch, a diet rich in nondigestible lactose and a diet rich in casein. Two-dimensional gel electrophoresis and electrospray-tandem mass spectrometry were used to identify differentially expressed proteins of bacteria recovered from small intestine and cecum. Oxidative stress response proteins such as AhpF, Dps and Fur, all of which belong to the oxyR regulon, were upregulated in E. coli of mice fed the lactose rich diet. Luciferase reporter-gene assays demonstrated that osmotic stress caused by carbohydrates leads to the expression of ahpCF and dps, which was not observed in E. coli ΛoxyR mutant. Growth of ahpCF and oxyR deletion mutants was strongly impaired when non-digestible sucrose was present in the medium. The wild-type phenotype could be restored by complementation of the deletions with plasmids containing the corresponding genes and promoters. The results indicate that some OxyR-dependent proteins play a major role in the adaptation of E. coli to osmotic stress. We conclude that there is an overlap of osmotic and oxidative stress responses. Mice fed the lactose-rich diet possibly had a higher intestinal osmolality leading to the upregulation of OxyR dependent proteins, which enable intestinal E. coli to better cope with diet-induced osmotic stress. [ABSTRACT FROM AUTHOR]