Impacts of pH-mediated EPS structure on probiotic bacterial pili–whey proteins interactions (original) (raw)
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Analysis of Homotypic Interactions of Lactococcus lactis Pili Using Single-Cell Force Spectroscopy
ACS Applied Materials & Interfaces, 2020
Cell surface proteins of Gram-positive bacteria play crucial roles in their adhesion to abiotic and biotic surfaces. Pili are long and flexible proteinaceous filaments known to enhance bacterial initial adhesion. They promote surface colonization and are thus considered as essential factors in biofilm cohesion. Our hypothesis is that pili mediate interactions between cells and may thereby directly affect biofilm formation. In this study, we use single-cell force spectroscopy (SCFS) to quantify the force of the homotypic pili interactions between individual bacterial cells, using different Lactococcus lactis strains producing pili or not as model bacteria. Moreover the force−distance curves were analyzed to determine the physical and nanomechanical properties of L. lactis pili. The results for pili-devoided strains showed a weak adhesion between cells (adhesion forces and work in the range of 100 pN and 7 × 10 −18 J, respectively). On the contrary, the piliated strains showed high adhesion levels with adhesion forces and adhesion work over 200 pN and 50 × 10 −18 J, respectively. The force−extension curves showed multiple adhesion events, typical of the unfolding of macromolecules. These unfolding force peaks were fitted using the physical worm-like chain model to get fundamental knowledge on the pili nanomechanical properties. In addition, SCFS applied to a L. lactis isolate expressing both pili and mucusbinding protein at its surface and two derivative mutants revealed the capacity of pili to interact with other surface proteins including mucus-binding proteins. This study demonstrates that pili are involved in L. lactis homotypic interactions and thus can influence biofilm structuring.
Scientific Reports
Lactic acid bacteria, in particular Lactococcus lactis, are widely used in the food industry, for the control and/or the protection of the manufacturing processes of fermented food. While L. lactis has been reported to form compact and uniform biofilms it was recently shown that certain strains able to display pili at their surface form more complex biofilms exhibiting heterogeneous and aerial structures. As the impact of those biofilm structures on the biomechanical properties of the biofilms is poorly understood, these were investigated using AFM force spectroscopy and imaging. Three types of strains were used i.e., a control strain devoid of pili and surface mucus-binding protein, a strain displaying pili but no mucus-binding proteins and a strain displaying both pili and a mucus-binding protein. To identify potential correlations between the nanomechanical measurements and the biofilm architecture, 24-h old biofilms were characterized by confocal laser scanning microscopy. Globa...
Food Hydrocolloids, 2014
Probiotic bacteria are being increasingly encapsulated to enhance their delivery in an active state at their preferred site of action. In this study, an encapsulation process based on emulsification and requiring only food grade components was used to protect wild-type Lactobacillus rhamnosus GG (LGG) and three of its surface mutants into dairy matrices. The mechanism of microencapsulation was studied at the molecular level by comparing the encapsulation efficiency of LGG wild type and three of its surface mutants with Atomic Force Microscopy. A significant decrease in the encapsulation efficiency was observed when the bacteria were depleted for pili, while the pilus also appeared to be crucial for location of LGG inside the microparticle. Hereto, the spaCBA mutant lacking pili), the welE mutant lacking long exopolysaccharides) and the dltD mutant having modified lipoteichoic acids were used. Atomic Force Microscopy enabled the confirmation of specific interactions between bacteria and whey proteins, in contrast to the observed nonspecific interactions with micellar casein. The role of the pili, i.e. multimeric appendages of several micrometers, was also modeled using WLC (Worm-Like Chain) or FJC (Freely Jointed Chain) models. This revealed that understanding molecular mechanisms of microencapsulation of probiotic bacteria should ultimately benefit their targeted application.
Analysis of Homotypic Interactions of Lactococcus lactis Pili Using Single-Cell Force Spectroscopy
Cell surface proteins of Gram-positive bacteria play crucial roles in their adhesion to abiotic and biotic surfaces. Pili are long and flexible proteinaceous filaments known to enhance bacterial initial adhesion. They promote surface colonization and are thus considered as essential factors in biofilm cohesion. Our hypothesis is that pili mediate interactions between cells and may thereby directly affect biofilm formation. In this study, we use single-cell force spectroscopy (SCFS) to quantify the force of the homotypic pili interactions between individual bacterial cells, using different Lactococcus lactis strains producing or not pili as model bacteria. Moreover the force-distance curves were analyzed to determine the physical and nanomechanical properties of L. lactis pili. The results for pili-devoided strainsshowed a weak adhesion between cells (adhesion forces and work in the range of 100 pN and 7.10-18 J respectively). On the contrary, the piliated strains showed high adhesion level with adhesion forces and adhesion work over 200 pN and 50.10-18 J respectively. The force-vs-extension curves showed multiple adhesion events, typical of the unfolding of macromolecules. These unfolding force peaks were fitted using the physical Worm Like-Chain (WLC) model to get fundamental knowledgeon the pili nanomechanical properties. In addition, SCFS applied to a L. lactis isolate expressing both pili and mucus-binding protein at its surface, and to derivative mutants revealed the capacity of pili to interact with other surface proteins including mucusbinding proteins. This study demonstrates that pili are involved in L. lactis homotypic interactions and thus can influence biofilm structuring.
pH-induced changes in adsorbed β-lactoglobulin molecules measured using atomic force microscopy
Soft Matter, 2009
We have used atomic force microscopy (AFM) imaging and force spectroscopy to study b-lactoglobulin (b-LG) food protein molecules adsorbed onto a mica surface. In particular, we have studied the effect of in situ changes in pH on several different properties: the topographical morphology of the adsorbed b-LG molecules, adhesion of the b-LG molecules to the underlying mica substrate, and the mechanical unfolding of the b-LG molecules. In AFM images, the structure of the adsorbed protein layer was observed to change dramatically with changes in pH. This result was consistent with the mechanical unfolding of single protein molecules within the adsorbed protein layer at different pH values performed using AFM. The short rupture length ($50 nm) measured for the fully unfolded protein at an acidic pH value of 2.5 is in good agreement with the dominant single molecule population measured previously for this pH value. Unfolding b-LG molecules from the same protein layer at a neutral value of pH ¼ 6.8 resulted primarily in longer rupture lengths, corresponding to dimers of b-LG. AFM force-distance curves collected at pH ¼ 9 were dominated by a large repulsion between the AFM tip and the adsorbed protein layer, which is likely due to the extended nature of the molecules because of irreversible denaturation for pH values greater than 9. This work provides a novel insight into the mechanisms of protein adsorption onto surfaces and shows that AFM force spectroscopy is a promising tool for probing in situ conformational changes in single molecules under various conditions.
Dynamic Cell Surface Hydrophobicity of Lactobacillus Strains with and without Surface Layer Proteins
Journal of Bacteriology, 2004
Variations in surface hydrophobicity of six Lactobacillus strains with and without an S-layer upon changes in ionic strength are derived from contact angle measurements with low- and high-ionic-strength aqueous solutions. Cell surface hydrophobicity changed in response to changes in ionic strength in three out of the six strains, offering these strains a versatile mechanism to adhere to different surfaces. The dynamic behavior of the cell surface hydrophobicity could be confirmed for two selected strains by measuring the interaction force between hydrophobic and hydrophilic tips with use of atomic force microscopy.
Single-Cell Force Spectroscopy of Probiotic Bacteria
Biophysical Journal, 2013
Single-cell force spectroscopy is a powerful atomic force microscopy modality in which a single living cell is attached to the atomic force microscopy cantilever to quantify the forces that drive cell-cell and cell-substrate interactions. Although various single-cell force spectroscopy protocols are well established for animal cells, application of the method to individual bacterial cells remains challenging, mainly owing to the lack of appropriate methods for the controlled attachment of single live cells on cantilevers. We present a nondestructive protocol for single-bacterial cell force spectroscopy, which combines the use of colloidal probe cantilevers and of a bioinspired polydopamine wet adhesive. Living cells from the probiotic species Lactobacillus plantarum are picked up with a polydopamine-coated colloidal probe, enabling us to quantify the adhesion forces between single bacteria and biotic (lectin monolayer) or abiotic (hydrophobic monolayer) surfaces. These minimally invasive single-cell experiments provide novel, to our knowledge, insight into the specific and nonspecific forces driving the adhesion of L. plantarum, and represent a generic platform for studying the molecular mechanisms of cell adhesion in probiotic and pathogenic bacteria.
PLoS ONE, 2011
The physicochemical properties and dynamics of bacterial envelope, play a major role in bacterial activity. In this study, the morphological, nanomechanical and electrohydrodynamic properties of Escherichia coli K-12 mutant cells were thoroughly investigated as a function of bulk medium ionic strength using atomic force microscopy (AFM) and electrokinetics (electrophoresis). Bacteria were differing according to genetic alterations controlling the production of different surface appendages (short and rigid Ag43 adhesins, longer and more flexible type 1 fimbriae and F pilus). From the analysis of the spatially resolved force curves, it is shown that cells elasticity and turgor pressure are not only depending on bulk salt concentration but also on the presence/absence and nature of surface appendage. In 1 mM KNO 3 , cells without appendages or cells surrounded by Ag43 exhibit large Young moduli and turgor pressures (,700-900 kPa and ,100-300 kPa respectively). Under similar ionic strength condition, a dramatic ,50% to ,70% decrease of these nanomechanical parameters was evidenced for cells with appendages. Qualitatively, such dependence of nanomechanical behavior on surface organization remains when increasing medium salt content to 100 mM, even though, quantitatively, differences are marked to a much smaller extent. Additionally, for a given surface appendage, the magnitude of the nanomechanical parameters decreases significantly when increasing bulk salt concentration. This effect is ascribed to a bacterial exoosmotic water loss resulting in a combined contraction of bacterial cytoplasm together with an electrostatically-driven shrinkage of the surface appendages. The former process is demonstrated upon AFM analysis, while the latter, inaccessible upon AFM imaging, is inferred from electrophoretic data interpreted according to advanced soft particle electrokinetic theory. Altogether, AFM and electrokinetic results clearly demonstrate the intimate relationship between structure/flexibility and charge of bacterial envelope and propensity of bacterium and surface appendages to contract under hypertonic conditions.
Atomic force microscopy study of Escherichia coli lactose permease proteolipid sheets
Biosensors & Bioelectronics, 2005
Proteolipid sheets (PLSs) obtained using the vesicle fusion technique on a convenient surface are the base to obtain transmembrane protein biosensors. In this preliminary work, we have screened several physicochemical conditions to optimize the visualization of proteolipid sheets formed between different phospholipid matrices and the membrane protein lactose permease (LacP) by atomic force microscopy (AFM).
Influence of csgD and ompR on Nanomechanics, Adhesion Forces, and Curli Properties of E. coli
Langmuir : the ACS journal of surfaces and colloids, 2016
Curli are bacterial appendages involved in the adhesion of cells to surfaces; their synthesis is regulated by many genes such as csgD and ompR. The expression of the two curli subunits (CsgA and CsgB) in Escherichia coli (E. coli) is regulated by CsgD; at the same time, csgD transcription is under the control of OmpR. Therefore, both genes are involved in the control of curli production. In this work, we elucidated the role of these genes in the nanomechanical and adhesive properties of E. coli MG1655 (a laboratory strain not expressing significant amount of curli) and its curli-producing mutants overexpressing OmpR and CsgD, employing atomic force microscopy (AFM). Nanomechanical analysis revealed that the expression of these genes gave origin to cells with a lower Young's modulus (E) and turgidity (P0), whereas the adhesion forces were unaffected when genes involved in curli formation were expressed. AFM was also employed to study the primary structure of the curli expressed t...