Interacción de un diramnolípido biotensioactivo con membranas fosfolipídicas modelo y membranas biológicas

Abstract

Rhamnolipids are glycolipids produced by the Gram-negative bacteria Pseudomonas aeruginosa formed through the linkage of one or two rhamnose rings to a hydrophobic region composed by one or two fatty acids, being called monorhamnolipids and dirhamnolipids (diRL) respectively. These molecules are biosurfactants, compounds produced by microorganisms with an amphipathic structure responsible for its surface activity over the interfaces. Surfactants are distinguished by their capacity to form structural aggregates when its concentration in an aqueous medium is high, which enables them to solubilize insoluble compounds. Other properties, such as their emulsifying, stabilizing or antioxidative actions, grants them a great relevance on biotechnological applications. Rhamnolipids are some of the most studied glycolipids, being highlighted by its very interesting biological activities, namely, their capacity to inhibit the growth of Bacillus subtilis, eliminate spores of several species among three genera of plant pathogens, or by their capability to inhibit the growth of the dimorphic fungus Mucor circinelloides. To characterize the physicochemical processes responsible for these activities many researchers have focused on the study of the interaction between rhamnolipids and different biological and model membranes, proteins, lipid membrane remodeling, permeabilization of membranes or the formation of pH dependent liposomes, among others. On the other hand, several works have previously studied the aggregation behavior of rhamnolipids in water, describing numerous structures which include micelles, and several types of vesicles or stacked membranes, indicating that rhamnolipids can form lamellar, vesicular, or micellar aggregates in a pH-dependent fashion. On the basis of the previous experience of our research group on the characterization of the interaction between rhamnolipids and membrane phospholipids and proteins, as well as on the study of the aggregation behavior of rhamnolipids in aqueous medium, it was considered to carry out a work focused on expanding knowledge within these lines of research. In the first study, the phase behavior of mixtures of a synthetic species of phosphatidylserine, 1,2-dimyristoylphosphatidylserine, with a diRL fraction produced by P. aeruginosa, was characterized. We have observed that increasing concentrations of diRL caused a progressive rise in the temperature of the gel to liquid-crystalline phase transition, as well as phase separation at high concentrations of biosurfactant. On the other hand, the study of the main functional groups unveiled an increase of both acyl chain disorder and the hydration of the polar head groups of the phospholipid. This increased hydration observed in both gel and liquid-crystalline phases, is due to a conformational change that leads to an augmented exposition of these regions to the water layer. Secondly, the interaction between diRL and the transmembrane model protein sarco/endoplasmic reticulum calcium ATPase (SERCA1a), an ion transport protein present in the membranes of intracellular calcium storages, was studied. As a result of this work, we demonstrated that the interaction with diRL causes conformational changes that make the enzyme unstable, facilitating its thermal denaturation, and inhibiting its ATPase activity at concentrations below the membrane solubilization threshold. Finally, the effect of various pH and temperature values on the stability of the different aggregation structures of the diRL was studied. Among the many results, it should be noted that diRL adopts fairly organized multilayered structures at low pH and temperature, which become highly disordered upon increasing any of these parameters. In addition, an endothermic thermotropic transition around 34ºC for the diRL aggregates formed by the protonated diRL is described. As a whole, this thesis provides new information on the physicochemical properties of the diRL in water, and its interactions with phospholipid model membranes. Each publication, taken individually, provides individual and necessary contributions to unveil the complex mechanism by which this biosurfactant affects biological membranes through its interaction with their different components.