Biofilms are a common life-styles for microbial cells embedded in a self-produced matrix composed of biopolymers and small molecules . Composition and morphology of biofilms influence microorganisms life. Among macromolecules, polysaccharides are considered the main contributors to the biofilm architecture .
Understanding the nature of the molecular interactions inside the biofilm is essential to understand their biology and towards the definition of novel anti-biofilm strategies. Cystic fibrosis constitutes an example of the presence of biofilm, where pathogens like those of the Burkholderia cepacia complex are involved. Among them, the C1576 strain of Burkholderia multivorans is interesting for the type of exopolysaccharide (named EpolC1576) found in biofilm. This polysaccharide, constituted by mannose and rhamnose, is characterized by the presence of a methoxy group substituting the C3 hydroxyl group of 50% of rhamnose units.
A previous investigation of EpolC1576 by fluorescence spectroscopy and molecular modelling established the non-polar characteristic of this polysaccharide and its ability to interact with hydrophobic molecules . To confirm the hydrophobic nature of EpolC1576 interactions, and establish its three-dimensional conformation, investigations by means of Surface Plasmon Resonance (SPR) and Atomic Force Microscopy (AFM) were carried out.
SPR indicated that EpolC1576 was able to interact with alkyl chains on a functionalized chip in a concentration dependent manner. It showed a higher binding affinity for alkyl chains with respect to dextran, used as a negative control. The formation of a stable complex between the polysaccharide and the alkyl chains was also detected, since its dissociation was not completely reversible.
The three-dimensional structure of EpolC1576 was investigated by means of AFM after spray-drying on mica surfaces from aqueous solutions. Single chains were characterized by a spherical compact morphology and the tendency of the polymer to self-aggregate was evidenced by the presence of aggregates upon increasing concentration. These findings are very different from previous AFM investigations on other polysaccharides, including Cepacian also produced by species of the B. cepacia complex, which showed an elongated chain morphology . To further characterize the EpolC1576 conformation, AFM experiments were carried out dissolving the polymer in mixed aqueous solvents composed of methanol and tetrahydrofuran. AFM images in these lower polarity solvents indicated a partial disruption of the aqueous conformation. This was especially evident in tetrahydrofuran where the measured height of the EpolC1576 was 0.5 nm compatible with a saccharidic chain lying on the mica surface.
In conclusion, the hydrophobic nature of intermolecular interactions, confirmed by SPR, and the aqueous conformation of EpolC1576 evidenced by AFM, suggest a novel structural role for EpolC1576 in the stabilization of the biofilm matrix through the formation of polysaccharides aggregates exhibiting a compact conformation. The non-polar nature of intermolecular interactions may explain the stability of the biofilm matrix in an aqueous environment. In addition, this kind of interactions may also play a role in the biochemistry of the matrix, as for example in the vehiculation of non-polar signalling molecules and adhesion of microbial cells to the biofilm matrix.
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