DOI: 10.1021/la904637k 8567 Langmuir 2010, 26(11), 8567–8572 Published on Web 02/10/2010 pubs.acs.org/Langmuir © 2010 American Chemical Society Hemolytic Activity of a Bacterial Trehalose Lipid Biosurfactant Produced by Rhodococcus sp.: Evidence for a Colloid-Osmotic Mechanism Ana Zaragoza, † Francisco J. Aranda, † Marı´a J. Espuny, ‡ Jos e A. Teruel, † Ana Marqu es, ‡  Angeles Manresa, ‡ and Antonio Ortiz* ,† † Departamento de Bioquı´mica y Biologı´a Molecular-A, Facultad de Veterinaria, Universidad de Murcia, E-30100 Murcia, Spain, and ‡ Laboratorio de Microbiologı´a, Facultad de Farmacia, Universidad de Barcelona, Joan XXIII s/n, E-08028 Barcelona, Spain. Received December 9, 2009. Revised Manuscript Received January 14, 2010 A succinoyl trehalose lipid produced by Rhodococcus sp. behaves as a biological surfactant and also displays various interesting biological activities. Trehalose lipid has been shown to have a great tendency to partition into phospholipid membranes; therefore, the characterization of its interaction with biological membranes is of central importance. In this work, human red blood cells have been used as an experimental model. Trehalose lipid causes the swelling of human erythrocytes followed by hemolysis at concentrations well below its critical micellar concentration. Kinetic measure- ments show that, upon addition of trehalose lipid, K þ release precedes that of hemoglobin. Osmotic protectants of the appropriate size added to the external medium make it possible to avoid hemolysis. The results indicate that trehalose lipid causes the hemolysis of human erythrocytes by a colloid-osmotic mechanism, most likely by formation of enhanced permeability domains, or “pores” enriched in the biosurfactant, within the erythrocyte membrane. Scanning electron microscopy shows trehalose lipid-induced spherocytosis and echinocytosis of red blood cells, which fits well within the framework of the bilayer-couple hypothesis. The presented results contribute to establishing a molecular basis for the biological properties of this trehalose lipid biosurfactant. 1. Introduction The production of amphiphilic compounds with surface acti- vity is widely spread among microorganisms. Thus, the term biosurfactant is commonly used to define surface-active amphi- philic compounds of biological origin. The intense investigation in this field is leading to the description of new chemical and biological properties; therefore, interesting potential applications are arising. 1-6 Biosurfactants present several advantages over surfactants of a chemical origin, particularly in relation to their biodegradability and environmental compatibility. An additional characteristic of some biosurfactants is the display of diverse biological activities, for instance, as antifungal or, in general, antimicrobial agents, which extends the field for future uses. 7-9 Some of these compounds can be produced through biotechno- logical processes in which the carbon source is provided by waste materials or side products from several industries, constituting an added value not only from an economic point of view but also for ecological reasons. 10 Thus, the identification of new biosurfactants and the characterization of their chemical and biological proper- ties are the subjects of intense research activity. 5,6,11 Among the various chemical structures found within biosur- factants, the most studied include lipopeptides, glycolipids, poly- mers, and others. 4 Because of their amphiphilic nature, biosurfac- tants, when added to biological systems, preferentially partition into biological membranes, altering their physicochemical prop- erties and function. 12-14 Much recent work has been devoted to the study of the membrane action of various biosurfactants, inclu- ding lipopeptides such as surfactin or iturin A 15-18 or glycolipids such as rhamnolipids 12,13,19,20 and trehalose lipids. 21-24 These compounds alter the physicochemical properties of the hydro- phobic core of the bilayer as well as perturb the hydration status *Corresponding author: Tel: þ34-868-884788. 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