Invasion of brain endothelial cells by C. neoformans

描述（由申请人提供）：该项目的长期目标是剖析隐球菌脑膜炎的发病机制。近年来，由新型隐球菌感染已大大增加，特别是在免疫组成的个人和艾滋病患者。这种病原体偏爱于大脑，导致毁灭性的脑膜脑炎。尚不清楚新生梭菌是如何穿过血脑屏障并引起中枢神经系统感染的。我们拟探讨新生C.入侵构成血脑屏障的人脑微血管内皮细胞（HBMEC）的机制。我们建立了体外和体内血脑屏障模型用于病原体入侵研究。使用这些模型系统，我们发现，无论是用透明质酸酶还是用4- methylumbellliferone（一种透明质酸合成酶抑制剂）处理，新生C.线虫都大大降低了其与HBMEC的结合能力。相比之下，外源性透明质酸被发现可以刺激C. neoformans的结合活性。CPS1蛋白序列与透明质酸合成酶具有同源性。cpsl被破坏的菌株失去了细胞表面的透明质酸，也大大降低了其与HBMEC的结合活性。最后，抗cd44阻断抗体可以中和新生C.与HBMEC的相互作用。综上所述，我们的初步研究表明，新生荚膜成分、透明质酸和HBMEC CD44参与了侵袭过程。在本研究中，我们将验证C. neoformes透明质酸和HBMEC CD44在C. neoformes发病机制中起关键作用的假设。我们将从以下几个方面探讨C. neoformans入侵HBMEC的机制：首先，我们将研究CPS1在透明质酸合成和C. neoformans入侵中的作用。其次，我们将确定新生C.透明质酸如何促进HBMEC的结合和侵袭。第三，我们将确定HBMEC CD44是否以及如何在新生C.侵袭过程中作为HA受体发挥作用。所提出的方法将使我们能够确定关键的新生芽胞杆菌成分、透明质酸和宿主在入侵过程中的反应的作用。从这一建议中得到的信息应该有助于制定新的策略来预防新型C.脑膜炎及其相关的发病率。

DESCRIPTION (provided by applicant): The long-term goal of this project is to dissect the pathogenesis of Cryptococcus meningitis. In recent years infection by Cryptococcus neoformans has increased considerably, particularly in immunocomprised individuals and AIDS patients. This pathogen has a predilection to the brain, resulting in devastating meningoencephalitis. It is unclear how C. neoformans traverses across the blood-brain barrier and causes the central nervous system infection. We propose to investigate the mechanisms of C. neoformans invasion in human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We have established in vitro and in vivo BBB models for the pathogen invasion studies. Using these model systems, we found that C. neoformans, either treated with hyaluronidase or with 4-methylumbelliferone (a hyaluronan synthase inhibitor) substantially reduced its binding ability to HBMEC. In contrast, exogenous hyaluronan was found to stimulate C. neoformans binding activity. The CPS1 protein sequence shares homology with hyaluronan synthase. The cpsl-disrupted strain lost its hyaluronan on the cell surface, and also substantially reduced its binding activity to HBMEC. Finally, anti-CD44 blocking antibody could neutralize the interaction between C. neoformans and HBMEC. Taken together, our preliminary studies suggested that the C. neoformans capsule component, hyaluronan, and HBMEC CD44 were involved in the invasion process. In this proposal, we will test the hypothesis that C. neoformans hyaluronan and HBMEC CD44 are crucial for C. neoformans pathogenesis. We will explore the mechanisms of C. neoformans invasion to HBMEC by the following Aims: First, we will examine the role of CPS1 in hyaluronan synthesis and C. neoformans invasion. Second, we will determine how C. neoformans hyaluronan contribute to the binding and invasion of HBMEC. Third, we will determine whether and how HBMEC CD44 functions as a HA receptor during C. neoformans invasion. The proposed approach will allow us to determine the role(s) of the crucial C. neoformans component, hyaluronan, and host responses during invasion. The information derived from this proposal should be helpful in the development of novel strategies to prevent C. neoformans meningitis and its associated morbidity.