Identification of surface proteins involved in the adhesion of a probiotic Bacillus cereus strain to mucin and fibronectin

Adhesion to the intestinal epithelium could constitute an essential mechanism of Bacillus cereus pathogenesis. However, the enterocytes are protected by mucus, a secretion composed mainly of mucin glycoproteins. These may serve as nutrients and sites of adhesion for intestinal bacteria. In this study, the food poisoning bacterium B. cereus NVH 0500/00 was exposed in vitro to gastrointestinal hurdles prior to evaluation of its attachment to mucin microcosms and its ability to produce nonhemolytic enterotoxin (Nhe). The persistence of mucin-adherent B. cereus after simulated gut emptying was determined using a mucin adhesion assay. The stability of Nhe toward bile and pancreatin (intestinal components) in the presence of mucin agar was also investigated. B. cereus could grow and simultaneously adhere to mucin during in vitro ileal incubation, despite the adverse effect of prior exposure to a low pH or intestinal components. The final concentration of B. cereus in the simulated lumen at 8 h of incubation was 6.62 ؎ 0.87 log CFU ml ؊1 . At that point, the percentage of adhesion was approximately 6%. No enterotoxin was detected in the ileum, due to either insufficient bacterial concentrations or Nhe degradation. Nevertheless, mucin appears to retain B. cereus and to supply it to the small intestine after simulated gut emptying. Additionally, mucin may play a role in the protection of enterotoxins from degradation by intestinal components.

Section: Discussionmentioning

confidence: 99%

Bacillus cereus NVH 0500/00 Can Adhere to Mucin but Cannot Produce Enterotoxins during Gastrointestinal Simulation

Tsilia

Kerckhof

Rajković

et al. 2016

“…Cells from 50 ml of culture were resuspended in 5 ml of 100 mM Na 2 CO 3 buffer, and cell suspensions were incubated for 1 h on ice and harvested by centrifugation (Na 2 CO 3 fraction) (35). Another washed cell pellet from 50 ml of culture was resuspended in 5 ml of 0.01 M NaOH, and an incubation at 37°C for 30 min under gentle agitation was performed (NaOH fraction) (36). The protease inhibitors EDTA and phenylmethylsulfonyl fluoride were added to all of the solutions at final concentrations of 5 and 1 mM, respectively.…”

Section: Methodsmentioning

confidence: 99%

Role of Extracellular Transaldolase from Bifidobacterium bifidum in Mucin Adhesion and Aggregation

González-Rodríguez

Sánchez

Ruíz

et al. 2012

106

ABSTRACTThe ability of bifidobacteria to establish in the intestine of mammals is among the main factors considered to be important for achieving probiotic effects. The role of surface molecules fromBifidobacterium bifidumtaxon in mucin adhesion capability and the aggregation phenotype of this bacterial species was analyzed. Adhesion to the human intestinal cell line HT29 was determined for a collection of 12B. bifidumstrains. In four of them—B. bifidumLMG13195, DSM20456, DSM20239, and A8—the involvement of surface-exposed macromolecules in the aggregation phenomenon was determined. The aggregation ofB. bifidumA8 and DSM20456 was abolished after treatment with proteinase K, this effect being more pronounced for the strain A8. Furthermore, a mucin binding assay ofB. bifidumA8 surface proteins showed a high adhesive capability for its transaldolase (Tal). The localization of this enzyme on the surface ofB. bifidumA8 was unequivocally demonstrated by immunogold electron microscopy experiments. The gene encoding Tal fromB. bifidumA8 was expressed inLactococcus lactis, and the protein was purified to homogeneity. The pure protein was able to restore the autoaggregation phenotype of proteinase K-treatedB. bifidumA8 cells. A recombinantL. lactisstrain, engineered to secrete Tal, displayed a mucin- binding level more than three times higher than the strain not producing the transaldolase. These findings suggest that Tal, when exposed on the cell surface ofB. bifidum, could act as an important colonization factor favoring its establishment in the gut.

“…lactis D1, and Lc. lactis ST to different matrices was performed as described before (30). Briefly, mucin (type II or type III; Sigma) or fibronectin (Sigma) at 20 mg/ml in PBS was coated onto Immuno MicroWell 96-well plates (Nunc, Roskilde, Denmark) at 4°C for 16 h. Just before their use, wells were blocked for 1 h with 300 l of 1% (wt/vol) bovine serum albumin and washed twice with PBS.…”

Section: Methodsmentioning

confidence: 99%

An Extracellular Serine/Threonine-Rich Protein from Lactobacillus plantarum NCIMB 8826 Is a Novel Aggregation-Promoting Factor with Affinity to Mucin

Hevia

Martínez

Ladero

et al. 2013

Autoaggregation in lactic acid bacteria is directly related to the production of certain extracellular proteins, notably, aggregation-promoting factors (APFs). Production of aggregation-promoting factors confers beneficial traits to probiotic-producing strains, contributing to their fitness for the intestinal environment. Furthermore, coaggregation with pathogens has been proposed to be a beneficial mechanism in probiotic lactic acid bacteria. This mechanism would limit attachment of the pathogen to the gut mucosa, favoring its removal by the human immune system. In the present paper, we have characterized a novel aggregation-promoting factor in Lactobacillus plantarum. A mutant with a knockout of the D1 gene showed loss of its autoaggregative phenotype and a decreased ability to bind to mucin, indicating an adhesion role of this protein. In addition, heterologous production of the D1 protein or an internal fragment of the protein, characterized by its abundance in serine/threonine, strongly induced autoaggregation in Lactococcus lactis. This result strongly suggested that this internal fragment is responsible for the bioactivity of D1 as an APF. To our knowledge, this is the first report on a gene coding for an aggregation-promoting factor in Lb. plantarum.