Adhesion of the genome-sequenced Lactococcus lactis subsp. cremoris IBB477 strain is mediated by specific molecular determinants.

Understanding the nature of mucus-microbe interactions will provide important information that can help to elucidate the mechanisms underlying probiotic adhesion. This study focused on the adhesive properties of the Lactococcus lactis subsp. cremoris IBB477 strain, previously shown to persist in the gastrointestinal tract of germ-free rats. The shear flow-induced detachment of L. lactis cells was investigated under laminar flow conditions. Such a dynamic approach demonstrated increased adhesion to bare and mucin-coated polystyrene for IBB477, compared to that observed for the MG1820 control strain. To identify potential genetic determinants giving adhesive properties to IBB477, the improved high-quality draft genome sequence comprising chromosome and five plasmids was obtained and analysed. The number of putative adhesion proteins was determined on the basis of surface/extracellular localisation and/or the presence of adhesion domains. To identify proteins essential for the IBB477 specific adhesion property, nine deletion mutants in chromosomal genes have been constructed and analysed using adhesion tests on bare polystyrene as well as mucin-, fibronectin- or collagen IV-coated polystyrene plates in comparison to the wild-type strain. These experiments demonstrated that gene AJ89_07570 encoding a protein containing DUF285, MucBP and four Big_3 domains is involved in adhesion to bare and mucin-coated polystyrene. To summarise, in the present work, we characterised the adhesion of IBB477 under laminar flow conditions; identified the putative adherence factors present in IBB477, which is the first L. lactis strain exhibiting adhesive and mucoadhesive properties to be sequenced and demonstrated that one of the proteins containing adhesion domains contributes to adhesion.

Real-time investigation of the muco-adhesive properties of Lactococcus lactis using a quartz crystal microbalance with dissipation monitoring.

This work was devoted to probe, at the entire population level, interactions between mucins and Lactococcus lactis, using QCM-D. Real-time monitoring of adsorption on polystyrene of PGM (Pig Gastric Mucin) and subsequent adhesion of L. lactis was performed for IBB477 and MG1820 strains. Measuring simultaneously shifts in resonance frequency and dissipation on the polystyrene-coated crystal demonstrated a two-phase process for PGM adsorption. XPS analysis confirmed the presence of adsorbed mucin. The Voigt-based model was used to describe the QCM-D outputs. The predicted thickness of the PGM layer was consistent with the AFM experimental value. Adhesion of L. lactis to bare or PGM-coated polystyrene was then monitored, in combination with DAPI cell counting. Positive frequency shifts were caused by adhering bacteria. The presence of adsorbed PGM strongly reduced bacterial adhesion. However, adhesion of IBB477 to the PGM coating was greatly increased in comparison with that of MG1820. Muco-adhesion may be a highly variable and valuable phenotypic trait among L. lactis strains.

Measuring kinetic dissociation/association constants between Lactococcus lactis bacteria and mucins using living cell probes.

In this work we focused on quantifying adhesion between Lactococcus lactis, the model for lactic acid bacteria (LAB) and mucins. Interactions between two strains of L. lactis (IBB477 and MG1820 as control) and pig gastric mucin-based coating were measured and compared with the use of atomic force microscopy. Analysis of retraction force-distance curves shed light on the differential contributions of nonspecific and specific forces. An increased proportion of specific adhesive events was obtained for IBB477 (20% vs. 5% for the control). Blocking assays with free pig gastric mucin and its O-glycan moiety showed that oligosaccharides play a major (but not exclusive) role in L. lactis-mucins interactions. Specific interactions were analyzed in terms of kinetic constants. An increase in the loading rate of atomic force microscope tip led to a higher force between interacting biological entities, which was directly linked to the kinetic dissociation constant (K(off)). Enhancing the contact time between the tip and the sample allowed an increase in the interaction probability, which can be related to the kinetic association constant (K(on)). Variations in the loading rate and contact time enabled us to determine K(on) (3.3 × 10(2) M(-1)·s(-1)) and K(off) (0.46 s(-1)), and the latter was consistent with values given in the literature for sugar-protein interactions.

Probing in vitro interactions between Lactococcus lactis and mucins using AFM.

This work was devoted to the first AFM investigation of the adhesion force to pig gastric mucin (PGM) using Lactococcus lactis as the model for lactic acid bacteria. The PGM coating on polystyrene was characterized using a complementary set of multiscale analytical methods, including AFM (HarmoniX mode), XPS, and the sessile drop method. The PGM layer, which was mainly composed of C-O, C-N, COOH, CONH, and sulfur-related species (protein core and oligosaccharide side chains), was quite homogeneous and hydrophilic, with an estimated thickness of 3.4 nm. L. lactis cells were immobilized on the AFM tip (lacto probe) and used as a force probe to measure the interaction forces between bacteria and PGM-coated polystyrene on the nanoscale. After mucin adsorption, adhesion force levels were lower because of the interplay of electrostatic, hydrophilic, and steric repulsions. For example, the adhesion forces of the lacto probe to bare and PGM-coated polymer were 0.74 +/- 0.10 and 0.12 +/- 0.06 nN, respectively. The shape analysis of retraction force-distance curves highlighted the contribution of both nonspecific and specific forces (ligand/receptor bonding). The lacto probe concept and the associated AFM measurements may now provide a powerful framework for understanding interaction mechanisms between mucins and lactic acid bacteria.

Unraveling the role of surface mucus-binding protein and pili in muco-adhesion of Lactococcus lactis.

Adhesion of bacteria to mucus may favor their persistence within the gut and their beneficial effects to the host. Interactions between pig gastric mucin (PGM) and a natural isolate of Lactococcus lactis (TIL448) were measured at the single-cell scale and under static conditions, using atomic force microscopy (AFM). In parallel, these interactions were monitored at the bacterial population level and under shear flow. AFM experiments with a L. lactis cell-probe and a PGM-coated surface revealed a high proportion of specific adhesive events (60%) and a low level of non-adhesive ones (2%). The strain muco-adhesive properties were confirmed by the weak detachment of bacteria from the PGM-coated surface under shear flow. In AFM, rupture events were detected at short (100-200 nm) and long distances (up to 600-800 nm). AFM measurements on pili and mucus-binding protein defective mutants demonstrated the comparable role played by these two surface proteinaceous components in adhesion to PGM under static conditions. Under shear flow, a more important contribution of the mucus-binding protein than the pili one was observed. Both methods differ by the way of probing the adhesion force, i.e. negative force contact vs. sedimentation and normal-to-substratum retraction vs. tangential detachment conditions, using AFM and flow chamber, respectively. AFM blocking assays with free PGM or O-glycan fractions purified from PGM demonstrated that neutral oligosaccharides played a major role in adhesion of L. lactis TIL448 to PGM. This study dissects L. lactis muco-adhesive phenotype, in relation with the nature of the bacterial surface determinants.