GLYCOCONJUGATES OF PARASITES & PATHOGENIC FUNGI

巴西副球藻是一种热二型真菌， 在37℃时以酵母形式生长，在23℃时以菌丝体形式生长。 年，巴西里氏肺吸虫感染在农村农场工人中普遍存在。 南美洲、中美洲和墨西哥，通常影响 肺、淋巴组织和皮肤粘膜组织。虽然病原学 目前仍不清楚，据推测患者感染了 菌丝体存在于土壤中。在患者中表现出 副球孢子菌病(PCM)，只检测到酵母菌。这个 酵母形态也是有传染性的，尤其是在培养过程中。 最近，我们描述了糖肌醇的分离。 酵母菌和菌丝体中的磷酰神经酰胺 包括与该病毒反应的酸性抗原(带1) 所有检测出PCM的患者的血清。中的末端半乳糖残基 它的呋喃西苷形式在血清学中被发现是免疫优势的。 反应。第二个酸性鞘糖脂(带2)，具有耐受性 薄层分析中的R迁移也是分离的，但不是反应性的 用PCM血清。频带1和频带2的完整表征 鞘糖脂的1H-和2D1H-和31P-核磁共振谱 光谱学；电喷雾质谱(ESI-MS)，包括 选择离子的低能CID-MS/MS实验；外切糖苷酶 消化后进行高效薄层层析； 通过GC-MS分析脂肪酸的甲酯、鞘氨醇 作为它们的N-乙酰-O-三甲基硅基衍生物，单糖作为 它们的Per-O-TMS甲基糖苷、过乙酰化肌醇和 部分甲基化的阿迪醇乙酸酯(PMAA)。标准一维和二维 进行了1H-核磁共振实验(DQF-COSY、TOCSY、NOESY) 尽可能多的质子共振，以建立身份和 所有单糖残基的异常形式，并建立 序列和尽可能多的连接位点通过糖苷类化合物 偶极相互作用。一维31P-核磁共振和1H-31P相关谱 用OPY方法建立了磷酸的连接位置 脂类中的基团。ESI-MS和-MS/MS分析(SCHEX API-III) 对天然的神经鞘糖脂进行检测以确认糖链序列 和磷酸肌酰胺的功能。每种脂类的等量(100(克)) 用Hakomori方法进行全甲基化、解聚和衍生 用于GC-MS分析的PMAAs(惠普5890 GC/5970 MSD，DB-5 列)。通过保留时间和EI对PMAAs进行了鉴定 与已知标准比较的碎片化模式；这些证实 所有己糖残基的同一性和连接形式 多聚糖。一份关于这部作品的手稿最近被 出版了。最近，我们已经开始研究糖基肌醇 另一种烟曲霉的磷酰神经酰胺抗原 真菌病原体在世界范围内的重要性越来越大。类似的说明 策略将用于这些抗原。一维数值模拟的初步结果 核磁共振氢谱表明存在一系列抗原 与已描述的巴西巴氏拟青霉相似；然而， A.似乎还合成了几种更复杂的抗原。 烟雾剂。这些目前正在研究中。最后，我们还 抗原单己糖神经酰胺结构的初步研究 烟曲霉(A.fumigatus)、巴西拟青霉(P.brasiliens)和其他多种 真菌病原体。在这两个方面都观察到了相似之处 糖和神经酰胺组分在主要烟曲霉菌和P. 巴西单己糖神经酰胺抗原。烟曲霉菌似乎是 能够合成葡萄糖和半乳糖神经酰胺，而 巴西假单胞菌只合成葡萄糖神经酰胺。两个展品都有 神经酰胺结构，在哺乳动物中没有发现，但已经 以前在各种真菌的神经鞘糖脂中发现过 以及海绵、海葵和海星等海洋无脊椎动物。 然而，有趣的是，这两种形式的主要葡萄糖神经酰胺 的不同之处在于存在单一不饱和 菌丝体的脂肪酸成分。一份手稿 描述这项工作正在准备中。

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.