Tomek Markus B, Maresch Daniel, Windwarder Markus, Friedrich Valentin, Janesch Bettina, Fuchs Kristina, Neumann Laura, Nimeth Irene, Zwickl Nikolaus F, Dohm Juliane C, Everest-Dass Arun, Kolarich Daniel, Himmelbauer Heinz, Altmann Friedrich, Schäffer Christina
NanoGlycobiology Unit, Department of NanoBiotechnology, Universität für Bodenkultur Wien, Vienna, Austria.
Division of Biochemistry, Department of Chemistry, Universität für Bodenkultur Wien, Vienna, Austria.
Front Microbiol. 2018 Aug 28;9:2008. doi: 10.3389/fmicb.2018.02008. eCollection 2018.
The cell surface of the oral pathogen is heavily glycosylated with a unique, complex decasaccharide that is -glycosidically linked to the bacterium's abundant surface (S-) layer, as well as other proteins. The S-layer glycoproteins are virulence factors of and there is evidence that protein glycosylation underpins the bacterium's pathogenicity. To elucidate the protein -glycosylation pathway, genes suspected of encoding pathway components were first identified in the genome sequence of the ATCC 43037 type strain, revealing a 27-kb gene cluster that was shown to be polycistronic. Using a gene deletion approach targeted at predicted glycosyltransferases (Gtfs) and methyltransferases encoded in this gene cluster, in combination with mass spectrometry of the protein-released glycans, we show that the gene cluster encodes the species-specific part of the ATCC 43037 decasaccharide and that this is assembled step-wise on a pentasaccharide core. The core was previously proposed to be conserved within the phylum, to which is affiliated, and its biosynthesis is encoded elsewhere on the bacterial genome. Next, to assess the prevalence of protein glycosylation among sp., the publicly available genome sequences of six strains were compared, revealing gene clusters of similar size and organization as found in the ATCC 43037 type strain. The corresponding region in the genome of a periodontal health-associated isolate showed a different gene composition lacking most of the genes commonly found in the pathogenic strains. Finally, we investigated whether differential cell surface glycosylation impacts 's overall immunogenicity. Release of proinflammatory cytokines by dendritic cells (DCs) upon stimulation with defined Gtf-deficient mutants of the type strain was measured and their T cell-priming potential post-stimulation was explored. This revealed that the glycan is pivotal to modulating DC effector functions, with the -specific glycan portion suppressing and the pentasaccharide core activating a Th17 response. We conclude that complex protein glycosylation is a hallmark of pathogenic strains and propose it as a valuable target for the design of novel antimicrobials against periodontitis.
口腔病原体的细胞表面高度糖基化，带有一种独特的、复杂的十糖，该十糖通过β-糖苷键与细菌丰富的表面（S-）层以及其他蛋白质相连。S层糖蛋白是该病原体的毒力因子，并且有证据表明蛋白质糖基化是该细菌致病性的基础。为了阐明蛋白质β-糖基化途径，首先在ATCC 43037型菌株的基因组序列中鉴定出怀疑编码途径成分的基因，发现了一个27 kb的基因簇，该基因簇被证明是多顺反子的。使用针对该基因簇中预测的糖基转移酶（Gtfs）和甲基转移酶的基因缺失方法，结合蛋白质释放聚糖的质谱分析，我们表明该基因簇编码ATCC 43037十糖的物种特异性部分，并且该十糖是在五糖核心上逐步组装而成的。先前有人提出该核心在该病原体所属的门内是保守的，其生物合成在细菌基因组的其他位置编码。接下来，为了评估该病原体中蛋白质糖基化的普遍性，比较了六个该病原体菌株的公开可用基因组序列，发现其基因簇的大小和组织与ATCC 43037型菌株中的相似。与牙周健康相关的该病原体分离株基因组中的相应区域显示出不同的基因组成，缺乏致病菌株中常见的大多数基因。最后，我们研究了细胞表面糖基化差异是否会影响该病原体的整体免疫原性。测量了树突状细胞（DCs）在用该型菌株特定的Gtf缺陷突变体刺激后促炎细胞因子的释放，并探索了刺激后它们的T细胞启动潜力。这表明聚糖对于调节DC效应功能至关重要，其中该病原体特异性聚糖部分抑制而五糖核心激活Th17反应。我们得出结论，复杂的蛋白质糖基化是致病该病原体菌株的标志，并提出将其作为设计针对牙周炎的新型抗菌药物的有价值靶点。
