Dataset Information

Identification of a Gene Cluster for the Biosynthesis of a Long, Galactose-Rich Exopolysaccharide in Lactobacillus rhamnosus GG and Functional Analysis of the Priming Glycosyltransferase.

ABSTRACT: Cell surface polysaccharides have an established role as virulence factors in human bacterial pathogens. Less documented are the biosynthesis and biological functions of surface polysaccharides in beneficial bacteria. We identified a gene cluster that encodes the enzymes and regulatory and transporter proteins for the different steps in the biosynthesis of extracellular polysaccharides (EPS) of the well-documented probiotic strain Lactobacillus rhamnosus GG. Subsequent mutation of the welE gene, encoding the priming glycosyltransferase within this cluster, and comparative phenotypic analyses of wild-type versus mutant strains confirmed the specific function of this gene cluster in the biosynthesis of high-molecular-weight, galactose-rich heteropolymeric EPS molecules. The phenotypic analyses included monomer composition determination, estimation of the polymer length of the isolated EPS molecules, and single-molecule force spectroscopy of the surface polysaccharides. Further characterization of the welE mutant also showed that deprivation of these long, galactose-rich EPS molecules results in an increased adherence and biofilm formation capacity of L. rhamnosus GG, possibly because of less shielding of adhesins such as fimbria-like structures.

SUBMITTER: Lebeer S

PROVIDER: S-EPMC2687306 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Identification of a Gene Cluster for the Biosynthesis of a Long, Galactose-Rich Exopolysaccharide in Lactobacillus rhamnosus GG and Functional Analysis of the Priming Glycosyltransferase.

Lebeer Sarah S Verhoeven Tine L A TL Francius Grégory G Schoofs Geert G Lambrichts Ivo I Dufrêne Yves Y Vanderleyden Jos J De Keersmaecker Sigrid C J SC

Applied and environmental microbiology 20090403 11

Cell surface polysaccharides have an established role as virulence factors in human bacterial pathogens. Less documented are the biosynthesis and biological functions of surface polysaccharides in beneficial bacteria. We identified a gene cluster that encodes the enzymes and regulatory and transporter proteins for the different steps in the biosynthesis of extracellular polysaccharides (EPS) of the well-documented probiotic strain Lactobacillus rhamnosus GG. Subsequent mutation of the welE gene, ...[more]

PMID: 19346339

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Project description:BACKGROUND:Acute gastroenteritis develops in millions of children in the United States every year, and treatment with probiotics is common. However, data to support the use of probiotics in this population are limited. METHODS:We conducted a prospective, randomized, double-blind trial involving children 3 months to 4 years of age with acute gastroenteritis who presented to one of 10 U.S. pediatric emergency departments. Participants received a 5-day course of Lactobacillus rhamnosus GG at a dose of 1×1010 colony-forming units twice daily or matching placebo. Follow-up surveys were conducted daily for 5 days and again 14 days after enrollment and 1 month after enrollment. The primary outcome was moderate-to-severe gastroenteritis, which was defined as an illness episode with a total score on the modified Vesikari scale of 9 or higher (scores range from 0 to 20, with higher scores indicating more severe disease), within 14 days after enrollment. Secondary outcomes included the duration and frequency of diarrhea and vomiting, the duration of day-care absenteeism, and the rate of household transmission (defined as the development of symptoms of gastroenteritis in previously asymptomatic household contacts). RESULTS:Among the 971 participants, 943 (97.1%) completed the trial. The median age was 1.4 years (interquartile range, 0.9 to 2.3), and 513 participants (52.9%) were male. The modified Vesikari scale score for the 14-day period after enrollment was 9 or higher in 55 of 468 participants (11.8%) in the L. rhamnosus GG group and in 60 of 475 participants (12.6%) in the placebo group (relative risk, 0.96; 95% confidence interval, 0.68 to 1.35; P=0.83). There were no significant differences between the L. rhamnosus GG group and the placebo group in the duration of diarrhea (median, 49.7 hours in the L. rhamnosus GG group and 50.9 hours in the placebo group; P=0.26), duration of vomiting (median, 0 hours in both groups; P=0.17), or day-care absenteeism (median, 2 days in both groups; P=0.67) or in the rate of household transmission (10.6% and 14.1% in the two groups, respectively; P=0.16). CONCLUSIONS:Among preschool children with acute gastroenteritis, those who received a 5-day course of L. rhamnosus GG did not have better outcomes than those who received placebo. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT01773967 .).

Project description:BackgroundLactobacillus rhamnosus GG (LGG) is the most widely used probiotic, but the mechanisms underlying its beneficial effects remain unresolved. Previous studies typically inoculated LGG in hosts with established gut microbiota, limiting the understanding of specific impacts of LGG on host due to numerous interactions among LGG, commensal microbes, and the host. There has been a scarcity of studies that used gnotobiotic animals to elucidate LGG-host interaction, in particular for gaining specific insights about how it modifies the metabolome. To evaluate whether LGG affects the metabolite output of pathobionts, we inoculated with LGG gnotobiotic mice containing Propionibacterium acnes, Turicibacter sanguinis, and Staphylococcus aureus (PTS).Results16S rRNA sequencing of fecal samples by Ion Torrent and MinION platforms showed colonization of germ-free mice by PTS or by PTS plus LGG (LTS). Although the body weights and feeding rates of mice remained similar between PTS and LTS groups, co-associating LGG with PTS led to a pronounced reduction in abundance of P. acnes in the gut. Addition of LGG or its secretome inhibited P. acnes growth in culture. After optimizing procedures for fecal metabolite extraction and metabolomic liquid chromatography-mass spectrometry analysis, unsupervised and supervised multivariate analyses revealed a distinct separation among fecal metabolites of PTS, LTS, and germ-free groups. Variables-important-in-projection scores showed that LGG colonization robustly diminished guanine, ornitihine, and sorbitol while significantly elevating acetylated amino acids, ribitol, indolelactic acid, and histamine. In addition, carnitine, betaine, and glutamate increased while thymidine, quinic acid and biotin were reduced in both PTS and LTS groups. Furthermore, LGG association reduced intestinal mucosal expression levels of inflammatory cytokines, such as IL-1α, IL-1β and TNF-α.ConclusionsLGG co-association had a negative impact on colonization of P. acnes, and markedly altered the metabolic output and inflammatory response elicited by pathobionts.

Dataset Information

Identification of a Gene Cluster for the Biosynthesis of a Long, Galactose-Rich Exopolysaccharide in Lactobacillus rhamnosus GG and Functional Analysis of the Priming Glycosyltransferase.

Publications

Identification of a Gene Cluster for the Biosynthesis of a Long, Galactose-Rich Exopolysaccharide in Lactobacillus rhamnosus GG and Functional Analysis of the Priming Glycosyltransferase.

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