Syndecan and Bacterial Translocation in Shock and Trauma

描述（由申请人提供）：正常的肠道细菌，如大肠杆菌和粪肠球菌，经常引起休克和创伤患者的并发感染。在这些患者中的常见发现是肠上皮通透性增加，并且培养的肠上皮细胞的实验表明，在肠上皮细胞紧密连接打开后，细菌粘附于肠上皮细胞并被肠上皮细胞内化，从而暴露肠上皮细胞的侧表面。Syndecan-1是一种表达于人肠上皮细胞基底外侧表面的跨膜蛋白聚糖，其胞外区表达硫酸乙酰肝素（HS）。我们的工作假设是，HS链的细胞表面蛋白聚糖，特别是syndecan-1，可能作为一个肠上皮细胞受体或共受体的各种肠道细菌。初步数据表明，像人类肠上皮细胞，HS和syndecan-1显着表达的培养HT-29肠上皮细胞的基底外侧表面，但不Caco-2肠上皮细胞。HT-29肠上皮细胞（旨在打开肠上皮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可能是革兰氏阳性菌而不是革兰氏阴性菌的受体。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细胞增生李斯特菌的粘附和内化，相关糖胺聚糖的实验表明这种抑制作用对HS具有特异性。用HT-29肠细胞进行的额外的初步实验表明，肝素和HS类似地抑制革兰氏阳性E.革兰氏阴性鼠伤寒沙门氏菌、奇异变形杆菌和大肠杆菌均未检出。杆菌使用表达低水平HS和多配体蛋白聚糖-1的Caco-2肠细胞，肝素对任何细菌物种的内化没有显著影响。单核细胞增多性李斯特菌在口服接种小鼠中的侵袭性比未处理的单核细胞增多性李斯特菌低。在这个建议中，几个实验工具被用来澄清培养的肠上皮细胞与各种革兰氏阴性细菌的相互作用，而重点是革兰氏阳性L。单核细胞增多症E. faecalis和S.金黄色。这些工具包括单克隆抗体、糖胺聚糖和肝素二糖，以及两种转染过表达多配体蛋白聚糖-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用于设计小鼠（远系繁殖和syndecan-1敲除）中的实验，以阐明HS和syndecan-1在肠细菌的肠定殖和肠外传播中的作用。来自这些实验的数据可能表明，肠上皮细胞具有参与多种革兰氏阳性细菌（包括大肠杆菌）的粘附和内化的受体（与细胞表面HS和可能的syndecan-1相关）。faecalis和S.金黄色。

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.