Syndecan and Bacterial Translocation in Shock and Trauma

描述（由申请人提供）：正常肠道细菌，如大肠杆菌和粪肠球菌，经常引起休克和创伤患者的并发症感染。这些患者的一个共同发现是肠上皮通透性增加，培养肠细胞的实验表明，随着肠细胞紧密连接打开，暴露肠细胞外侧表面，细菌对肠细胞的粘附和内化增加。Syndecan-1表达于人肠细胞基底外侧表面，是一种细胞表面跨膜蛋白多糖，在其胞外结构域表达硫酸肝素（HS）。我们的工作假设是细胞表面蛋白聚糖的HS链，特别是syndecan-1，可能作为多种肠道细菌的肠细胞受体或共受体。初步数据表明，HS和syndecan-1与人肠细胞一样，在培养的HT-29肠细胞基底外侧表面显著表达，而Caco-2肠细胞不表达。对HT-29肠细胞（旨在打开肠细胞紧密连接并干扰细菌与syndecan-1上HS链的结合）的实验表明，HS可能是革兰氏阳性细菌的受体，而不是革兰氏阴性细菌的受体。HS类似物肝素和HS本身抑制HT-29肠细胞对革兰氏阳性单核增生李斯特菌的粘附和内化，与相关糖胺聚糖的实验表明，这种抑制作用对HS是特异性的。另外对HT-29肠细胞进行的初步实验表明，肝素和HS同样能抑制革兰氏阳性的粪肠球菌和金黄色葡萄球菌的内化，但对革兰氏阴性的鼠伤寒沙门菌、变形杆菌和大肠杆菌没有抑制作用。肝素对Caco-2肠细胞内化任何细菌没有明显影响，Caco-2肠细胞表达低水平的HS和syndecan-1。其他初步实验表明，肝素处理的单核增生乳杆菌在口服接种小鼠中的侵袭性小于未处理的单核增生乳杆菌。在本研究中，我们使用了几种实验工具来阐明培养的肠细胞与多种革兰氏阴性细菌的相互作用，同时重点研究革兰氏阳性的单核细胞增生乳杆菌、粪肠球菌和金黄色葡萄球菌。这些工具包括单克隆抗体、糖胺聚糖和肝素双糖，以及转染过表达syndecan-1的两种细胞系，即ARH-77骨髓瘤细胞和Caco-2肠细胞。来自体外研究的数据用于设计小鼠实验（近亲繁殖和syndecan-1敲除），以阐明HS和syndecan-1在肠道细菌定植和肠外传播中的作用。这些实验数据可能表明，肠细胞有一种受体（与细胞表面HS有关，可能与syndecan-1有关）参与多种革兰氏阳性细菌（包括粪肠球菌和金黄色葡萄球菌）的粘附和内化。

DESCRIPTION (provided by applicant): Normal enteric bacteria, such as Escherichia coli and Enterococcus faecalis, frequently cause complicating infections in patients with shock and trauma. A common finding in these patients is increased intestinal epithelial permeability, and experiments with cultured enterocytes have shown that bacterial adherence to and internalization by enterocytes is increased following opening of enterocyte tight junctions, exposing the enterocyte lateral surface. Syndecan-1, expressed on the basolateral surface of human enterocytes, is a cell surface transmembrane proteoglycan that expresses heparan sulfate (HS) on its extracellular domain. Our working hypothesis is that HS chains of cell surface proteoglycans, and specifically syndecan-1, may act as an enterocyte receptor or co-receptor for a variety of enteric bacteria. Preliminary data indicated that,like human enterocytes, HS and syndecan-1 are prominently expressed on the basolateral surface of cultured HT-29 enterocytes but not Caco-2 enterocytes. Experiments with HT-29 enterocytes (designed to open enterocyte tight junctions and interfere with bacterial binding to the HS chains on syndecan-1) suggested that HS may be a receptor for gram-positive but not gram-negative bacteria. The HS analog heparin, and HS itself, inhibited adherence and internalization of gram-positive Listeria monocytogenes by HT-29 enterocytes, and experiments with related glycosaminoglycans indicated that this inhibition was specific for HS. Additional preliminary experiments with HT-29 enterocytes indicated that heparin and HS similarly inhibited internalization of gram-positive E. faecalis and Staphylococcus aureus, but not gram-negative Salmonella typhimurium, Proteus mirabilis, and E. coli. Heparin did not have a noticeable effect on internalization of any bacterial species using Caco-2 enterocytes, which express low levels of HS and syndecan-1 Other preliminary experiments indicated that heparin-treated L. monocytogenes was less invasive in orally inoculated mice than was untreated L monocytogenes. In this proposal several experimental tools are used to clarify the interactions of cultured enterocytes with a variety of gram-negative bacteria, while focusing on gram-positive L. monocytogenes, E. faecalis, and S. aureus. These tools include monoclonal antibodies, glycosamino glycans, and heparin disaccharides, and two cell lines transfected to over express syndecan-1, namely ARH-77 myeloma cells and Caco-2 enterocytes. Data from in vitro studies are used to design experiments in mice (outbred and syndecan-1 knockout) to clarify the role of HS and syndecan-1 in intestinal colonization and extra intestinal dissemination of enteric bacteria. Data from these experiments may indicate that enterocytes have a receptor (related to cell surface HS and perhaps syndecan-1) involved in adherence and internalization of a variety of gram-positive bacteria including E. faecalis and S. aureus.