Invasion of C. neoformans into brain endothelial cells

新型隐球菌侵入脑内皮细胞

• 批准号: 7625341
• 负责人: AMBROSE Y JONG
• 金额: $ 37.94万
• 依托单位: CHILDREN'S HOSPITAL OF LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2009-07-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7625341
• 关键词: Abbreviations; Actins; Adhesions; Animals; Attenuated; Binding; Blood; Blood - brain barrier anatomy; Brain; CD44 gene; CYP21A2 gene; Caveolins; Central Nervous System Infections; Cerebrospinal Fluid; Cholesterol; Clinical; Colony-forming units; Cryptococcal Meningitis; Cytochalasin D; Cytoskeleton; Development; Dominant-Negative Mutation; Electron Microscope; Endothelial Cells; Event; F-Actin; Filipin; GF109203X; Goals; Grant; Human; Hyaluronan; Hyaluronic Acid; Immune response; In Vitro; Infection; Knock-out; Knockout Mice; Membrane; Meningitis; Modeling; Molecular; Morbidity - disease rate; Mus; Mutation; Neuraxis; Numbers; Patients; Process; Protein Kinase C; Protein Kinase C Inhibitor; Reagent; Role; Scanning; Severities; Signal Transduction; Site; Stream; Testing; Virulence; Yeasts; base; caveolin 1; cellular microvillus; citrate carrier; gastrointestinal microvillus; mouse model; novel strategies; pathogen; prevent; receptor; transmission process

Pathogenic yeast C. neoformans can disseminate through the blood stream and cause devastating meningitis. The mechanism transversal of C. neoformans across the blood-brain barrier (BBB) to cause the central nervous system (CNS) infection is largely unknown. The goal of this project is to continue investigating how C. neoformans enters into human brain microvascular endothelial cells (HBMEC), which constitute the BBB. In the last grant period, we found that C. neoformans could induce morphological changes in HBMEC via cytoskeleton reorganization. We demonstrated that C. neoformans CPS1 encoded hyaluronic acid synthase. We also verified that CPS1 was required for C. neoformans binding to HBMEC using an in vitro BBB model. Furthermore, our studies showed that CD44 was the primary receptor on HBMEC for C. neoformans adhesion. Upon C. neoformans binding to the HBMEC, host CD44 translocated to the membrane rafts and surrounded the yeast entry site. Either CPS1 deletion in C. neoformans or CD44- knockout on HBMEC significantly impaired the yeast infection. We also observed that yeast binding and/or invasion was considerably reduced in the filipin-, GF109203X-, cytochalasin D- treated HBMEC. Filipin extracts cholesterol and caveolin on the membrane rafts, GF109203X is a Protein Kinase C (PKC) inhibitor, and cytochalasin D is a F-actin disrupting reagent. Dominant-negative PKCa also inhibited yeast invasion into HBMEC. The results suggest that the integrity of membrane rafts, functional PKCa, and F-actin were necessary for yeast invasion. Based on the above observations, we hypothesize that CD44-elicited signaling and induced cytoskeleton reorganization are required for yeast entry into HBMEC. We will explore the mechanisms of C. neoformans invasion by the following Aims: (1) To determine the CD44-elicited signaling during the C. neoformans invasion, (2) To examine how C. neoformans induces cytoskeleton reorganization on HBMEC and its relationship to yeast infection, and (3) To evaluate the role of CD44 during C. neoformans invasion in mouse models. In our previous grant period, we have demonstrated the adhesion step of pathogen-host interaction. In this grant period, we will further characterize the molecular events at the internalization step. These studies are related to the clinical observations that a significant number of patients suffer severe meningitis and eventually succumb to this pathogen. The information derived from the studies is expected to be helpful in the development of novel strategies to prevent cryptococcal meningitis and its associated morbidity.

致病酵母C.新型人可以通过血液传播， 脑膜炎C.新生儿穿过血脑屏障（BBB）， 中枢神经系统（CNS）感染在很大程度上是未知的。该项目的目标是继续 调查C.新生儿进入人脑微血管内皮细胞（HBMEC）， 构成了BBB。 在上一个资助期，我们发现C。新生儿可引起HBMEC的形态学改变 通过细胞骨架重组。我们证明了C.新生儿CPS 1编码的透明质酸 合成酶我们还验证了CPS 1是C.新生儿与HBMEC结合的体外试验 BBB模型。此外，我们的研究表明，CD 44是C. 新生儿粘连。在C。新生儿与HBMEC结合，宿主CD 44易位至 膜筏和周围的酵母入口网站。C.新生儿或CD 44- HBMEC的敲除显著削弱了酵母菌的感染。我们还观察到酵母结合和/或 在菲律宾肽、GF 109203 X、细胞松弛素D处理的HBMEC中，侵袭显著降低。菲律宾菌素 提取膜筏上的胆固醇和小窝蛋白，GF 109203 X是蛋白激酶C（PKC）抑制剂， 细胞松弛素D是F-肌动蛋白破坏剂。显性负性PKCa也抑制酵母的侵袭 进入HBMEC。结果表明，细胞膜筏的完整性、功能性PKCa和F-肌动蛋白的表达， 酵母入侵所必需的。基于上述观察，我们假设CD 44-诱导的 信号传导和诱导的细胞骨架重组是酵母进入HBMEC所必需的。我们将探讨 C.通过以下目的检测新生儿的侵袭：（1）确定CD 44-诱导的 在C.（2）探讨C.新生儿诱导细胞骨架 CD 44在HBMEC中的表达及其与酵母菌感染的关系;（3）探讨CD 44在HBMEC中的作用 在C.小鼠模型中的新生动物侵袭。 在我们之前的资助期间，我们已经证明了病原体-宿主相互作用的粘附步骤。在 在此期间，我们将进一步描述内化步骤中的分子事件。这些研究 与临床观察结果有关，即大量患者患有严重脑膜炎， 最终会被这种病原体感染从研究中获得的信息预计将有助于 制定新的策略来预防隐球菌脑膜炎及其相关的发病率。