Molecular Characterization of N-glycan Degradation and Transport in Streptococcus pneumoniae and Its Contribution to Virulence.
Molecular Characterization of N-glycan Degradation and Transport in Streptococcus pneumoniae and Its Contribution to Virulence.
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DOI：
10.1371/journal.ppat.1006090
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发表时间：
2017-01
期刊：
影响因子：
6.7
通讯作者：
Boraston AB
中科院分区：
文献类型：
--
作者：
Robb M;Hobbs JK;Woodiga SA;Shapiro-Ward S;Suits MD;McGregor N;Brumer H;Yesilkaya H;King SJ;Boraston AB
人体组织和细胞的富含碳水化合物的涂层为细菌的定居和入侵提供了第一个接触点。与N-糖基化相适应，N-糖基化是一种丰富的蛋白质糖基化形式，在宿主中具有关键的功能作用，一些宿主适应的细菌拥有处理N-连接的多糖的机制。人类病原体肺炎链球菌用酶解聚复杂的N-糖链，在内切N-乙酰氨基葡萄糖苷酶(Endo-β-N-Acc)从蛋白质中释放糖之前，酶会顺序地将复杂的N-糖切下到Man3GlcNac2的核心。在这里，我们检查肺炎链球菌处理高甘露糖N-聚糖和运输产品的能力。通过生化和结构分析，我们证明肺炎链球菌也具有α-(1，2)-甘露糖苷酶(SpGH92)。这种酶能够切割高甘露糖N-聚糖的末端α-(1，2)连接的甘露糖残基，生成Man5GlcNAc2。通过这种活性，SpGH92能够产生一种内切酶底物，这种底物在含有α-(1，2)连接的甘露糖残基的高甘露糖上不起作用。结合研究和X射线结晶学研究表明，ABC转运蛋白(ABCNG)的溶质结合蛋白NGTS能够高亲和力地结合EndoD活性产物Man5GlcNAc。最后，我们评估了EndoD和ABCNG在N-糖基化糖蛋白模型上对肺炎链球菌生长的贡献，以及这些酶和SpGH92在小鼠模型中对毒力的贡献。我们发现EndoD和ABCNG对肺炎链球菌的生长都有贡献，但只有SpGH92和EndoD对毒力有贡献。因此，肺炎链球菌的完全毒力需要N-糖链的加工，而不是释放的糖链的运输。总之，我们将我们的发现综合到肺炎链球菌处理N-糖链的模型中，在该模型中，复杂的和高甘露糖的N-糖链都是靶向的，并且这一降解途径的两个臂在ABCNG处汇合。肺炎链球菌(简称肺炎球菌)是一种在人类中引起广泛发病率和死亡率的细菌。疫苗和抗生素分别是有效的预防和治疗形式，但也面临着挑战，因为在抗生素耐药性继续上升的同时，要针对庞大且不断演变的不同血清型细菌池进行疫苗接种是一场持续的竞赛。因此，有必要更好地了解宿主-肺炎球菌相互作用的分子方面，以便为潜在的替代治疗策略的产生提供信息。肺炎链球菌依靠其处理寄主细胞表面的碳水化合物的能力来实现完全毒力。在这项研究中，我们检测了该细菌处理高甘露糖N-连接糖的能力，这是迄今为止对肺炎链球菌未知的能力。结果表明，肺炎球菌基因组编码能够处理这些糖的酶，值得注意的是，由一种酶执行的启动反应去除末端α-(1，2)连接的甘露糖残基对小鼠模型的毒力至关重要。这项研究揭示了肺炎链球菌中针对N-连接糖的广泛途径，这是宿主-病原体相互作用的关键，因此揭示了治疗干预的潜在靶点。
The carbohydrate-rich coating of human tissues and cells provide a first point of contact for colonizing and invading bacteria. Commensurate with N-glycosylation being an abundant form of protein glycosylation that has critical functional roles in the host, some host-adapted bacteria possess the machinery to process N-linked glycans. The human pathogen Streptococcus pneumoniae depolymerizes complex N-glycans with enzymes that sequentially trim a complex N-glycan down to the Man3GlcNAc2 core prior to the release of the glycan from the protein by endo-β-N-acetylglucosaminidase (EndoD), which cleaves between the two GlcNAc residues. Here we examine the capacity of S. pneumoniae to process high-mannose N-glycans and transport the products. Through biochemical and structural analyses we demonstrate that S. pneumoniae also possesses an α-(1,2)-mannosidase (SpGH92). This enzyme has the ability to trim the terminal α-(1,2)-linked mannose residues of high-mannose N-glycans to generate Man5GlcNAc2. Through this activity SpGH92 is able to produce a substrate for EndoD, which is not active on high-mannose glycans with α-(1,2)-linked mannose residues. Binding studies and X-ray crystallography show that NgtS, the solute binding protein of an ABC transporter (ABCNG), is able to bind Man5GlcNAc, a product of EndoD activity, with high affinity. Finally, we evaluated the contribution of EndoD and ABCNG to growth of S. pneumoniae on a model N-glycosylated glycoprotein, and the contribution of these enzymes and SpGH92 to virulence in a mouse model. We found that both EndoD and ABCNG contribute to growth of S. pneumoniae, but that only SpGH92 and EndoD contribute to virulence. Therefore, N-glycan processing, but not transport of the released glycan, is required for full virulence in S. pneumoniae. To conclude, we synthesize our findings into a model of N-glycan processing by S. pneumoniae in which both complex and high-mannose N-glycans are targeted, and in which the two arms of this degradation pathway converge at ABCNG. Streptococcus pneumoniae (pneumococcus) is a bacterium that causes extensive morbidity and mortality in humans. Vaccines and antibiotics are effective forms of prevention and treatment, respectively, but present challenges as it is a constant race to vaccinate against the enormous and ever evolving pool of different serotypes of the bacterium while resistance to antibiotics continues to trend upwards. It is thus necessary to better understand the molecular aspects of the host-pneumococcus interaction in order to inform the potential generation of alternative treatment strategies. S. pneumoniae relies on its ability to process the carbohydrates presented on the surface of host cells for full-virulence. In this study, we examine the capability of the bacterium to process high-mannose N-linked sugars, a heretofore unknown ability for S. pneumoniae. The results show that the pneumococcal genome encodes enzymes capable of processing these sugars and that, remarkably, the initiating reaction performed by an enzyme that removes terminal α-(1,2)-linked mannose residues is critical to virulence in a mouse model. This study illuminates an extensive pathway in S. pneumoniae that targets N-linked sugars and is key to the host-pathogen interaction, therefore revealing a potential target for therapeutic intervention.
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DOI：
10.1107/s0907444905036693
发表时间：
2006-01-01
影响因子：
2.2
作者：
Evans, P
通讯作者：
Evans, P
影响因子：
5.6
作者：
Higgins, Melanie A.;Abbott, D. Wade;Boulanger, Martin J.;Boraston, Alisdair B.
通讯作者：
Boraston, Alisdair B.
影响因子：
4.8
作者：
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通讯作者：
Boraston, Alisdair B.
影响因子：
6.7
作者：
Greene NG;Narciso AR;Filipe SR;Camilli A
通讯作者：
Camilli A
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K