Wasylnka Julie A, Simmer Megan I, Moore Margo M
Department of Molecular Biology and Biochemistry1 and Department of Biological Sciences2, Simon Fraser University, Burnaby, B.C., Canada V5A 1S6.
Microbiology (Reading). 2001 Apr;147(Pt 4):869-877. doi: 10.1099/00221287-147-4-869.
fumigatus is a ubiquitous soil fungus that causes invasive lung disease in the immunocompromised host. The structure of the conidial wall has not been well characterized although it is thought that adhesins present on the surface are involved in attachment of the conidia to host lung cells and proteins, which is a prerequisite for the establishment of infection. Negatively charged carbohydrates on the conidial surface have been previously identified as the molecules responsible for attachment of conidia to extracellular matrix proteins. The aim of this research was to identify carbohydrates on the conidial surface that contribute to its negative charge. Direct chemical analysis and indirect binding assays have demonstrated that A. fumigatus possesses sialic acids on the conidial surface. Pre-treatment of A. fumigatus conidia with sialidase decreased binding of a sialic acid-specific lectin, Limax flavus agglutinin (LFA), to the conidial surface and decreased adhesion of conidia to the positively charged polymer poly L-lysine. Two other sialic acid-specific lectins, Maackia amurensis agglutinin and Sambucus nigra agglutinin, exhibited negligible binding to A. fumigatus conidia indicating that 2,3-alpha- and 2,6-alpha-linked sialic acids are not the major structures found on the conidial surface. Mild acid hydrolysis and purification of conidial wall carbohydrates yielded a product that had the same R(F) as the Neu5Ac standard when analysed by high-performance thin-layer chromatography. A density of 6.7 x 10(5) sialic acid residues per conidium was estimated using a colorimetric assay. Conidia grown on a minimal medium lacking sialic acid also reacted with LFA, indicating that sialic acid biosynthesis occurs de novo. Sialic acid biosynthesis was shown to be regulated by nutrient composition: the density of sialic acids on the surface of conidia grown in minimal media was lower than that observed when conidia were grown on rich, complex media. It has previously been shown that pathogenic Aspergillus species adhere to basal lamina proteins to a greater extent than non-pathogenic Aspergillus species. To determine whether the expression of sialic acid on the conidial surface was correlated with adhesion to basal lamina, conidia from other non-pathogenic Aspergillus species were tested for their reactivity towards LFA. Flow cytometric analysis demonstrated that A. fumigatus had a significantly greater sialic acid density than three non-pathogenic Aspergillus species. Sialic acids on the conidial wall may be involved in adhesion to fibronectin, a component of the basal lamina, as binding of A. fumigatus conidia to fibronectin was strongly inhibited in the presence of a sialylated glycoprotein.
烟曲霉是一种广泛存在于土壤中的真菌，可在免疫功能低下的宿主中引起侵袭性肺部疾病。尽管人们认为分生孢子表面存在的粘附素参与了分生孢子与宿主肺细胞及蛋白质的附着过程，而这是感染发生的先决条件，但分生孢子壁的结构尚未得到很好的表征。分生孢子表面带负电荷的碳水化合物此前已被确定为负责分生孢子与细胞外基质蛋白附着的分子。本研究的目的是鉴定分生孢子表面有助于其带负电荷的碳水化合物。直接化学分析和间接结合试验表明，烟曲霉在分生孢子表面含有唾液酸。用唾液酸酶预处理烟曲霉分生孢子可降低唾液酸特异性凝集素黄蛞蝓凝集素（LFA）与分生孢子表面的结合，并降低分生孢子与带正电荷的聚合物聚L-赖氨酸的粘附。另外两种唾液酸特异性凝集素，即黑荆树凝集素和黑接骨木凝集素，与烟曲霉分生孢子的结合可忽略不计，这表明2,3-α-和2,6-α-连接的唾液酸不是分生孢子表面的主要结构。对分生孢子壁碳水化合物进行温和酸水解和纯化后，得到的产物在通过高效薄层色谱分析时，其比移值（R(F)）与Neu5Ac标准品相同。使用比色法估计每个分生孢子表面唾液酸残基的密度为6.7×10(5) 。在缺乏唾液酸的基本培养基上生长的分生孢子也能与LFA发生反应，这表明唾液酸的生物合成是从头开始的。唾液酸的生物合成显示受营养成分的调节：在基本培养基上生长的分生孢子表面唾液酸的密度低于在丰富、复杂培养基上生长的分生孢子。此前已经表明，致病性曲霉菌种比非致病性曲霉菌种更能粘附于基膜蛋白。为了确定分生孢子表面唾液酸的表达是否与对基膜的粘附相关，对其他非致病性曲霉菌种的分生孢子进行了LFA反应性测试。流式细胞术分析表明，烟曲霉的唾液酸密度明显高于三种非致病性曲霉菌种。分生孢子壁上的唾液酸可能参与了与纤连蛋白（基膜的一种成分）的粘附，因为在存在唾液酸化糖蛋白的情况下，烟曲霉分生孢子与纤连蛋白的结合受到强烈抑制。
