Syndecan and Bacterial Translocation in Shock and Trauma

描述(申请人提供)：正常的肠道细菌，如大肠埃希菌和粪肠球菌，经常导致休克和创伤患者的并发感染。这些患者的常见发现是肠上皮通透性增加，培养的肠细胞实验表明，随着肠细胞紧密连接的开放，暴露出肠细胞的侧面，细菌对肠细胞的黏附和内化增加。Syndecan-1表达于人肠上皮细胞的基底外侧表面，是一种细胞表面的跨膜蛋白多糖，在其胞外区表达硫酸乙酰肝素(HS)。我们的工作假设是，细胞表面蛋白多糖的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与人的肠细胞一样，在培养的HT-29肠上皮细胞的基底外侧表面显著表达，而Caco-2肠上皮细胞则不表达。用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本身可抑制HT-29肠上皮细胞对革兰氏阳性单核细胞增生性李斯特菌的黏附和内化，与相关糖胺多聚糖的实验表明这种抑制是HS所特有的。另外对HT-29肠道细胞的初步实验表明，肝素和HS类似地抑制革兰氏阳性粪肠球菌和金黄色葡萄球菌的内化，但不抑制革兰氏阴性鼠伤寒沙门氏菌、奇异变形杆菌和大肠杆菌的内化。肝素对使用表达低水平HS和Syndecan-1的Caco-2肠道细胞的任何细菌物种的内化都没有显著影响。其他初步实验表明，经肝素处理的单核细胞增多性乳杆菌在口服接种的小鼠中比未经处理的L单核细胞增多症小鼠侵袭性更小。在这项建议中，使用了几种实验工具来阐明培养的肠细胞与各种革兰氏阴性细菌的相互作用，同时重点研究了革兰氏阳性单核细胞增生菌、粪肠球菌和金黄色葡萄球菌。这些工具包括单抗、糖胺聚糖和肝素二糖，以及两种过表达syndecan-1的细胞系，即ARH-77骨髓瘤细胞和Caco-2肠细胞。来自体外研究的数据被用来设计在小鼠身上的实验(杂交和Syndecan-1基因敲除)，以阐明HS和Syndecan-1在肠道定植和肠道外传播肠道细菌中的作用。这些实验的数据可能表明，肠细胞有一个受体(与细胞表面HS有关，也许还与Syndecan-1有关)，参与包括粪肠球菌和金黄色葡萄球菌在内的各种革兰氏阳性细菌的黏附和内化。

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.