Role of TraJ in Neonatal E. coli Sepsis and Meningitis

描述(申请人提供)：尽管使用了现代治疗和诊断措施，但新生儿细菌性败血症和脑膜炎仍然是一种发病率和死亡率不可接受的疾病。这种疾病的死亡率为15%-75%；此外，约50%的脑膜炎幸存者有严重的神经和发育异常。大肠杆菌K1(革兰氏阴性脑膜炎最常见的病原体)的主要感染途径是口服。肠道定植后，细菌通过胃肠道转移到肠系膜淋巴结(MLN)、肝、脾和血液的肠外部位。然后，大肠杆菌K1在血液中系统繁殖，达到菌血症的必要阈值，从而进入中枢神经系统(CNS)。最近，我们发现了一个编码血脑屏障(BBB)侵袭基因的E.ColiK1，TRAJ与多种F-样质粒trA操纵子的TRAJ同源。我们的初步数据表明，在新生大鼠中，TRAJ突变特异性地减弱了MLN向肝、脾、血液和中枢神经系统的扩散。此外，尽管口服感染TRAJ突变体的动物显示肝或脾中可恢复的细菌减少或没有恢复，但这些组织显示出显著的炎症反应。体外研究表明，TRAJ突变体较少被巨噬细胞摄取，并丢失了一种55 kDa的分泌蛋白。这一应用的中心假设是TRAJ控制一组基因的表达，这些基因的产物(即55 kDa分泌蛋白)参与了大肠杆菌K1的传播、系统感染和跨越BBB，这些事件在疾病过程中通过TRAJ依赖的与专业吞噬细胞的相互作用而发生。以下提出的实验旨在测试和证实我们的假设。我们的目标是：1)阐明TRAJ区和质粒区的遗传和分子特征，评价内源性TRAJ的自我转移能力，并确定含有TRAJ的质粒在E.ColiK1新生大鼠毒力中的潜在作用；2)评价TRAJ调控蛋白(即55 kDa分泌蛋白)在E.ColiK1全身播散和脑膜炎中的功能；3)确定新生大鼠依赖TRAJ的宿主炎症反应的机制。提高对早期全身传播的分子机制的了解，以及在这些事件期间宿主炎症反应的准确相互作用，将有助于实现我们的长期目标，即确定新的合理方法来开发新的治疗和预防策略，以治疗大肠杆菌K1败血症和脑膜炎。

DESCRIPTION (provided by applicant): Despite the use of modern therapeutics and diagnostic measures, neonatal bacterial sepsis and meningitis continues to be a disease with unacceptable rates of morbidity and mortality. The fatality rate of this disease is 15-75 percent; furthermore, approximately 50 percent of meningitis survivors have significant neurological and developmental abnormalities. The primary, route of infection for E. coli K1 (the most common causative agent of gram-negative meningitis) is oral. Following intestinal colonization, bacteria translocate through the GI tract to extra-intestinal sites of mesenteric lymph nodes (MLN), liver, spleen, and the blood. E. coli K1 then multiply systemically within the bloodstream reaching a necessary threshold of bacteremia to gain access to the central nervous system (CNS). Recently we identified an E. coli K1 plasmid-encoded blood-brain barrier (BBB) invasion gene, traJ, with homology to traJ of various F-like plasmid tra operons. Our preliminary data indicate that the traJ mutation specifically attenuates dissemination from the MLN to the liver, spleen, blood, and the CNS in the neonatal rat. In addition, although animals orally infected with the traJ mutant demonstrated a decrease or no recoverable bacteria in the liver or spleen, these tissues showed a significant inflammatory response. In vitro studies determined that the traJ mutant is taken-up less by macrophages and shows a loss of a 55 kDa-secreted protein. The central hypothesis of this application is that TraJ controls the expression of a set of genes whose products (i.e., 55 kDa secreted protein) are involved in E. coli K1 dissemination, systemic infection and crossing the BBB, and these events within the disease process occur via TraJ-dependent interactions with professional phagocytes. The following proposed experiments are designed to test and substantiate our hypotheses. We aim to 1) Elucidate the genetic and molecular characteristics of the traJ region and plasmid, evaluate the ability of the endogenous plasmid to self-transfer, and determine the potential role of the traJ-containing plasmid in E. coli K1 neonatal rat virulence, 2) Evaluate the function of the TraJ-regulated proteins (i.e., 55 kDa-secreted protein) in E. coli K1 systemic dissemination and meningitis, and 3) Determine the mechanism of TraJ-dependent host inflammatory response in the neonatal rat. Improved knowledge of molecular mechanisms for early systemic dissemination and the exact interplay of the host inflammatory response during these events will assist in achieving our long-term goal of identifying novel rational approaches to development of new treatments and preventive strategies for E. coli K1 sepsis and meningitis.