Biological Physics Group, Physics Department, Carnegie Mellon University, Pittsburgh, Pennsylvania; Instituto Superior de Investigaciones Biológicas (INSIBIO) CONICET-UNT and Instituto de Química Biológica "Dr Bernabé Bloj", Facultad de Bioquímica, Química y Farmacia, UNT, San Miguel de Tucumán, Argentina.
Biological Physics Group, Physics Department, Carnegie Mellon University, Pittsburgh, Pennsylvania.
Biophys J. 2018 Feb 27;114(4):919-928. doi: 10.1016/j.bpj.2017.12.027.
Although colistin's clinical use is limited due to its nephrotoxicity, colistin is considered to be an antibiotic of last resort because it is used to treat patients infected with multidrug-resistant bacteria. In an effort to provide molecular details about colistin's ability to kill Gram-negative (G(-)) but not Gram-positive (G(+)) bacteria, we investigated the biophysics of the interaction between colistin and lipid mixtures mimicking the cytoplasmic membrane of G(+), G(-) bacteria as well as eukaryotic cells. Two different models of the G(-) outer membrane (OM) were assayed: lipid A with two deoxy-manno-octulosonyl sugar residues, and Escherichia coli lipopolysaccharide mixed with dilaurylphosphatidylglycerol. We used circular dichroism and x-ray diffuse scattering at low and wide angle in stacked multilayered samples, and neutron reflectivity of single, tethered bilayers mixed with colistin. We found no differences in secondary structure when colistin was bound to G(-) versus G(+) membrane mimics, ruling out a protein conformational change as the cause of this difference. However, bending modulus K perturbation was quite irregular for the G(-) inner membrane, where colistin produced a softening of the membranes at an intermediate lipid/peptide molar ratio but stiffening at lower and higher peptide concentrations, whereas in G(+) and eukaryotic mimics there was only a slight softening. Acyl chain order in G(-) was perturbed similarly to K. In G(+), there was only a slight softening and disordering effect, whereas in OM mimics, there was a slight stiffening and ordering of both membranes with increasing colistin. X-ray and neutron reflectivity structural results reveal colistin partitions deepest to reach the hydrocarbon interior in G(-) membranes, but remains in the headgroup region in G(+), OM, and eukaryotic mimics. It is possible that domain formation is responsible for the erratic response of G(-) inner membranes to colistin and for its deeper penetration, which could increase membrane permeability.
虽然多粘菌素由于其肾毒性而临床应用受限，但由于其用于治疗感染多重耐药菌的患者，因此被认为是抗生素的最后手段。为了提供关于多粘菌素杀死革兰氏阴性（G(-)）但不杀死革兰氏阳性（G(+)）细菌的分子细节，我们研究了多粘菌素与模拟 G(+)、G(-)细菌以及真核细胞细胞质膜的脂质混合物相互作用的生物物理学。我们检测了两种不同的 G(-)外膜（OM）模型：带有两个去氧甘露糖辛醣基的脂质 A，以及与双十四烷酰基磷脂酰甘油混合的大肠杆菌脂多糖。我们使用圆二色性和低角度及广角的 X 射线漫散射在堆积的多层样品中，以及与多粘菌素混合的单、连接双层的中子反射率。当多粘菌素与 G(-)与 G(+)膜模拟物结合时，我们没有发现二级结构的差异，从而排除了这种差异是由于蛋白质构象变化引起的可能性。然而，对于 G(-)内膜，弯曲模量 K 的扰动相当不规则，其中多粘菌素在中间脂质/肽摩尔比下使膜变软，但在较低和较高的肽浓度下使膜变硬，而在 G(+)和真核模拟物中只有轻微的软化。G(-)中的酰基链有序性也受到类似的 K 扰动。在 G(+)中，只有轻微的软化和无序效应，而在 OM 模拟物中，随着多粘菌素的增加，两个膜都有轻微的变硬和有序化。X 射线和中子反射率结构结果表明，多粘菌素在 G(-)膜中最深地分配以到达烃内部，但在 G(+)、OM 和真核模拟物中仍留在头基区域。有可能是域形成导致 G(-)内膜对多粘菌素的不规则反应及其更深的渗透，这可能增加膜通透性。
