Mechanism of Brain Invasion by Cryptococcus neoformans

新型隐球菌脑侵袭机制

• 批准号: 8745269
• 负责人: Kyung Kwon-Chung
• 金额: $ 27.63万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8745269
• 关键词: Adhesions; Affect; Alveolar; Alveolar Macrophages; Apoenzymes; Bacteria; Bacterial Proteins; Binding; Biochemical; Biological Assay; Birds; Blood - brain barrier anatomy; Brain; Breathing; CD44 Antigens; CD44 gene; CYP21A2 gene; California; Cell membrane; Cells; Cessation of life; Collaborations; Columbidae; Coupled; Cryptococcus; Cryptococcus neoformans; Cytochalasin D; Cytoskeleton; Data; Development; Disease; Encapsulated; Endocytosis; Endothelial Cells; Environment; Enzymes; Frequencies; Ganglioside GM1; Genes; Genetic; Genome; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; HIV; Histidine; Homologous Gene; Human; Hyaluronic Acid; Immunocompetent; Immunocompromised Host; In Vitro; Individual; Infection; Invaded; Ligands; Lung; Mass Spectrum Analysis; Mediating; Membrane Microdomains; Meningoencephalitis; Modeling; Nickel; Nitrogen; Nocodazole; Nuclear Envelope; Organism; Oxygen; PTK2 gene; Pathway interactions; Patients; Penetration; Phagocytosis; Phospholipase; Phosphotransferases; Plants; Plasma; Play; Polysaccharides; Prevention; Prevention approach; Process; Protein Kinase C Alpha; Proteins; Reporting; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Sister; Small Interfering RNA; Soil; Source; Structure; Structure of thyroid parafollicular cell; System; Tree Bark; Two-Hybrid System Techniques; Universities; Urea; Urease; Yeasts; base; bikunin; burden of illness; enzyme activity; extracellular; ezrin; factor C; fungus; insight; monolayer; mouse model; mutant; novel strategies; pathogen; response

Cryptococcus neoformans is an environmental fungal pathogen that causes fatal meningoencephalitis and the global burden of the disease is estimated to be annual one million cases with 600,000 death primarily in HIV infected patients. However, the fungus also causes infection in otherwise normal patients at a low frequency. The disease is 100% fatal unless treated and even with the most effective antimycotic agents, the fatality rate is about 25%. C. neoformans is commonly found in the human environment world-wide. In previous years, we discovered that C. neoformans yeast cells invaded the brain by crossing the blood-brain barrier transcellularly. We also, reported that the product of CPS1 gene that plays an important role in association and trversal of C. neoformans yeast cells across the blood-brain barrier (BBB)is hyaluronic acid which is located at the base of the extracellular polysaccharide that surrounds the yeast cells. We also showed, in collaboration with Dr. Jong's group, that protein kinase C-alpha activation is required for the yeast to cross the BBB and CD44 is involved in the association of the yeast cells with the brain microvasular endothelial cells. In order to gain better insight into the invasion process, we have taken a genetic approach during 2008-2009 period. During 2009-2010, in collaboration with Dr. Kim at Johns Hopkins University, we found that the host cell Rac1 GTPase contributes to C. neoformans traversal of the blood-brain barrier and pharmacological inhibition of Rac1 was efficient in inhibiting C. neoformans traversal of the blood-brain barrier, the essential step for the development of C. neoformans meningoencephalitis. This was shown by our demonstrations that (a) Rac1 activation occurs in response to C. neoformans in human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, (b) inhibition of Rac1 was efficient in prevention of HBMEC traversal for both encapsulated and acapsular strains of C. neoformans, and (c) pharmacological inhibition of Rac1 significantly decreased C. neoformans penetration into the brain. These findings demonstrate that targeting host cell signaling molecules essential to C. neoformans traversal of the blood-brain barrier, as shown here with Rac1 GTPase, provides a novel approach to prevention of C. neoformans meningoencephalitis. During 2010-2011,in collaboration with Dr. Jong at the University of Southern California,we found several lines of evidence indicating that C. neoformans invasion is mediated through the endocytic pathway via lipid rafts. Human CD44 molecules from lipid rafts can directly interact with hyaluronic acid, the fungal ligand. Bikunin, which perturbs CD44 function in the lipid raft, can block fungal adhesion and invasion of HBMEC. The lipid raft marker, ganglioside GM1, colocalizes with CD44 on the plasma membrane, and fungal cells can adhere to the host cells where GM1 is enriched. The results suggest the fungal entry is taking place on the lipid rafts. Upon fungal engagement, GM1 was internalized through vesicular structures to the nuclear membrane. This endocytic redistribution process can be perturbed by cytochalasin D, nocodazole, and anti-DYRK3 siRNA. Concomitantly, the knockdown of DYRK3 significantly reduces fungal invasion across the HBMEC monolayer in vitro. Our data demonstrated that the lipid-raft dependent endocytosis process mediates C. neoformans internalization into HBMEC, in which the hosts CD44 protein, cytoskeleton, and intracellular kinase-DYRK3 are involved.During 2011-2012, we investigated the cryptococcal factors that can activate the host signaling pathways that enhance C. neoformans crossing of the blood-brain barrier by using the HBMEC and found that phospholipase secreted by yeast cells of C. neoformans first activates RhoGTPases followed by PKC-alpha, FAK and ezrin. Since cryptococcal urease is reported to be an important factor required for brain invasion and yet the urease activation system has not been studied, we characterized all the factors required for converting apourease into active urease in 2012-2013.Although urea is not required for synthesis of apourease encoded by URE1, the available nitrogen source affected the expression of URE1 as well as the level of the enzyme activity. Activation of the apoenzyme required three accessory proteins,Ure4, Ure6, and Ure7, which are homologs of the bacterial urease accessory proteins UreD, UreF, and UreG, respectively. A yeast two-hybrid assay showed positive interaction of Ure1 with the three accessory proteins encoded by URE4, URE6, and URE7. Metalloproteomic analysis of cryptococcal lysates using inductively coupled plasma mass spectrometry (ICP-MS) and a biochemical assay of urease activity showed that, as in many other organisms, urease is a metallocentric enzyme that requires nickel transported by Nic1 for its catalytic activity. The Ure7 accessory protein (bacterial UreG homolog) binds nickel likely via its conserved histidine-rich domain and appears to be responsible for the incorporation of Ni2 into the apourease. Although the cryptococcal genome lacks the bacterial UreE homolog, Ure7 appeared to combine the functions of bacterial UreE and UreG, thus making this pathogen more similar to that seen with the plant system. Brain invasion by the ure1, ure7, and nic1 mutant strains that lack urease activity was significantly less effective in a mouse model. This indicated that an activated urease and not the Ure1 protein was responsible for enhancement of brain invasion and that the factors required for urease activation in C. neoformans resemble those of plants more than those of bacteria.

隐型新外身是一种环境真菌病原体，会导致致命的脑膜脑炎，据估计，该疾病的全球负担为每年一百万例，主要在受HIV感染的患者中死亡600,000例。但是，真菌还会在低频中引起正常患者的感染。除非治疗，否则该疾病是100％致命的，即使使用最有效的抗虫质药物，死亡率也约为25％。 Neoformans C.通常在全球人类环境中发现。在过去的几年中，我们发现Neoformans酵母菌细胞通过跨细胞跨越血脑屏障而入侵了大脑。我们还报道说，CPS1基因的产物在跨血脑屏障（BBB）的新生孢菌酵母细胞的关联和曲折中起重要作用，它位于围绕酵母细胞的细胞外多糖的底部。我们还与Jong博士的小组合作表明，酵母越过BBB需要蛋白激酶C-α激活，而CD44与酵母细胞与脑微度内皮细胞的关联涉及。为了更好地了解入侵过程，我们在2008 - 2009年期间采取了一种遗传方法。 During 2009-2010, in collaboration with Dr. Kim at Johns Hopkins University, we found that the host cell Rac1 GTPase contributes to C. neoformans traversal of the blood-brain barrier and pharmacological inhibition of Rac1 was efficient in inhibiting C. neoformans traversal of the blood-brain barrier, the essential step for the development of C. neoformans meningoencephalitis. This was shown by our demonstrations that (a) Rac1 activation occurs in response to C. neoformans in human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, (b) inhibition of Rac1 was efficient in prevention of HBMEC traversal for both encapsulated and acapsular strains of C. neoformans, and (c) pharmacological inhibition of Rac1 significantly decreased C. neoformans穿透大脑。这些发现表明，靶向宿主细胞信号分子对血脑屏障的新外形遍历必不可少的，如Rac1 GTPase所示，为预防Neoformans脑膜脑炎的预防提供了一种新颖的方法。在2010-2011期间，与南加州大学的Jong博士合作，我们发现了几条证据，表明C. Neoformans入侵是通过脂质筏通过内吞途径介导的。 来自脂质筏的人CD44分子可以直接与真菌配体透明质酸直接相互作用。 Bikunin在脂质筏中的CD44功能可阻止真菌粘附和侵袭HBMEC。 脂质筏标记GM1 GM1与质膜上的CD44共定位，真菌细胞可以粘附在富含GM1的宿主细胞上。结果表明，真菌进入是在脂质筏上进行的。 真菌参与后，GM1通过囊泡结构内化为核膜。 这种内吞再分配过程可能会受到细胞切拉斯蛋白D，诺科唑和抗DyRK3 siRNA的干扰。 同时，DYRK3的敲低显着降低了体外HBMEC单层的真菌侵袭。 Our data demonstrated that the lipid-raft dependent endocytosis process mediates C. neoformans internalization into HBMEC, in which the hosts CD44 protein, cytoskeleton, and intracellular kinase-DYRK3 are involved.During 2011-2012, we investigated the cryptococcal factors that can activate the host signaling pathways that enhance C. neoformans crossing of the blood-brain barrier by using the HBMEC，发现由新近梭菌的酵母细胞分泌的磷脂酶首先激活Rhogtpases，然后激活PKC-Alpha，Fak和Ezrin。 Since cryptococcal urease is reported to be an important factor required for brain invasion and yet the urease activation system has not been studied, we characterized all the factors required for converting apourease into active urease in 2012-2013.Although urea is not required for synthesis of apourease encoded by URE1, t​​he available nitrogen source affected the expression of URE1 as well as the level of the enzyme activity.中酶的激活需要三种辅助蛋白URE4，URE6和URE7，它们分别是细菌尿素辅助蛋白蛋白，UREF和UREG的同源物。酵母两杂化测定法显示了URE1与由URE4，URE6和URE7编码的三种辅助蛋白的阳性相互作用。使用电感耦合的血浆质谱法（ICP-MS）和尿素活性活性的生化测定法表明，与许多其他生物体一样，尿素是一种金属中心的酶，需要通过NIC1运输的NIC1的镍型硫化酶。 URE7辅助蛋白（细菌UREG同源物）可能通过其保守的组氨酸结构域结合镍，并且似乎是将Ni2掺入Apourease中的原因。尽管隐球菌基因组缺乏细菌Ure同源物，但URE7似乎结合了细菌Uree和Ureg的功能，因此使该病原体与植物系统所见的病原体更相似。在小鼠模型中，缺乏尿素酶活性的URE1，URE7和NIC1突变菌株的脑侵袭明显降低。这表明激活的尿素酶而不是URE1蛋白是 负责增强大脑侵袭的能力，而新甲状腺梭菌激活所需的因素比细菌的植物比植物的因素更类似于植物。