Ability of Lactobacillus fermentum to overcome host α-galactosidase deficiency, as evidenced by reduction of hydrogen excretion in rats consuming soya α-galacto-oligosaccharides

<p>Abstract</p> <p>Background</p> <p>Soya and its derivatives represent nutritionally high quality food products whose major drawback is their high content of α-galacto-oligosaccharides. These are not digested in the small intestine due to the natural absence of tissular α-galactosidase in mammals. The passage of these carbohydrates to the large intestine makes them available for fermentation by gas-producing bacteria leading to intestinal flatulence. The aim of the work reported here was to assess the ability of α-galactosidase-producing lactobacilli to improve the digestibility of α-galacto-oligosaccharides <it>in situ</it>.</p> <p>Results</p> <p>Gnotobiotic rats were orally fed with soy milk and placed in respiratory chambers designed to monitor fermentative gas excretion. The validity of the animal model was first checked using gnotobiotic rats monoassociated with a <it>Clostridium butyricum </it>hydrogen (H₂)-producing strain. Ingestion of native soy milk by these rats caused significant H₂emission while ingestion of α-galacto-oligosaccharide-free soy milk did not, thus validating the experimental system. When native soy milk was fermented using the α-galactosidase-producing <it>Lactobacillus fermentum </it>CRL722 strain, the resulting product failed to induce H₂emission in rats thus validating the bacterial model. When <it>L. fermentum </it>CRL722 was coadministered with native soy milk, a significant reduction (50 %, <it>P </it>= 0.019) in H₂emission was observed, showing that α-galactosidase from <it>L. fermentum </it>CRL722 remained active <it>in situ</it>, in the gastrointestinal tract of rats monoassociated with <it>C. butyricum</it>. In human-microbiota associated rats, <it>L. fermentum </it>CRL722 also induced a significant reduction of H₂emission (70 %, <it>P </it>= 0.004).</p> <p>Conclusion</p> <p>These results strongly suggest that <it>L. fermentum </it>α-galactosidase is able to partially alleviate α-galactosidase deficiency in rats. This offers interesting perspectives in various applications in which lactic acid bacteria could be used as a vector for delivery of digestive enzymes in man and animals.</p

Functionality of Sortase A in Lactococcus lactis▿

Lactococcus lactis IL1403 harbors a putative sortase A (SrtA) and 11 putative sortase substrates that carry the canonical LPXTG signature of such substrates. We report here on the functionality of SrtA to anchor five LPXTG substrates to the cell wall, thus suggesting that SrtA is the housekeeping sortase in L. lactis IL1403

Pilus Biogenesis in Lactococcus lactis: molecular characterization and role in aggregation and biofilm formation

The genome of Lactococcus lactis strain IL1403 harbors a putative pilus biogenesis cluster consisting of a sortase C gene flanked by 3 LPxTG protein encoding genes (yhgD, yhgE, and yhhB), called here pil. However, pili were not detected under standard growth conditions. Over-expression of the pil operon resulted in production and display of pili on the surface of lactococci. Functional analysis of the pilus biogenesis machinery indicated that the pilus shaft is formed by oligomers of the YhgE pilin, that the pilus cap is formed by the YhgD pilin and that YhhB is the basal pilin allowing the tethering of the pilus fibers to the cell wall. Oligomerization of pilin subunits was catalyzed by sortase C while anchoring of pili to the cell wall was mediated by sortase A. Piliated L. lactis cells exhibited an auto-aggregation phenotype in liquid cultures, which was attributed to the polymerization of major pilin, YhgE. The piliated lactococci formed thicker, more aerial biofilms compared to those produced by non-piliated bacteria. This phenotype was attributed to oligomers of YhgE. This study provides the first dissection of the pilus biogenesis machinery in a non-pathogenic Gram-positive bacterium. Analysis of natural lactococci isolates from clinical and vegetal environments showed pili production under standard growth conditions. The identification of functional pili in lactococci suggests that the changes they promote in aggregation and biofilm formation may be important for the natural lifestyle as well as for applications in which these bacteria are used

Bacterial strains and plasmids used in this study.

*<p>ColE1 and pAMβ1 refer to the replicon; Tet^r, tetracycline resistance; Em^r, erythromycin resistance; Kan^r, kanamycin resistance; <i>srtC</i>*, mutated <i>srtC</i> gene encoding an inactive sortase C; plasmid and strain designations used in the text are indicated in parentheses.</p>$<p>Christine Delorme, INRA, Micalis-UMR1319, F78350-Jouy-en-Josas.</p

Immunolocalization of the YhhB basal pilin.

<p>Negative staining of <i>L. lactis</i> strains was performed using phosphotungstic acid. Strains were immobilized on Formvar-carbon-coated nickel grids and pilins were detected using as primary antibodies, guinea-pig anti-YhhB polyclonal antibodies. The preparations were treated with secondary antibodies consisting of anti-guinea-pig conjugated to 15 nm gold beads. The negatively stained pili are indicated by black arrows and the YhhB pilin is indicated with purple arrowheads. Control refers to <i>L. lactis</i> IL1403 strain harboring pIL253 plasmid and IL pPil to <i>L. lactis</i> IL1403 strain in which the <i>pil</i> operon is overexpressed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone-0050989-t001" target="_blank">Table 1</a>). (Scale bars, 200 nm).</p

Pili display and related auto-aggregation and biofilm phenotypes in <i>L. lactis</i> IL pPil and derivatives.

*<p>for strain designation, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone-0050989-t001" target="_blank">Table 1</a>.</p

Ability of to overcome host α-galactosidase deficiency, as evidenced by reduction of hydrogen excretion in rats consuming soya α-galacto-oligosaccharides-1

<p><b>Copyright information:</b></p><p>Taken from "Ability of to overcome host α-galactosidase deficiency, as evidenced by reduction of hydrogen excretion in rats consuming soya α-galacto-oligosaccharides"</p><p>http://www.biomedcentral.com/1471-2180/8/22</p><p>BMC Microbiology 2008;8():22-22.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2270848.</p><p></p

Maximum height of biofilms obtained with <i>L. lactis</i> strains.

<p>Strains that yielded biofilms whose maximum height measured at 4 and 15 h of growth was significantly different (<i>P</i><0.05) to that of control <i>L. lactis</i> IL1403 are marked by asterisks. Standard error is indicated. For strain designation, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone-0050989-t001" target="_blank">Table 1</a>. Indicated values are the mean of 3 determinations per experiment.</p

Auto-aggregation phenotype of <i>L. lactis</i> cultures.

<p>Strains over-expressing all or parts of the <i>pil</i> operon as indicated above the pictures were grown overnight under static conditions. Control refers to <i>L. lactis</i> IL1403 strain harboring the pIL253 plasmid. For strain designation, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone-0050989-t001" target="_blank">Table 1</a>.</p

Western blot analysis of cell wall-anchored proteins of <i>L. lactis</i> strains using anti-YhgE antibodies.

<p>Equivalent protein amounts from <i>L. lactis</i> control strain and from derivatives expressing all or parts of the <i>pil</i> operon were separated on 3–8% gradient Tris-acetate Criterion XT SDS-PAGE gel and were detected by immunoblotting. Control refers to <i>L. lactis</i> IL1403 strain harboring pIL253 plasmid. For strain designation, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050989#pone-0050989-t001" target="_blank">Table 1</a>. The positions of molecular mass standards (in kilodaltons) are indicated and the YhgE monomer is shown by a black arrowhead.</p