Contribution of plasmid-encoded peptidase S8 (PrtP) to adhesion and transit in the gut of Lactococcus lactis IBB477 strain

Abstract

The ability of Lactococcus lactis to adhere to the intestinal mucosa can potentially prolong the contact with the host, and therefore favour its persistence in the gut. In the present study, the contribution of plasmid-encoded factors to the adhesive and transit properties of the L. lactis subsp. cremoris IBB477 strain was investigated. Plasmid-cured derivatives as well as deletion mutants were obtained and analysed. Adhesion tests were performed using non-coated polystyrene plates, plates coated with mucin or fibronectin and mucus-secreting HT29-MTX intestinal epithelial cells. The results indicate that two plasmids, pIBB477a and b, are involved in adhesion of the IBB477 strain. One of the genes localised on plasmid pIBB477b (AJ89_14230), which encodes cell wall-associated peptidase S8 (PrtP), mediates adhesion of the IBB477 strain to bare, mucin- and fibronectin-coated polystyrene, as well as to HT29-MTX cells. Interactions between bacteria and mucus secreted by HT29-MTX cells were further investigated by fluorescent staining and confocal microscopy. Confocal images showed that IBB477 forms dense clusters embedded in secreted mucus. Finally, the ability of IBB477 strain and its ΔprtP deletion mutant to colonise the gastrointestinal tract of conventional C57Bl/6 mice was determined. Both strains were present in the gut for up to 72 h. In summary, adhesion and persistence of IBB477 were analysed by in vitro and in vivo approaches, respectively. Our studies revealed that plasmidic genes encoding cell surface proteins are more involved in the adhesion of IBB477 strain than in the ability to confer a selective advantage in the gut.

Surface Proteins of Lactococcus lactis: Bacterial Resources for Muco-adhesion in the Gastrointestinal Tract

“…In L. lactis plasmid free laboratory strains IL1403 and MG1363, only one MUB protein with four Mub domains and devoid of signal sequence, is encoded in the chromosome sequence ( Boekhorst et al, 2006 ). However, these strains do not exhibit muco-adhesive properties ( Radziwill-Bienkowska et al, 2017 ), suggesting that this MUB protein is not expressed or not functional. In contrast, three other MUB proteins found in dairy or vegetal strains were shown to contribute to bacterial adhesion to mucins as tested by different methods and tools ( Table 1 ).…”

“…The presence of PrtP at the surface of L. lactis was shown to modify the cell surface physico-chemical properties, leading to a greater hydrophobicity and increased adhesion to abiotic surfaces ( Habimana et al, 2007 ). In L. lactis IBB477, the cell surface PrtP was shown to contribute to mucin adhesion ( Radziwill-Bienkowska et al, 2017 ) ( Table 1 ).…”

“…In vitro models that are most commonly used for evaluation of L. lactis adhesive properties, involve static microtiter plate assays using immobilized commercially available mucin (pig gastric mucin, PGM) and/or gut-related epithelial cell lines ( Figure 1 ). Adherent bacterial cells are quantified using various approaches such as culturing/microscopical enumeration ( Kimoto et al, 1999 ; Meyrand et al, 2013 ), radiolabeling ( Rintahaka et al, 2014 ), crystal violet staining ( Lukic et al, 2012 ; Radziwill-Bienkowska et al, 2016 ), or FISH ( Radziwill-Bienkowska et al, 2017 ). The two most commonly used intestinal epithelial cell lines to study LAB adhesion are Caco-2 and HT29, which are originally derived from a human colorectal adenocarcinoma.…”

“…Their major disadvantage is the lack of substantial mucus production. However, a mucus-secreting subpopulation of goblet cells from HT29 cell population (HT29-MTX) has been obtained after growth adaptation of HT29 cells to anticancer drug methotrexate (MTX) ( Lesuffleur et al, 1990 ) and this cell line is increasingly used to investigate the muco-adhesive properties of L. lactis ( Lukic et al, 2012 ; Radziwill-Bienkowska et al, 2017 ).…”

“…Furthermore, the high adhesion of IBB477 vs. MG1820 to PGM was confirmed at the multicellular level and under flow field conditions, using real-time quartz crystal microbalance with dissipation monitoring (QCM-D) ( Le et al, 2012 ) and shear stress flow chamber ( Radziwill-Bienkowska et al, 2016 ). Later studies revealed the combined contribution of chromosomal and plasmid-encoded cell-wall proteins (MucBP-Big_3 containing protein and proteinase PrtP respectively) in the muco-adhesive properties of IBB477 ( Radziwill-Bienkowska et al, 2016 , 2017 ).…”