GLYCOCONJUGATES OF PARASITES & PATHOGENIC FUNGI

寄生虫的糖缀合物

• 批准号: 6493674
• 负责人: STEVEN B LEVERY
• 金额: $ 28.8万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6493674
• 关键词: biological products; biomedical resource; carbohydrates; immunology; infection; inflammation; international health /scientific organization; lipids; plants; respiratory system

Paracoccidioides brasiliensis is a thermally dimorphic fungus, growing in its yeast form at 37(C and in mycelium form at 23(C. Infection by P. brasiliensis is prevalent among rural farm workers in South America, Central America, and Mexico, commonly affecting the lung, lymphoid, and mucocutaneous tissues. Although the etiology remains unclear, it is assumed that patients are infected by the mycelium form present in soil. In patients exhibiting paracoccidioidomycosis (PCM), only the yeast form is detected. The yeast form is also infectious, particularly when handled in culture. Recently, we described the isolation of glycosylinositol phosphorylceramides from the yeast and mycelium forms of P. brasiliensis, including an acidic antigen (band 1) reactive with the sera of all patients tested exhibiting PCM. A terminal Gal residue in its furanosidic form was found to be immunodominant in the serologic reaction. A second acidic glycosphingolipid (band 2), having a faste r migration in TLC analysis, was also isolated but was not reactive with PCM sera. Complete characterization of both band 1 and band 2 glycosphingolipids was undertaken by 1- and 2-D 1H- and 31P-NMR spectroscopy; electrospray mass spectrometry (ESI-MS), including low-energy CID MS/MS experiments on selected ions; exoglycosidase digestion followed by high-performance thin layer chromatography; and by GC-MS analysis of fatty acids as their methyl esters, sphingosines as their N-acetyl-O-trimethylsilyl derivatives, monosaccharides as their per-O-TMS methylglycosides, peracetylated inositols, and partially methylated alditol acetates (PMAAs). Standard 1- and 2-D 1H-NMR experiments (DQF-COSY, TOCSY, NOESY) were performed to assign as many proton resonances as possible, to establish identity and anomeric form of all monosaccharide residues, and to establish sequence and as many linkage sites as possible via interglycosidic dipolar interactions. 1-D 31P-NMR and 1H-31P correlation spectrosc opy was performed to establish the linkage positions of the phosphate groups in the lipids. ESI-MS and -MS/MS analysis (Sciex API-III) was performed on the native glycosphingolipids to confirm glycan sequence and phosphoceramide features. An aliquot (100 (g) of each lipid was permethylated by the Hakomori method, depolymerized, and derivatized to PMAAs for GC-MS analysis (Hewlett-Packard 5890 GC/5970 MSD, DB-5 column). Identification of PMAAs was made by retention times and EI fragmentation patterns compared with known standards; these confirmed both the identity and linkage form of all hexose residues in the glycans. A manuscript concerning this work has recently been published. More recently, we have begun work on glycosylinositol phosphorylceramide antigens from Aspergillus fumigatus, another mycopathogen with growing importance worldwide. A similar elucidation strategy will be used for these antigens. Preliminary results of 1-D 1H-NMR spectroscopy indicate the presence of a series of antigens similar to those already described for P. brasiliensis; however, several more complex antigens appear to be synthesized as well by A. fumigatus. These are currently under study. Finally, we have also initiated studies of the structures of antigenic monohexosylceramides of A. fumigatus, P. brasiliensis, and a variety of other mycopathogens. Similarities have been observed, with respect to both sugar and ceramide components, between the major A. fumigatus and P. brasiliensis monohexosylceramide antigens. A. fumigatus appears to be capable of synthesizing both glucosyl- and galactosylceramides, while P. brasiliensis synthesizes only glucosyl ceramides. Both exhibit ceramide structures that are not found in mammals but have been demonstrated previously in glycosphingolipids from a variety of fungi and marine invertebrates such as sponges, anemones, and starfish. Interestingly, however, the major glucosylceramides of the two forms of P. brasiliensis differ by the presence of a single unsaturation in the fatty acid component of the mycelium form. A manuscript describing this work is in preparation.

巴拉西氏菌巴西氏菌是一种热二态真菌， 以酵母形式生长在37（c和菌丝形式），分别为23（c。 巴西疟原虫的感染在农村工人中普遍存在 南美，中美洲和墨西哥通常会影响 肺，淋巴样和粘膜皮组织。 虽然病因 尚不清楚，假设患者被感染 菌丝形成土壤中。 在表现出的患者中 副霉菌病（PCM），仅检测到酵母状形式。 这 酵母菌形式也具有感染性，尤其是在文化中处理时。 最近，我们描述了糖水肌醇的分离 酵母和菌丝形式的磷酸氯脲。 巴西龙，包括酸性抗原（带1）与 所有测试的患者的血清展示了PCM。 末端gal残留物 发现其呋喃糖苷形式在血清学中是免疫主导的 反应。 第二个酸性糖磷脂（带2），具有速度 在TLC分析中迁移的R迁移也分离出来，但不是反应性的 与PCM血清。 频段1和带2的完全表征 糖磷脂由1-D和2-D 1H-和31P-NMR进行 光谱；电喷雾质谱法（ESI-MS），包括 对选定离子的低能CID MS/MS实验；外糖苷酶 消化，然后是高性能薄层色谱；和 通过对脂肪酸作为甲基酯的GC-MS分析，鞘氨酸 作为其N-乙酰基-O-三甲基硅烷基衍生物，单糖为 它们的甲基糖苷，过乙酰化肌醇和 部分甲基化醛醇乙酸盐（PMAA）。 标准1和2-D 进行了1H-NMR实验（DQF-Cosy，Tocsy，Noesy）进行分配 尽可能多的质子共振，建立身份和 所有单糖残基的异构形式，并建立 序列和尽可能多的链接位点通过糖层间 偶极相互作用。 1-D 31P-NMR和1H-31P相关光谱 进行OPY以建立磷酸盐的连锁位置 脂质中的组。 ESI-MS和-MS/MS分析（SCIEX API-III）为 在天然糖磷脂上进行以确认聚糖序列 和磷酸氧化物特征。 每个脂质的等分试样（100（g） 通过hakomori方法二苄甲基化，解聚并衍生化 到PMAA进行GC-MS分析（Hewlett-Packard 5890 GC/5970 MSD，DB-5 柱子）。 通过保留时间和EI识别PMAA的识别 与已知标准相比，分裂模式；这些证实 在 聚糖。 关于这项工作的手稿最近已经 出版。 最近，我们已经开始从事糖基辛醇 来自曲霉的磷酸甘油酰胺抗原，另一种 全世界的分枝杆菌原子越来越重要。 类似的阐明 这些抗原将使用策略。 1-D的初步结果 1H-NMR光谱表明存在一系列抗原 类似于已经描述的巴西疟原虫；然而， A.似乎也合成了几种更复杂的抗原。 富马图斯。 这些目前正在研究中。 最后，我们也有 开始研究抗原单己糖基酰胺的结构 A. fumigatus，P。Brasiliensis和其他各种 分枝杆菌。 已经观察到相似之处 糖和神经酰胺成分，主要烟曲霉和P. 巴西单己糖基酰胺抗原。 A. Fumigatus似乎是 能够合成葡萄糖基和半乳糖基酰胺，而 巴西疟原虫仅合成葡萄糖基神经酰胺。 两者都表现出来 在哺乳动物中未发现但已经存在的神经酰胺结构 先前在各种真菌的糖磷脂中展示 以及海洋无脊椎动物，例如海绵，海葵和海星。 然而，有趣的是，这两种形式的主要葡萄糖基酰胺 巴西假单胞菌的存在因单一不饱和的存在而不同 菌丝形式的脂肪酸成分。 手稿 描述这项工作正在准备。