Delivery of the non-membrane-permeative antibiotic gentamicin into mammalian cells by using Shigella flexneri membrane vesicles
Delivery of the non-membrane-permeative antibiotic gentamicin into mammalian cells by using Shigella flexneri membrane vesicles
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DOI：
10.1128/aac.42.6.1476
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发表时间：
1998-06-01
影响因子：
4.9
通讯作者：
Beveridge, TJ
中科院分区：
文献类型：
--
作者：
Kadurugamuwa, JL;Beveridge, TJ
我们开发了一个模型来测试是否可以通过细菌膜囊泡将非膜渗透性治疗剂如庆大霉素递送到哺乳动物细胞中。许多革兰氏阴性菌在正常生长过程中会产生膜泡（MV），这些膜泡的数量可以通过短暂暴露于庆大霉素而增加（J. L。Kadurugamuwa和T. J. Beveridge，J. Bacteriol. 177：3998-4008，1995），其可被截留在MV内。庆大霉素诱导的MV（g-MW）从福氏志贺菌中分离，并且含有85 +/-2ng庆大霉素/μ g MV蛋白。免疫金电镜下用抗沙门氏菌抗体标记薄切片。福氏脂多糖（LPS）的粘附和随后的人Henle 407肠上皮细胞系对MV的吞噬。g-MV与S.福氏杆菌感染的Henle细胞显示，在孵育的第一个小时内，g-MV渗透到整个感染的细胞中，并将细胞内病原体减少了约1.5 log（10）CFU。在宿主细胞的胞浆中检测到抗生素，表明药物在g-MV渗透后的细胞内定位。可溶性抗生素作为液体加入组织培养生长培养基中，对细胞内细菌生长没有影响，证实了组织细胞膜对庆大霉素的不渗透性。用S.福氏Ipa特异性抗体表明MV中存在侵袭蛋白抗原IpaB、IpaC和iPad。由于是双层的，外表面由LPS和Ipa蛋白组成，这些MV很容易被其他不可渗透的膜吞没，并最终将其内容物释放到宿主组织的细胞质中。
We developed a model to test whether non-membrane-permeative therapeutic agents such as gentamicin could be delivered into mammalian cells by means of bacterial membrane vesicles. Many gram-negative bacteria bleb off membrane vesicles (MVs) during normal growth, and the quantity of these vesicles can be increased by brief exposure to gentamicin (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995), which can be entrapped within the MVs. Gentamicin-induced MVs (g-MWs) were isolated from Shigella flexneri and contained 85 +/- 2 ng of gentamicin per mu g of MV protein. Immunogold electron microscopic labeling of thin sections with antibodies specific to S. flexneri lipopolysaccharide (LPS) demonstrated the adherence and subsequent engulfment of MVs by the human Henle 407 intestinal epithelial cell line. Further incubation of g-MVs with S. flexneri-infected Henle cells revealed that the g-MVs penetrated throughout the infected cells and reduced the intracellular pathogen by similar to 1.5 log(10) CFU in the first hour of incubation. Antibiotic was detected in the cytoplasms of host cells, indicating the intracellular placement of the drug following the penetration of g-MVs. Soluble antibiotic, added as a fluid to the tissue culture growth medium, had no effect on intracellular bacterial growth, confirming the impermeability of the cell membranes of the tissue to gentamicin. Western blot analysis of MVs with S. flexneri Ipa-specific antibodies demonstrated that the invasion protein antigens IpaB, IpaC, and IpaD were present in MVs. Being bilayered, with outer faces composed of LPS and Ipa proteins, these MVs were readily engulfed by the otherwise impermeable membranes and eventually liberated their contents into the cytoplasmic substance of the host tissue.