Molecular Mass and Localization of α-1,3-Glucan in Cell Wall Control the Degree of Hyphal Aggregation in Liquid Culture of Aspergillus nidulans.
Molecular Mass and Localization of α-1,3-Glucan in Cell Wall Control the Degree of Hyphal Aggregation in Liquid Culture of Aspergillus nidulans.
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DOI：
10.3389/fmicb.2018.02623
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发表时间：
2018
影响因子：
5.2
通讯作者：
Abe K
中科院分区：
文献类型：
--
作者：
Miyazawa K;Yoshimi A;Kasahara S;Sugahara A;Koizumi A;Yano S;Kimura S;Iwata T;Sano M;Abe K
α-1,3-葡聚糖是丝状真菌细胞壁中的主要多糖之一。构巢曲霉有两个α-1,3-葡聚糖合酶基因，agsA 和agsB。我们之前揭示了AgsB是营养菌丝中主要的α-1,3-葡聚糖合酶，但AgsA的功能仍然未知，因为它的表达水平低且在基因破坏时缺乏表型改变。为了阐明α-1,3-葡聚糖在菌丝聚集中的作用，我们构建了过表达agsA（agsAOE）或agsB（agsBOE）的菌株，其中另一个α-1,3-葡聚糖合酶基因被破坏。在液体培养中，野生型和agsBOE菌株形成紧密聚集的菌丝团，而agsAOE菌丝聚集较弱。我们分析了 agsAOE 和 agsBOE 菌株细胞壁 α-1,3-葡聚糖的化学性质。 agsAOE 菌株的 α-1,3-葡聚糖峰值分子量 (1,480 ± 80 kDa) 远大于野生型 (147 ± 52 kDa) 和 agsBOE (372 ± 47 kDa)；然而，α-1,3-葡聚糖中重复亚基的峰值分子量几乎相同（Smith 降解后：agsAOE，41.6 ± 5.8 kDa；agsBOE，38.3 ± 3.0 kDa）。我们还通过使用 α-1,3-葡聚糖结合域融合 GFP (AGBD-GFP) 进行荧光标记，分析了 α-1,3-葡聚糖在两种菌株细胞壁中的定位。 agsBOE细胞的α-1,3-葡聚糖明显位于最外层，而agsAOE细胞中检测到弱标记。然而，用β-1,3-葡聚糖酶处理的agsAOE细胞明显被AGBD-GFP标记。这些观察结果表明，β-1,3-葡聚糖覆盖了 AgsA 合成的大部分 α-1,3-葡聚糖，尽管少量 α-1,3-葡聚糖仍存在于外层。我们还构建了 amyG 基因被破坏的菌株，该基因编码一种细胞内 α-淀粉酶，可合成 α-1,4-低聚葡萄糖作为 α-1,3-葡聚糖生物合成的引物。该菌株的菌丝团和α-1,3-葡聚糖的峰值分子量（94.5±1.4 kDa）均小于野生型菌株，且α-1,3-葡聚糖最外层仍被AGBD-GFP标记。总体而言，这些结果表明菌丝团的形成取决于 α-1,3-葡聚糖的分子质量和空间定位以及构巢曲霉细胞壁中 α-1,3-葡聚糖的量。
α-1,3-Glucan is one of the main polysaccharides in the cell wall of filamentous fungi. Aspergillus nidulans has two α-1,3-glucan synthase genes, agsA and agsB. We previously revealed that AgsB is a major α-1,3-glucan synthase in vegetative hyphae, but the function of AgsA remained unknown because of its low expression level and lack of phenotypic alteration upon gene disruption. To clarify the role of α-1,3-glucan in hyphal aggregation, we constructed strains overexpressing agsA (agsAOE) or agsB (agsBOE), in which the other α-1,3-glucan synthase gene was disrupted. In liquid culture, the wild-type and agsBOE strains formed tightly aggregated hyphal pellets, whereas agsAOE hyphae aggregated weakly. We analyzed the chemical properties of cell wall α-1,3-glucan from the agsAOE and agsBOE strains. The peak molecular mass of α-1,3-glucan from the agsAOE strain (1,480 ± 80 kDa) was much larger than that from the wild type (147 ± 52 kDa) and agsBOE (372 ± 47 kDa); however, the peak molecular mass of repeating subunits in α-1,3-glucan was almost the same (after Smith degradation: agsAOE, 41.6 ± 5.8 kDa; agsBOE, 38.3 ± 3.0 kDa). We also analyzed localization of α-1,3-glucan in the cell wall of the two strains by fluorescent labeling with α-1,3-glucan-binding domain–fused GFP (AGBD-GFP). α-1,3-Glucan of the agsBOE cells was clearly located in the outermost layer, whereas weak labeling was detected in the agsAOE cells. However, the agsAOE cells treated with β-1,3-glucanase were clearly labeled with AGBD-GFP. These observations suggest that β-1,3-glucan covered most of α-1,3-glucan synthesized by AgsA, although a small amount of α-1,3-glucan was still present in the outer layer. We also constructed a strain with disruption of the amyG gene, which encodes an intracellular α-amylase that synthesizes α-1,4-glucooligosaccharide as a primer for α-1,3-glucan biosynthesis. In this strain, the hyphal pellets and peak molecular mass of α-1,3-glucan (94.5 ± 1.4 kDa) were smaller than in the wild-type strain, and α-1,3-glucan was still labeled with AGBD-GFP in the outermost layer. Overall, these results suggest that hyphal pellet formation depends on the molecular mass and spatial localization of α-1,3-glucan as well as the amount of α-1,3-glucan in the cell wall of A. nidulans.
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通讯作者：
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