Backer Marina V, Patel Vimal, Jehning Brian T, Claffey Kevin P, Karginov Vladimir A, Backer Joseph M
SibTech, Inc., 705 North Mountain Road, Newington, CT 06111, USA.
Antimicrob Agents Chemother. 2007 Jan;51(1):245-51. doi: 10.1128/AAC.00983-06. Epub 2006 Oct 30.
In the course of Bacillus anthracis infection, B. anthracis lethal factor (LF) and edema factor bind to a protective antigen (PA) associated with cellular receptors ANTXR1 (TEM8) or ANTXR2 (CMG2), followed by internalization of the complex via receptor-mediated endocytosis. A new group of potential antianthrax drugs, beta-cyclodextrins, has recently been described. A member of this group, per-6-(3-aminopropylthio)-beta-cyclodextrin (AmPrbetaCD), was shown to inhibit the toxicity of LF in vitro and in vivo. In order to determine which steps in lethal factor trafficking are inhibited by AmPrbetaCD, we developed two targeted fluorescent tracers based on LFn, a catalytically inactive fragment of LF: (i) LFn site specifically labeled with the fluorescent dye AlexaFluor-594 (LFn-Al), and (ii) LFn-decorated liposomes loaded with the fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid (LFn-Lip). Both tracers retained high affinity to PA/ANTXR complexes and were readily internalized via receptor-mediated endocytosis. Using fluorescent microscopy, we found that AmPrbetaCD inhibits receptor-mediated cell uptake but not the binding of LFn-Al to PA/ANTXR complexes, suggesting that AmPrbetaCD works outside the cell. Moreover, AmPrbetaCD and LFn-Al synergistically protect RAW 264.7 cells from PA-mediated LF toxicity, confirming that AmPrbetaCD did not affect the binding of LFn-Al to receptor-associated PA. In contrast, AmPrbetaCD did not inhibit PA-mediated internalization of LFn-Lip, suggesting that multiplexing of LFn on the liposomal surface overcomes the inhibiting effects of AmPrbetaCD. Notably, internalized LFn-Al and LFn-Lip protected cells that overexpressed anthrax receptor TEM8 from PA-induced, LF-independent toxicity, suggesting an independent mechanism for PA inhibition inside the cell. These data suggest the potential for the use of beta-cyclodextrins in combination with LFn-Lip loaded with antianthrax drugs against intracellular targets.
在炭疽芽孢杆菌感染过程中，炭疽芽孢杆菌致死因子（LF）和水肿因子与一种与细胞受体ANTXR1（TEM8）或ANTXR2（CMG2）相关的保护性抗原（PA）结合，随后该复合物通过受体介导的内吞作用内化。最近描述了一组新的潜在抗炭疽药物，即β-环糊精。该组中的一个成员，全-6-（3-氨丙基硫基）-β-环糊精（AmPrβCD），已显示在体外和体内均能抑制LF的毒性。为了确定AmPrβCD抑制致死因子运输的哪些步骤，我们基于LF的催化无活性片段LFn开发了两种靶向荧光示踪剂：（i）用荧光染料AlexaFluor-594特异性标记的LFn（LFn-Al），以及（ii）装载有荧光染料8-羟基芘-1,3,6-三磺酸的LFn修饰脂质体（LFn-Lip）。两种示踪剂对PA/ANTXR复合物均保持高亲和力，并通过受体介导的内吞作用容易地内化。使用荧光显微镜，我们发现AmPrβCD抑制受体介导的细胞摄取，但不抑制LFn-Al与PA/ANTXR复合物的结合，这表明AmPrβCD在细胞外起作用。此外，AmPrβCD和LFn-Al协同保护RAW 264.7细胞免受PA介导的LF毒性，证实AmPrβCD不影响LFn-Al与受体相关PA的结合。相反，AmPrβCD不抑制PA介导的LFn-Lip内化，这表明LFn在脂质体表面的多重化克服了AmPrβCD的抑制作用。值得注意的是，内化的LFn-Al和LFn-Lip保护过表达炭疽受体TEM8的细胞免受PA诱导的、LF非依赖性毒性，这表明细胞内PA抑制存在独立机制。这些数据表明β-环糊精与装载抗炭疽药物的LFn-Lip联合用于对抗细胞内靶点的潜力。
