The pathogenic fungus Aspergillus fumigatus contains galactomannans localized on the surface layer of its cell walls, which are involved in various biological processes. Galactomannans comprise α-(1→2)-/α-(1→6)-mannan and β-(1→5)-/β-(1→6)-galactofuranosyl chains. We previously revealed that GfsA is a β-galactofuranoside β-(1→5)-galactofuranosyltransferase involved in the biosynthesis of β-(1→5)-galactofuranosyl chains. In this study, we clarified the biosynthesis of β-(1→5)-galactofuranosyl chains in A. fumigatus Two paralogs exist within A. fumigatus: GfsB and GfsC. We show that GfsB and GfsC, in addition to GfsA, are β-galactofuranoside β-(1→5)-galactofuranosyltransferases by biochemical and genetic analyses. GfsA, GfsB, and GfsC can synthesize β-(1→5)-galactofuranosyl oligomers at up to lengths of 7, 3, and 5 galactofuranoses within an established in vitro highly efficient assay of galactofuranosyltransferase activity. Structural analyses of galactomannans extracted from ΔgfsB, ΔgfsC, ΔgfsAC, and ΔgfsABC strains revealed that GfsA and GfsC synthesized all β-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans and that GfsB exhibited limited function in A. fumigatus The loss of β-(1→5)-galactofuranosyl residues decreased the hyphal growth rate and conidium formation ability and increased the abnormal hyphal branching structure and cell surface hydrophobicity, but this loss is dispensable for sensitivity to antifungal agents and virulence toward immunocompromised mice.IMPORTANCE β-(1→5)-Galactofuranosyl residues are widely distributed in the subphylum Pezizomycotina of the phylum Ascomycota. Pezizomycotina includes many plant and animal pathogens. Although the structure of β-(1→5)-galactofuranosyl residues of galactomannans in filamentous fungi was discovered long ago, it remains unclear which enzyme is responsible for biosynthesis of this glycan. Fungal cell wall formation processes are complicated, and information concerning glycosyltransferases is essential for understanding them. In this study, we showed that GfsA and GfsC are responsible for the biosynthesis of all β-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans. The data presented here indicate that β-(1→5)-galactofuranosyl residues are involved in cell growth, conidiation, polarity, and cell surface hydrophobicity. Our new understanding of β-(1→5)-galactofuranosyl residue biosynthesis provides important novel insights into the formation of the complex cell wall structure and the virulence of the members of the subphylum Pezizomycotina.

中文翻译：

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烟曲霉烟曲霉内真菌型和O-甘露糖型半乳甘露聚糖β-（1→5）-半乳糖呋喃糖基链的生物合成。

病原性真菌烟曲霉含有位于其细胞壁表层的半乳甘露聚糖，其参与各种生物过程。半乳甘露聚糖包含α-（1→2）-/α-（1→6）-甘露聚糖和β-（1→5）-/β-（1→6）-半呋喃呋喃糖基链。我们先前发现，GfsA是参与生物合成β-（1→5）-半乳糖呋喃糖基链的β-半呋喃糖苷β-（1→5）-半呋喃糖基转移酶。在这项研究中，我们阐明了烟曲霉中β-（1→5）-半乳糖呋喃糖基链的生物合成。烟曲霉中存在两个旁系同源物：GfsB和GfsC。我们通过生化和遗传分析表明，除GfsA以外，GfsB和GfsC均为β-半乳糖呋喃糖苷β-（1→5）-半呋喃糖苷转移酶。GfsA，GfsB和GfsC可以合成β-（1→5）-半乳糖呋喃糖基低聚物，其长度可达7、3，在已建立的体外半乳糖呋喃糖基转移酶活性的高效测定法中，检测了5种半乳糖呋喃糖酶。从ΔgfsB，ΔgfsC，ΔgfsAC和ΔgfsABC菌株中提取的半乳甘露聚糖的结构分析显示，GfsA和GfsC合成了真菌型和O-甘露糖型半乳甘露聚糖的所有β-（1→5）-半乳糖呋喃糖基残基，且功能有限烟曲霉β-（1→5）-半乳糖呋喃糖基残基的丢失降低了菌丝的生长速度和分生孢子形成能力，并增加了异常的菌丝分支结构和细胞表面疏水性，但是这种损失对于抗真菌剂的敏感性和对沙门氏菌的毒性是必不可少的。重要的是，β-（1→5）-半乳糖呋喃糖基残基广泛分布在子囊门的Pezizomycotina门下。Pezizomycotina包括许多动植物病原体。尽管很早以前就发现了丝状真菌中半乳甘露聚糖的β-（1→5）-半乳糖呋喃糖基残基的结构，但尚不清楚哪种酶负责这种聚糖的生物合成。真菌细胞壁的形成过程很复杂，有关糖基转移酶的信息对于理解它们至关重要。在这项研究中，我们表明GfsA和GfsC负责真菌型和O-甘露糖型半乳甘露聚糖的所有β-（1→5）-半乳糖呋喃糖基残基的生物合成。此处提供的数据表明，β-（1→5）-半乳糖呋喃糖基残基与细胞生长，分生，极性和细胞表面疏水性有关。