Unique Organization of Extracellular Amylases into Amylosomes in the Resistant Starch-Utilizing Human Colonic Firmicutes Bacterium Ruminococcus bromii.
Unique Organization of Extracellular Amylases into Amylosomes in the Resistant Starch-Utilizing Human Colonic Firmicutes Bacterium Ruminococcus bromii.
复制标题
DOI：
10.1128/mbio.01058-15
复制
发表时间：
2015-09-29
期刊：
影响因子：
6.4
通讯作者：
Flint HJ
中科院分区：
文献类型：
--
作者：
Ze X;Ben David Y;Laverde-Gomez JA;Dassa B;Sheridan PO;Duncan SH;Louis P;Henrissat B;Juge N;Koropatkin NM;Bayer EA;Flint HJ
溴瘤胃球菌是人类肠道微生物群的主要成员，其在从膳食淀粉释放能量中起关键作用，膳食淀粉通过其对颗粒状“抗性”淀粉的特殊活性而逃避宿主酶的消化。基因组分析表明，它是高度专业化的，其21个糖苷水解酶中有15个属于一个家庭（GH 13）。我们发现，在R. bromii中的淀粉酶活性是组成型表达的，其中在以果糖作为能量来源的生长期间观察到的活性与以淀粉作为能量来源观察到的活性相似。通过蛋白质组学分析在溴化罗斯酵母培养物中检测到六种携带信号肽的GH 13淀粉酶。这些酶中有四种是预测携带锚定蛋白模块的26种布氏杆菌蛋白质中的一种，Amy 4，也携带粘附蛋白模块。由于已知粘附素-锚定蛋白相互作用介导纤维素分解反刍球菌中蛋白质复合物的形成，因此在将来自布氏杆菌的4种粘附素和11种锚定蛋白作为重组融合蛋白过表达后，研究了它们的结合相互作用。预计酶Amy 4和Amy 9所具有的停靠蛋白结合蛋白支架蛋白2（Sca 2）中存在的粘附蛋白，其类似于纤维素分解相关物（R. flavefaciens）的ScaE细胞壁锚定蛋白。进一步的复合物之间的锚定蛋白携带淀粉酶Amy 4，Amy 9，Amy 10，和Amy 12和其他两个cohesin携带蛋白质，而Amy 4具有自聚集的能力，因为它的锚定蛋白可以识别自己的cohesin。这种淀粉降解酶的组织是前所未有的，并提供了第一个例子，被卷入淀粉分解系统，我们称之为“淀粉体”的粘着蛋白-锚定蛋白的相互作用。人类结肠微生物群对饮食中不可消化的碳水化合物的发酵提供了支持肠道中微生物生长的大部分能量。这种活性通过调节微生物群组成以及微生物代谢物的生理和营养作用，包括从短链脂肪酸向宿主提供能量，对健康产生重要影响。最近的证据表明，某些人类结肠细菌在降解不可消化的底物中起关键作用，其中占优势但很少研究的物种溴化瘤胃球菌（Ruminococcus bromii）显示出降解膳食抗性淀粉的特殊能力（即，由于其它聚合物、颗粒结构、凝沉或化学交联提供的保护而逃避上胃肠道中宿主酶消化的膳食淀粉）。在这份报告中，我们揭示了独特的组织的淀粉分解酶系统的R. bromii，涉及组件蛋白质之间的cohesin-dockerin相互作用。虽然dockerins和cohesins是纤维素分解瘤胃球菌的纤维素酶系统的组织的基础，但它们对淀粉酶的组织的贡献以前没有被认识到，并且可能有助于解释R. bromii的淀粉降解能力。
Ruminococcus bromii is a dominant member of the human gut microbiota that plays a key role in releasing energy from dietary starches that escape digestion by host enzymes via its exceptional activity against particulate “resistant” starches. Genomic analysis of R. bromii shows that it is highly specialized, with 15 of its 21 glycoside hydrolases belonging to one family (GH13). We found that amylase activity in R. bromii is expressed constitutively, with the activity seen during growth with fructose as an energy source being similar to that seen with starch as an energy source. Six GH13 amylases that carry signal peptides were detected by proteomic analysis in R. bromii cultures. Four of these enzymes are among 26 R. bromii proteins predicted to carry dockerin modules, with one, Amy4, also carrying a cohesin module. Since cohesin-dockerin interactions are known to mediate the formation of protein complexes in cellulolytic ruminococci, the binding interactions of four cohesins and 11 dockerins from R. bromii were investigated after overexpressing them as recombinant fusion proteins. Dockerins possessed by the enzymes Amy4 and Amy9 are predicted to bind a cohesin present in protein scaffoldin 2 (Sca2), which resembles the ScaE cell wall-anchoring protein of a cellulolytic relative, R. flavefaciens. Further complexes are predicted between the dockerin-carrying amylases Amy4, Amy9, Amy10, and Amy12 and two other cohesin-carrying proteins, while Amy4 has the ability to autoaggregate, as its dockerin can recognize its own cohesin. This organization of starch-degrading enzymes is unprecedented and provides the first example of cohesin-dockerin interactions being involved in an amylolytic system, which we refer to as an “amylosome.” Fermentation of dietary nondigestible carbohydrates by the human colonic microbiota supplies much of the energy that supports microbial growth in the intestine. This activity has important consequences for health via modulation of microbiota composition and the physiological and nutritional effects of microbial metabolites, including the supply of energy to the host from short-chain fatty acids. Recent evidence indicates that certain human colonic bacteria play keystone roles in degrading nondigestible substrates, with the dominant but little-studied species Ruminococcus bromii displaying an exceptional ability to degrade dietary resistant starches (i.e., dietary starches that escape digestion by host enzymes in the upper gastrointestinal tract because of protection provided by other polymers, particle structure, retrogradation, or chemical cross-linking). In this report, we reveal the unique organization of the amylolytic enzyme system of R. bromii that involves cohesin-dockerin interactions between component proteins. While dockerins and cohesins are fundamental to the organization of cellulosomal enzyme systems of cellulolytic ruminococci, their contribution to organization of amylases has not previously been recognized and may help to explain the starch-degrading abilities of R. bromii.