Panjwani N, Zaidi T S, Gigstad J E, Jungalwala F B, Barza M, Baum J
Department of Ophthalmology, Tufts University School of Medicine, New England Medical Center, Boston, Massachusetts 02111.
Infect Immun. 1990 Jan;58(1):114-8. doi: 10.1128/iai.58.1.114-118.1990.
35S-labeled Pseudomonas aeruginosa isolates were shown to bind to neutral glycosphingolipids (NGSLs) of rabbit corneal epithelia in culture by a thin-layer chromatogram overlay procedure. The lipids of the corneal epithelial cells grown in culture were extracted and partitioned into a chloroform-rich lower phase containing NGSLs and an aqueous upper phase containing gangliosides. By using a dot-blot assay, at least six times more radiolabeled P. aeruginosa isolates were shown to bind to the lipids in the lower phase compared with those in the upper phase. Thin-layer chromatography of the lower-phase lipids followed by staining with an orcinol spray revealed at least 10 NGSL components and several fast-migrating, nonglycosylated neutral lipid components (including cholesterol). 35S-labeled P. aeruginosa was shown to bind to NGSL components 1, 2, 5, 6, and 9. P. aeruginosa-reactive NGSL components 6 and 9 migrated with chromatographic mobilities similar to those of the standards ceramide trihexoside (CT) and ceramide monohexoside, respectively. Components 1 and 2 migrated slightly ahead of asialo GM1, and component 5 migrated faster than globoside but slower than CT. Among the various standards tested, P. aeruginosa bound to asialo GM1 and, to a lesser extent, to ceramide dihexoside and CT but not to GM1, GD1A, GM3, or ceramide monohexoside. It remains to be determined whether any of the five P. aeruginosa-reactive NGSL components of corneal epithelium identified in this study plays a role in the development of corneal infection. However, we have previously shown that component 9, one of the five P. aeruginosa-reactive NGSL components identified in this study, is present in significantly greater amounts in migrating epithelia than it is in nonmigrating epithelia (N. Panjwani, G. Michalopoulos, J. Song, G. Yogeeswaran, and J. Baum, Invest. Ophthalmol. Vis. Sci., in press). This may prove to be of biological significance because it is generally believed that traumatized (migrating) epithelia are more susceptible to infection than normal (nonmigrating) epithelia are.
通过薄层层析图覆盖法显示，35S标记的铜绿假单胞菌分离株在培养物中可与兔角膜上皮的中性糖鞘脂（NGSLs）结合。提取培养的角膜上皮细胞的脂质，并分成富含氯仿的下层相（含有NGSLs）和水相上层相（含有神经节苷脂）。通过斑点印迹分析，结果显示与上层相脂质相比，至少6倍以上的放射性标记铜绿假单胞菌分离株可与下层相脂质结合。下层相脂质的薄层层析，然后用苔黑酚喷雾染色，显示至少有10种NGSL成分和几种快速迁移的、非糖基化的中性脂质成分（包括胆固醇）。35S标记的铜绿假单胞菌显示可与NGSL成分1、2、5、6和9结合。与铜绿假单胞菌反应的NGSL成分6和9的色谱迁移率分别与标准品神经酰胺三己糖苷（CT）和神经酰胺单己糖苷相似。成分1和2的迁移略先于脱唾液酸GM1，成分5的迁移速度比红细胞糖苷脂快，但比CT慢。在测试的各种标准品中，铜绿假单胞菌与脱唾液酸GM1结合，在较小程度上与神经酰胺二己糖苷和CT结合，但不与GM1、GD1A、GM3或神经酰胺单己糖苷结合。本研究中鉴定出的角膜上皮的五种与铜绿假单胞菌反应的NGSL成分中是否有任何一种在角膜感染的发生中起作用，仍有待确定。然而，我们之前已经表明，本研究中鉴定出的五种与铜绿假单胞菌反应的NGSL成分之一的成分9，在迁移上皮中的含量明显高于非迁移上皮（N. Panjwani、G. Michalopoulos、J. Song、G. Yogeeswaran和J. Baum，《Invest. Ophthalmol. Vis. Sci.》，即将发表）。这可能具有生物学意义，因为一般认为受创伤的（迁移的）上皮比正常的（非迁移的）上皮更容易受到感染。
