早川 枝李

Ionic liquids (ILs) are room-temperature molten salts that have applications in both physical sciences and more recently in the purification of proteins and lipids, gene transfection and sample preparation for electron microscopy (EM) studies. Transfection of genes into cells requires membrane fusion between the cell membrane and the transfection reagent, thus, ILs may be induce a membrane fusion event. To clarify the behavior of ILs with cell membranes the effect of ILs on model membranes, i.e., liposomes, were investigated. We used two standard ILs, 1-ethyl-3-methylimidazolium lactate ([EMI][Lac]) and choline lactate ([Ch][Lac]), and focused on whether these ILs can induce lipid vesicle fusion. Fluorescence resonance energy transfer and dynamic light scattering were employed to determine whether the ILs induced vesicle fusion. Vesicle solutions at low IL concentrations showed negligible fusion when compared with the controls in the absence of ILs. At concentrations of 30% (v/v), both types of ILs induced vesicle fusion up to 1.3 and 1.6 times the fluorescence intensity of the control in the presence of [Ch][Lac] and [EMI] [Lac], respectively. This is the first demonstration that [EMI][Lac] and [Ch][Lac] induce vesicle fusion at high IL concentrations and this observation should have a significant influence on basic biophysical studies. Conversely, the ability to avoid vesicle fusion at low IL concentrations is clearly advantageous for EM studies of lipid samples and cells. This new information describing IL-lipid membrane interactions should impact EM observations examining cell morphology. © 2013 Hayakawa et al.

Tuberculosis is an infectious and potentially fatal disease caused by the acid-fast bacillus Mycobacterium tuberculosis (MTB). One hallmark of a tuberculosis infection is the ability of the bacterium to subvert the normal macrophage defense mechanism of the host immune response. Lipoarabinomannan ( LAM), an integral component of the MTB cell wall, is released when MTBs are taken into phagosomes and has been reported to be involved in the inhibition of phago-lysosomal (P-L) fusion. However, the physical chemistry of the effects of LAM on lipid membrane structure relative to P-L fusion has not been studied. We produced membranes in vitro composed of dioleoylphosphatidylcholine, sphingomyelin, and cholesterol to simulate phagosomal lipid membranes and quantified the effects of the addition of LAM to these membranes, using fluorescence resonance energy transfer assays and atomic force microscopy. We found that LAM inhibits vesicle fusion and markedly alters lipid membrane domain morphology and sphingomyelin-chollesterol/dioleoylphosphatidylcholine ratios. These data demonstrate that LAM induces a dramatic reorganization of lipid membranes in vitro and clarifies the role of LAM in the inhibition of P-L fusion and the survival of the MTB within the macrophage.