Regulation Of Sugar Transport And Metabolism In Oral Bac

Oral Bac 中糖转运和代谢的调节

• 批准号: 7318442
• 负责人: john thompson
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7318442
• 关键词: Regulation; Sugar; Transport; Metabolism; Oral

Dietary sucrose is a major contributor to the etiology of dental caries. This disaccharide, (comprised of glucose and fructose molecules) provides the precursors for glycan synthesis, that facilitates adherence of Streptococci to the tooth surface. The localized production of lactic acid (via the microbial fermentation of sucrose) causes demineralization of surface enamel, and initiation of tooth decay. Positional change of the O-glycosidic linkage between C1 of the glucose moiety and the remaining five -OH groups of the fructose ring, yields the five stereo- isomers of sucrose: trehalulose, turanose, maltulose, leucrose and palatinose. Prior to commencement of our research program, it was generally assumed the microorganisms were unable to metabolize the isomers of sucrose. This belief, and the fact that the isomers are comparatively sweet, has encouraged the use of these ?non-cariogenic? compounds as substitutes for sucrose in various food products. Surprisingly, and contrary to expectation, we have found that many bacterial species from both Gram-positive and Gram-negative genera (including, Klebsiella pneumoniae, Bacillus subtilis, Clostridium acetobutylicum and Fusobacterium mortiferum) readily grow on these five isomeric disaccharides. Bacteria with the capacity to metabolize the isomers of sucrose invariably possess two chromosomal genes that encode: (i) a membrane-localized phosphoenolpyruvate (PEP)-dependent transporter that facilitates the simultaneous translocation and phosphorylation of the isomers, and (ii) a unique NAD/Mn(2+) -dependent phospho- alpha-glucosidase that catalyzes the intracellular hydrolysis of phosphorylated isomers to yield glucose 6-phosphate and fructose. In the case of Klebsiella pneumoniae, the two genes (aglA, aglB) and their products are designated AglA and AglB, respectively. Although prerequisite for growth of K. pneumoniae and other species, it was unclear whether expression of these two proteins alone, was sufficient for growth of microorganisms on the isomeric disaccharides. Of particular concern was the fact that these particular species lack a gene encoding EIIA(Agl), a third phosphorylatable protein required for functional activity of a PEP-dependent Agl: phosphotransferase system (PTS). Previously (but without proof), we suggested that an EIIA from a different PTS must substitute for the ?missing? EIIA(Agl) in order to complement activity of the PEP-dependent Agl transport system. To address this hypothesis, a collaborative investigation was initiated in 2006 with researchers at the University of Berne, Switzerland. For our studies, Escherichia coli K-12 was the organism of choice, because this enteric bacterium is unable to metabolize sucrose or its isomers, and is readily amenable to genetic manipulation. Importantly, strains of E. coli K-12 containing the necessary mutations, and purified components of the phosphotransferase system (HPr and Enzyme I) were made available by our collaborators. Our experimental approach was to first clone both aglA and aglB genes into an expression plasmid (pAP2). When transformed with this plasmid, E. coli K-12 grew readily on a wide variety of O-alpha-linked glucosides, including all isomers of sucrose. Subsequently, a mutant strain of E. coli K-12 lacking EIIA(Glc) of the glucose: PTS was also transformed with pAP2. This transformant failed to grow on any of the alpha-glucosides tested, thus suggestive of EIIA(Glc) participation in the transport of sucrose isomers. Confirmation for our hypothesis was obtained from in vitro experiments conducted with membrane preparations of the E. coli K-12 containing the AglA transporter from K. pneumoniae. When supplemented with the high-energy phosphoryl donor (phosphoenolpyruvate), HPr and EI, these cytoplasmic membranes failed to catalyze the phosphorylation of the isomeric compounds. However, upon addition of purified EIIA(Glc) to the reaction mixture, an immediate and rapid phosphorylation of the disaccharides was observed. We believe that AglA (transporter), AglB (phospho-alpha -glucosidase), and EIIA(Glc) of the glucose-PTS are necessary and sufficient, for the growth of microorganisms on sucrose isomers and related alpha-D-glucopyranosides.

膳食蔗糖是导致龋齿的主要原因。这种二糖（由葡萄糖和果糖分子组成）提供聚糖合成的前体，促进链球菌粘附到牙齿表面。乳酸的局部产生（通过蔗糖的微生物发酵）导致表面牙釉质脱矿，并引发蛀牙。葡萄糖部分的C1和果糖环的其余五个-OH基团之间的O-糖苷键的位置变化产生蔗糖的五种立体异构体：海藻酮糖、松二糖、麦芽酮糖、明串珠菌糖和帕拉金糖。在我们的研究计划开始之前，人们普遍认为微生物无法代谢蔗糖的异构体。这种信念以及异构体相对较甜的事实鼓励了这些“非致龋性”的使用。在各种食品中作为蔗糖替代品的化合物。令人惊讶的是，与预期相反，我们发现来自革兰氏阳性和革兰氏阴性属的许多细菌物种（包括肺炎克雷伯氏菌、枯草芽孢杆菌、丙酮丁醇梭菌和腐烂梭杆菌）很容易在这五种异构二糖上生长。具有代谢蔗糖异构体能力的细菌总是拥有两个编码的染色体基因：(i) 膜定位的磷酸烯醇丙酮酸 (PEP) 依赖性转运蛋白，促进异构体的同时易位和磷酸化，以及 (ii) 独特的 NAD /Mn(2+) 依赖性磷酸-α-葡萄糖苷酶，催化磷酸化物质的细胞内水解异构体产生葡萄糖 6-磷酸和果糖。就肺炎克雷伯菌而言，两个基因（aglA、aglB）及其产物分别指定为 AglA 和 AglB。尽管这是肺炎克雷伯菌和其他物种生长的先决条件，但尚不清楚这两种蛋白质的单独表达是否足以使微生物在异构二糖上生长。特别值得关注的是，这些特定物种缺乏编码 EIIA(Agl) 的基因，EIIA(Agl) 是 PEP 依赖性 Agl 功能活性所需的第三种可磷酸化蛋白：磷酸转移酶系统 (PTS)。之前（但没有证据），我们建议来自不同 PTS 的 EIIA 必须替代“缺失”的内容。 EIIA(Agl) 以补充 PEP 依赖性 Agl 转运系统的活性。为了验证这一假设，瑞士伯尔尼大学的研究人员于 2006 年发起了一项合作调查。对于我们的研究，大肠杆菌 K-12 是选择的生物体，因为这种肠道细菌无法代谢蔗糖或其异构体，并且很容易进行基因操作。重要的是，我们的合作者提供了含有必要突变的大肠杆菌 K-12 菌株以及磷酸转移酶系统的纯化成分（HPr 和酶 I）。我们的实验方法是首先将 aglA 和 aglB 基因克隆到表达质粒 (pAP2) 中。当用该质粒转化时，大肠杆菌 K-12 可以在多种 O-α 连接糖苷上轻松生长，包括蔗糖的所有异构体。随后，缺乏葡萄糖的EIIA(Glc)的大肠杆菌K-12突变株：PTS也用pAP2转化。该转化体无法在任何测试的 α-葡萄糖苷上生长，因此表明 EIIA(Glc) 参与了蔗糖异构体的转运。我们的假设得到了体外实验的证实，该实验使用含有肺炎克雷伯菌 AglA 转运蛋白的大肠杆菌 K-12 膜制剂进行。当补充高能磷酰基供体（磷酸烯醇丙酮酸）、HPr 和 EI 时，这些细胞质膜无法催化异构化合物的磷酸化。然而，在将纯化的 EIIA(Glc) 添加到反应混合物中后，观察到二糖立即快速磷酸化。我们相信，葡萄糖-PTS 的 AglA（转运蛋白）、AglB（磷酸-α-葡萄糖苷酶）和 EIIA（Glc）对于微生物在蔗糖异构体和相关 α-D-吡喃葡萄糖苷上的生长是必要且充分的。