Lactobacillus reuteri 100-23 modulates urea hydrolysis in the murine stomach.

Comparisons of in vivo (mouse stomach) and in vitro (laboratory culture) transcriptomes of Lactobacillus reuteri strain 100-23 were made by microarray analysis. These comparisons revealed the upregulation of genes associated with acid tolerance, including urease production, in the mouse stomach. Inactivation of the ureC gene reduced the acid tolerance of strain 100-23 in vitro, and the mutant was outcompeted by the wild type in the gut of ex-Lactobacillus-free mice. Urine analysis showed that stable isotope-labeled urea, administered by gavage, was metabolized to a greater extent in Lactobacillus-free mice than animals colonized by strain 100-23. This surprising observation was associated with higher levels of urease activity and fecal-type bacteria in the stomach digesta of Lactobacillus-free mice. Despite the modulation of urea hydrolysis in the stomach, recycling of urea nitrogen in the murine host was not affected since the essential amino acid isoleucine, labeled with a stable isotope, was detected in the livers of both Lactobacillus-free and 100-23-colonized animals. Therefore, our experiments reveal a new and unexpected impact of Lactobacillus colonization on urea hydrolysis in the murine gut.

Altered transcription of murine genes induced in the small bowel by administration of probiotic strain Lactobacillus rhamnosus HN001.

Lactobacillus rhamnosus HN001 is a probiotic strain reported to increase resistance to epithelium-adherent and -invasive intestinal pathogens in experimental animals. To increase understanding of the relationship between strain HN001 and the bowel, transcription of selected genes in the mucosa of the murine small bowel was measured. Mice previously naive to lactobacilli (Lactobacillus-free mice) were examined after daily exposure to HN001 in drinking water. Comparisons were made to results from matched Lactobacillus-free mice. Infant and adult mice were investigated to provide a temporal view of gene expression in response to exposure to HN001. Genes sgk1, angptl4, and hspa1b, associated with the apoptosis pathway, were selected for investigation by reverse transcription-quantitative PCR on the basis of a preliminary duodenal DNA microarray screen. Normalized to gapdh gene transcription, these three genes were upregulated after 6 to 10 days exposure of adult mice to HN001. Angptl4 was shown by immunofluorescence to be upregulated in duodenal epithelial cells of mucosal samples. Epithelial cell migration was faster in HN001-exposed mice than in the Lactobacillus-free controls. Transcriptional responses in infant mice differed according to bowel region and age. For example, sgk1 was upregulated in duodenal, jejunal, and ileal mucosa of mice less than 25 days old, whereas angptl4 and hspa1b were upregulated at 10 days in the duodenum but downregulated in the jejunal mucosa until mice were 25 days old. Overall, the results provide links between a probiotic strain, mucosal gene expression, and host phenotype, which may be useful in delineating mechanisms of probiotic action.

Resource partitioning in relation to cohabitation of Lactobacillus species in the mouse forestomach.

Phylogenetic analysis of gut communities of vertebrates is advanced, but the relationships, especially at the trophic level, between commensals that share gut habitats of monogastric animals have not been investigated to any extent. Lactobacillus reuteri strain 100-23 and Lactobacillus johnsonii strain 100-33 cohabit in the forestomach of mice. According to the niche exclusion principle, this should not be possible because both strains can utilise the two main fermentable carbohydrates present in the stomach digesta: glucose and maltose. We show, based on gene transcription analysis, in vitro physiological assays, and in vivo experiments that the two strains can co-exist in the forestomach habitat because 100-23 grows more rapidly using maltose, whereas 100-33 preferentially utilises glucose. Mutation of the maltose phosphorylase gene (malA) of strain 100-23 prevented its growth on maltose-containing culture medium, and resulted in the numerical dominance of 100-33 in the forestomach. The fundamental niche of L. reuteri 100-23 in the mouse forestomach can be defined in terms of 'glucose and maltose trophism'. However, its realised niche when L. johnsonii 100-33 is present is 'maltose trophism'. Hence, nutritional adaptations provide niche differentiation that assists cohabitation by the two strains through resource partitioning in the mouse forestomach. This real life, trophic phenomenon conforms to a mathematical model based on in vitro bacterial doubling times, in vitro transport rates, and concentrations of maltose and glucose in mouse stomach digesta.

The evolution of host specialization in the vertebrate gut symbiont Lactobacillus reuteri.

Recent research has provided mechanistic insight into the important contributions of the gut microbiota to vertebrate biology, but questions remain about the evolutionary processes that have shaped this symbiosis. In the present study, we showed in experiments with gnotobiotic mice that the evolution of Lactobacillus reuteri with rodents resulted in the emergence of host specialization. To identify genomic events marking adaptations to the murine host, we compared the genome of the rodent isolate L. reuteri 100-23 with that of the human isolate L. reuteri F275, and we identified hundreds of genes that were specific to each strain. In order to differentiate true host-specific genome content from strain-level differences, comparative genome hybridizations were performed to query 57 L. reuteri strains originating from six different vertebrate hosts in combination with genome sequence comparisons of nine strains encompassing five phylogenetic lineages of the species. This approach revealed that rodent strains, although showing a high degree of genomic plasticity, possessed a specific genome inventory that was rare or absent in strains from other vertebrate hosts. The distinct genome content of L. reuteri lineages reflected the niche characteristics in the gastrointestinal tracts of their respective hosts, and inactivation of seven out of eight representative rodent-specific genes in L. reuteri 100-23 resulted in impaired ecological performance in the gut of mice. The comparative genomic analyses suggested fundamentally different trends of genome evolution in rodent and human L. reuteri populations, with the former possessing a large and adaptable pan-genome while the latter being subjected to a process of reductive evolution. In conclusion, this study provided experimental evidence and a molecular basis for the evolution of host specificity in a vertebrate gut symbiont, and it identified genomic events that have shaped this process.

Structure and functions of exopolysaccharide produced by gut commensal Lactobacillus reuteri 100-23.

Lactobacillus reuteri strain 100-23 together with a Lactobacillus-free mouse model, provides a system with which the molecular traits underpinning bacterial commensalism in vertebrates can be studied. A polysaccharide was extracted from sucrose-containing liquid cultures of strain 100-23. Chemical analysis showed that this exopolysaccharide was a levan (ß-2, 6-linked fructan). Mutation of the fructosyl transferase (ftf) gene resulted in loss of exopolysaccharide production. The ftf mutant was able to colonise the murine gastrointestinal tract in the absence of competition, but colonisation was impaired in competition with the wild type. Biofilm formation by the mutant on the forestomach epithelial surface was not impaired and the matrix between cells was indistinguishable from that of the wild type in electron micrographs. Colonisation of the mouse gut by the wild-type strain led to increased proportions of regulatory T cells (Foxp3+) in the spleen, whereas colonisation by the ftf mutant did not. Survival of the mutant in sucrose-containing medium was markedly reduced relative to the wild type. Comparison of the genomic ftf loci of strain 100-23 with other L. reuteri strains suggested that the ftf gene was acquired by lateral gene transfer early in the evolution of the species and subsequently diversified at accelerated rates. Levan production by L. reuteri 100-23 may represent a function acquired by the bacterial species for life in moderate to high-sucrose extra-gastrointestinal environments that has subsequently been diverted to novel uses, including immunomodulation, that aid in colonisation of the murine gut.

Gut commensal Lactobacillus reuteri 100-23 stimulates an immunoregulatory response.

Lactobacillus reuteri 100-23 is a bacterial commensal of the gastrointestinal tract of mice. Previous studies have shown that colonization of the murine gut by this strain stimulates small-bowel enterocytes to produce proinflammatory cytokines. This is associated with a mild, transitory inflammatory response 6 days after inoculation of formerly Lactobacillus-free animals. The inflammation subsides by 21 days after colonization, although lactobacilli continue to be present in the bowel. To determine the immunological mechanisms that underpin tolerance to bowel commensals, we investigated cytokine responses of dendritic cells and T cells after exposure to cells of L. reuteri 100-23. Interleukin-10 (IL-10), IL-2 and transforming growth factor-beta (TGF-beta) concentrations in supernatants of cultured immune cells, as well as the results of proliferative assays of mesenteric lymph node (MLN) cells and quantification of Foxp3-positive cells in MLN and spleen, indicated that L. reuteri 100-23 stimulated the development of an increased number of regulatory T cells.

Lactobacillus reuteri 100-23 transiently activates intestinal epithelial cells of mice that have a complex microbiota during early stages of colonization.

Monoassociations of germ-free animals with colitogenic and probiotic bacterial strains trigger intestinal epithelial cell (IEC) activation and host-derived feedback mechanisms. To characterize the impact of a single nonpathogenic bacterial strain on the intestinal epithelium in the presence of an established microbiota, we inoculated reconstituted Lacotobacillus-free (RLF) mice at 8 wk of age with Lactobacillus reuteri 100-23. Primary IEC from the small intestine of L. reuteri-inoculated and control RLF mice were isolated 2, 6, and 21 d after inoculation followed by gene expression analysis (real-time PCR; Affymetrix microarrays) as well as 2-dimensional-gel electrophoreses (2D SDS-PAGE) and peptide mass fingerprinting via matrix-assisted laser desorption/ionization time of flight MS. At d 6, gene expression of proinflammatory cytokines and chemokines including interleukin (IL)-1alpha, IL-6, interferon-gamma-inducible protein 10, and macrophage inflammatory protein 2 was transiently induced, whereas gene expression levels of regulatory proteins A20 and Toll-interacting protein decreased. In addition, 8 target proteins with changes in the steady-state protein expression levels were identified at d 2 and 6 of L. reuteri colonization. Consistent with the absence of histopathology, L. reuteri-induced activation of primary IEC returned to control levels by d 21 after inoculation of RLF mice. The capability of L. reuteri 100-23 to directly trigger epithelial cell activation was confirmed in small IEC cultures using the murine cell line Mode-K. These results clearly indicate that the intestinal epithelium is reactive toward environmental changes induced by the commensal bacterial strain L. reuteri even in the presence of an already-established microbiota. The induction of transient IEC activation may help to maintain mucosal homeostasis.

Glucosyltransferase A (GtfA) and inulosucrase (Inu) of Lactobacillus reuteri TMW1.106 contribute to cell aggregation, in vitro biofilm formation, and colonization of the mouse gastrointestinal tract.

Members of the genus Lactobacillus are common inhabitants of the proximal gastrointestinal tract of animals such as mice, rats, chickens and pigs, where they form epithelial biofilms. Little is known about the traits that facilitate biofilm formation and gut colonization. This study investigated the ecological role of a glucosyltransferase (GtfA) and inulosucrase (Inu) of Lactobacillus reuteri TMW1.106 and a fructosyltransferase (FtfA) of L. reuteri LTH5448. In vitro experiments using isogenic mutants revealed that GtfA was essential for sucrose-dependent autoaggregation of L. reuteri TMW1.106 cells under acidic conditions, while inactivation of Inu slowed the formation of cell aggregates. Experiments using an in vitro biofilm assay showed that GtfA and Inu contributed to biofilm formation of L. reuteri TMW1.106. Experiments using ex-Lactobacillus-free mice revealed that the ecological performance of the inu mutant, but not of the gtfA or ftfA mutant, was reduced in the gastrointestinal tract when in competition with the parental strain. In the absence of competition, the gtfA mutant showed delayed colonization of the murine gut relative to the wild-type. In addition, the gtfA mutant showed reduced ecological performance in competition experiments with Lactobacillus johnsonii #21. From the evidence provided in this study we conclude that GtfA and Inu confer important ecological attributes of L. reuteri TMW1.106 and contribute to colonization of the mouse gastrointestinal tract.

D-alanyl ester depletion of teichoic acids in Lactobacillus reuteri 100-23 results in impaired colonization of the mouse gastrointestinal tract.

The dlt operon of Gram-positive bacteria encodes proteins required for the incorporation of D-alanine esters into cell wall-associated teichoic acids (TA). D-alanylation of TA has been shown to be important for acid tolerance, resistance to antimicrobial peptides, adhesion, biofilm formation, and virulence of a variety of pathogenic organisms. The aim of this study was to determine the importance of D-alanylation for colonization of the gastrointestinal tract by Lactobacillus reuteri 100-23. Insertional inactivation of the dltA gene resulted in complete depletion of D-alanine substitution of lipoteichoic acids. The dlt mutant had similar growth characteristics as the wild type under standard in vitro conditions, but formed lower population sizes in the gastrointestinal tract of ex-Lactobacillus-free mice, and was almost eliminated from the habitat in competition experiments with the parental strain. In contrast to the wild type, the dlt mutant was unable to form a biofilm on the forestomach epithelium during gut colonization. Transmission electron microscope observations showed evidence of cell wall damage of mutant bacteria present in the forestomach. The dlt mutant had impaired growth under acidic culture conditions and increased susceptibility to the cationic peptide nisin relative to the wild type. Ex vivo adherence of the dlt mutant to the forestomach epithelium was not impaired. This study showed that D-alanylation is an important cell function of L. reuteri that seems to protect this commensal organism against the hostile conditions prevailing in the murine forestomach.

Ecological behavior of Lactobacillus reuteri 100-23 is affected by mutation of the luxS gene.

The luxS gene of Lactobacillus reuteri 100-23C was amplified by PCR, cloned, and then sequenced. To define a physiological and ecological role for the luxS gene in L. reuteri 100-23C, a luxS mutant was constructed by insertional mutagenesis. The luxS mutant did not produce autoinducers AI-2 or AI-3. Complementation of the luxS mutation by a plasmid construct containing luxS restored AI-2 and AI-3 synthesis. In vitro experiments revealed that neither the growth rate, nor the cell yield, nor cell survival in the stationary phase were compromised in the luxS mutant relative to the wild type and complemented mutant. The ATP content of exponentially growing cells of the luxS mutant was, however, 65% of that of wild-type cells. Biofilms formed by the luxS mutant on plastic surfaces in a bioreactor were thicker than those formed by the wild type. Biofilm thickness was not restored to wild-type values by the addition of purified AI-2 to the culture medium. In vivo experiments, conducted with ex-Lactobacillus-free mice, showed that biofilms formed by the mutant strain on the epithelial surface of the forestomach were approximately twice as thick as those formed by the wild type. The ecological performance of the luxS mutant, when in competition with L. reuteri strain 100-93 in the mouse cecum, was reduced compared to that of a xylA mutant of 100-23C. These results demonstrate that LuxS influences important ecological attributes of L. reuteri 100-23C, the consequences of which are niche specific.

A high-molecular-mass surface protein (Lsp) and methionine sulfoxide reductase B (MsrB) contribute to the ecological performance of Lactobacillus reuteri in the murine gut.

Members of the genus Lactobacillus are common inhabitants of the gut, yet little is known about the traits that contribute to their ecological performance in gastrointestinal ecosystems. Lactobacillus reuteri 100-23 persists in the gut of the reconstituted Lactobacillus-free mouse after a single oral inoculation. Recently, three genes of this strain that were specifically induced (in vivo induced) in the murine gut were identified (38). We report here the detection of a gene of L. reuteri 100-23 that encodes a high-molecular-mass surface protein (Lsp) that shows homology to proteins involved in the adherence of other bacteria to epithelial cells and in biofilm formation. The three in vivo-induced genes and lsp of L. reuteri 100-23 were inactivated by insertional mutagenesis in order to study their biological importance in the murine gastrointestinal tract. Competition experiments showed that mutation of lsp and a gene encoding methionine sulfoxide reductase (MsrB) reduced ecological performance. Mutation of lsp impaired the adherence of the bacteria to the epithelium of the mouse forestomach and altered colonization dynamics. Homologues of lsp and msrB are present in the genomes of several strains of Lactobacillus and may play an important role in the maintenance of these bacteria in gut ecosystems.

Identification of Lactobacillus reuteri genes specifically induced in the mouse gastrointestinal tract.

Lactobacilli are common inhabitants of the gastrointestinal tracts of mammals and have received considerable attention due to their putative health-promoting properties. Little is known about the traits that enhance the ability of these bacteria to inhabit the gastrointestinal tract. In this paper we describe the development and application of a strategy based on in vivo expression technology (IVET) that enables detection of Lactobacillus reuteri genes specifically induced in the murine gut. A plasmid-based system was constructed containing 'ermGT (which confers lincomycin resistance) as the primary reporter gene for selection of promoters active in the gastrointestinal tract of mice treated with lincomycin. A second reporter gene, 'bglM (beta-glucanase), allowed differentiation between constitutive and in vivo inducible promoters. The system was successfully tested in vitro and in vivo by using a constitutive promoter. Application of the IVET system with chromosomal DNA of L. reuteri 100-23 and reconstituted lactobacillus-free mice revealed three genes induced specifically during colonization. Two of the sequences showed homology to genes encoding xylose isomerase (xylA) and peptide methionine sulfoxide reductase (msrB), which are involved in nutrient acquisition and stress responses, respectively. The third locus showed homology to the gene encoding a protein whose function is not known. Our IVET system has the potential to identify genes of lactobacilli that have not previously been functionally characterized but which may be essential for growth of these bacteria in the gastrointestinal ecosystem.