Regulation Of Sugar Transport And Metabolism In Oral Bacteria

口腔细菌中糖运输和代谢的调节

• 批准号: 8553317
• 负责人: john thompson
• 金额: $ 40.51万
• 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/8553317
• 关键词: Acute Pneumonia; Address; Adherence; Affinity; Arbutin; Area; Bacillus subtilis; Bacteria; Belief; Benign; Binding; Biological Assay; Calorimetry; Carbohydrates; Carrier Proteins; Cataloging; Catalogs; Cellobiose; Cells; Cessation of life; Characteristics; Chinese People; Complex; Computer Simulation; DNA; Data; Databases; Dental Enamel; Dental caries; Deposition; Dietary Sucrose; Dietary Sugars; Disaccharides; Discrimination; Enzymes; Epithelial Cells; Escherichia coli; Etiology; Exhibits; Family; Fermentation; Fructose; Future; Gene Deletion; Genes; Genetic; Genome; Glucose; Glycoside Hydrolases; Growth; Hexoses; Host Defense; Human; Hydrolase; Hydrolysis; Infection; Ions; Isomerism; Journals; Kinetics; Klebsiella pneumonia bacterium; Laboratories; Lactic acid; Leptothrix; Leptotrichia; Ligands; Link; Meningitis; Metabolic; Metabolism; Microbe; Microbial Physiology; Mining; Minor; Modification; Molecular; Monosaccharides; Mucins; Mutation; Operon; Oral; Oral Microbiology; Otitis Media; Pathogenicity; Peer Review; Phase; Play; Polysaccharides; Procedures; Property; Proteins; Proteomics; Publishing; Pyruvate; Recombinant DNA; Regulation; Research; Research Personnel; Resolution; Roentgen Rays; Role; Science; Septicemia; Sequence Alignment; Series; Site-Directed Mutagenesis; Streptococcus; Streptococcus mutans; Streptococcus pneumoniae; Structure; Substrate Specificity; Sucrose; Surface; Synchrotrons; Taxon; Technology; Testing; Thermodynamics; Titrations; Translations; Universities; Variant; Virulence; Virulence Factors; Virulent; analog; base; beta Glucosidases; beta-Glucosidase; cofactor; comparative; demineralization; enthalpy; enzyme structure; expression cloning; expression vector; gene cloning; genetic analysis; genetic regulatory protein; genome sequencing; gentiobiose; glucosidase; inorganic phosphate; interest; member; microorganism; novel; oral bacteria; oral biofilm; oral microbiome; pathogen; polypeptide; programs; research study; salicin; stem; stoichiometry; sugar; tooth surface; trait

FERMENTATION OF SUCROSE ISOMERS BY ORAL LEPTOTRICHIA The sequenced genome of L. buccalis ATCC 14201 revealed three contiguous genes at loci: Lebu_1525,1526 and 1527. The translation products of these genes exhibited significant homology with phospho-α-glucosidase (Pagl), a regulatory protein (GntR) and a phosphoenol pyruvate-dependent sugar transport protein (EIICB), respectively. In non-oral bacterial species, these genes comprise the sim operon that facilitates sucrose isomer metabolism. Growth studies showed that L. buccalis fermented a wide variety of carbohydrates, including four of the five isomers of sucrose. Growth on the isomeric disaccharides elicited expression of a 50kDa polypeptide comparable in size to that encoded by Lebu_1525. The latter gene was cloned, and the expressed protein was purified to homogeneity from Escherichia coli TOP 10 cells. In the presence of two essential cofactors (NAD+ and Mn2+ ion) the enzyme readily hydrolyzed p-nitrophenyl-alpha-glucopyranoside 6-phosphate (pNPalphaG6P), a chromogenic analog of the phosphorylated isomers of sucrose. By comparative sequence alignment, immuno- reactivity and signature motifs, the enzyme was assigned to the phospho-α-glucosidase (Pagl) clade of glycosylhydrolase Family 4. It is our contention that the products of Lebu_1527 and 1525, catalyze the phosphorylative translocation and hydrolysis of sucrose isomers in L. buccalis, respectively. Four genetically diverse, but 16S rDNA related species of Leptotrichia have recently been described: L. goodfellowii, L. hofstadii, L. shahii and L. wadei. Our determination of the phenotypic traits of these new species with respect to carbohydrate utilization yielded some unexpected findings. Whereas non-oral bacterial species invariably grow on all sucrose isomers, considerable variation was noted in the number(s) of isomers that supported growth of a particular species of Leptotrichia. For example, L. shahii metabolized all five isomeric compounds, L. buccalis fermented four of the sucro-disaccharides whereas, under the same conditions, L.wadei failed to grow on either maltulose or leucrose. At the molecular level, such isomeric discrimination presumably reflects subtle species-specific mutations or conformational changes in either the transport or intracellular Pagl proteins. The genetic basis for the variability of metabolic traits may be revealed by future comparative analyses when the genomes of the different species of Leptotrichia have been sequenced in their entirety. As genetic information accumulates in the Human Oral Microbiome Database (HOMD), it likely that genetic loci similar to the sim operon in Leptotrichia will be discovered in presently un-sequenced genomes of oral bacteria. In this context, the fermentation of presumed non-cariogenic disaccharides by oral microorganisms may be more widespread than currently envisaged. Our findings have been published in the peer-reviewed journal, Molecular Oral Microbiology. STRUCTURE AND FUNCTION OF PHOSPHO-BETA-GLUCOSIDASE (BGLA-2) FROM STREPTOCOCCUS PNEUMONIAE TIGR4. Streptococcus pneumonia is the major causative agent of acute pneumonia, otitis media, meningitis, and septicemia, which annually result in the deaths of millions worldwide. In the human host, S. pneumoniae encounters a variety of glyco-conjugates, including mucins, host defense molecules, and surface exposed glycans on epithelial cells. In common with other pathogenic microbes, S. pneumonia produces a variety of secreted or surface-associated glycosidases whose function(s) include the modification and hydrolysis of host glyco-conjugates. Genome sequencing, in combination with exploration of new virulence factors, suggests that a large number of glycosidases are necessary for maximum virulence of S. pneumoniae. BglA-2 is encoded by the gene Sp_0578 in the chromosomal DNA of S. pneumoniae TIGR4. After cloning of the gene in a high expression vector, BglA-2 (471 residues, Mr = 54,361) was purified to electrophoretic homogeneity. The natural substrates of BglA-2 include: cellobiose-6-phosphate, gentiobiose-6P, arbutin-6P, salicin-6P and related O-beta-linked disaccharide phosphates. These compounds are not commercially available, and accordingly were prepared enzymatically by procedures developed previously in our laboratory (J. Biol. Chem. 277: 34310-34321, 2002). Use of these novel compounds permitted substrate specificity and kinetic analyses to be conducted. Thermodynamic parameters including enthalpy changes, ligand affinity and stoichiometry of binding between substrates and BglA-2 were determined by isothermal titration calorimetry (ITC). Crystals of BglA-2 in native form, and in complex with a non-hydrolyzable analog (thio-cellobiose-6-phosphate), have been prepared, and the structure of the enzyme has been solved by X-ray (synchrotron) diffraction and resolution of phase by molecular replacement. Future experiments, involving site-directed mutagenesis and gene deletion, will address the pathogenic contribution(s) of BglA-2 to S. pneumoniae infection.

口服纤毛虫发酵蔗糖异构体 颊乳杆菌 ATCC 14201 的基因组测序揭示了 Lebu_1525、1526 和 1527 位点上的三个连续基因。这些基因的翻译产物与磷酸-α-葡萄糖苷酶 (Pagl)、调节蛋白 (GntR) 和磷酸烯醇表现出显着的同源性分别是丙酮酸依赖性糖转运蛋白（EIICB）。在非口腔细菌物种中，这些基因包含促进蔗糖异构体代谢的 sim 操纵子。生长研究表明，颊乳杆菌发酵多种碳水化合物，包括蔗糖五种异构体中的四种。在异构二糖上的生长引发了 50kDa 多肽的表达，其大小与 Lebu_1525 编码的多肽相当。后一个基因被克隆，表达的蛋白质从大肠杆菌TOP 10细胞中纯化至同质。在两种必需辅因子（NAD+ 和 Mn2+ 离子）存在的情况下，该酶很容易水解对硝基苯基-α-吡喃葡萄糖苷 6-磷酸 (pNPalphaG6P)，一种蔗糖磷酸化异构体的显色类似物。通过比较序列比对、免疫反应性和特征基序，该酶被归属于糖基水解酶家族 4 的磷酸-α-葡萄糖苷酶 (Pagl) 进化枝。我们认为 Lebu_1527 和 1525 的产物催化磷酸化易位和水解分别是 L. buccalis 中的蔗糖异构体。最近描述了四种遗传多样性但 16S rDNA 相关的纤毛菌属物种：L. goodfellowii、L. hofstadii、L. shahii 和 L. wadei。我们对这些新物种在碳水化合物利用方面的表型特征的确定产生了一些意想不到的发现。尽管非口腔细菌物种总是在所有蔗糖异构体上生长，但支持细毛菌特定物种生长的异构体数量存在相当大的变化。例如，L. shahii 代谢所有五种异构化合物，L. buccalis 发酵四种蔗糖二糖，而在相同条件下，L.wadei 无法在麦芽糖或明串珠菌糖上生长。在分子水平上，这种异构体区分可能反映了转运蛋白或细胞内 Pagl 蛋白中微妙的物种特异性突变或构象变化。当纤毛虫不同物种的基因组得到完整测序后，未来的比较分析可能会揭示代谢性状变异的遗传基础。随着人类口腔微生物组数据库 (HOMD) 中遗传信息的积累，在目前尚未测序的口腔细菌基因组中，很可能会发现与细毛菌中 sim 操纵子相似的遗传位点。在这种情况下，口腔微生物对假定的非致龋性二糖的发酵可能比目前设想的更为广泛。我们的研究结果发表在同行评审期刊《分子口腔微生物学》上。 肺炎链球菌 TiGR4 的磷酸-β-葡萄糖苷酶 (BGLA-2) 的结构和功能。 肺炎链球菌是急性肺炎、中耳炎、脑膜炎和败血症的主要病原体，每年导致全世界数百万人死亡。在人类宿主中，肺炎链球菌遇到多种糖缀合物，包括粘蛋白、宿主防御分子和上皮细胞上表面暴露的聚糖。与其他病原微生物一样，肺炎链球菌产生多种分泌型或表面相关糖苷酶，其功能包括修饰和水解宿主糖缀合物。基因组测序与新毒力因子的探索相结合表明，肺炎链球菌的最大毒力需要大量糖苷酶。 BglA-2 由肺炎链球菌 TIGR4 染色体 DNA 中的基因 Sp_0578 编码。将基因克隆到高表达载体中后，BglA-2（471 个残基，Mr = 54,361）被纯化至电泳均质。 BglA-2的天然底物包括：纤维二糖-6-磷酸、龙胆二糖-6P、熊果苷-6P、水杨苷-6P和相关的O-β-连接二糖磷酸酯。这些化合物不可商购，因此是通过我们实验室先前开发的程序酶促制备的（J. Biol. Chem. 277: 34310-34321, 2002）。使用这些新型化合物可以进行底物特异性和动力学分析。通过等温滴定量热法(ITC)测定热力学参数，包括热函变化、配体亲和力和底物与BglA-2之间结合的化学计量。已制备出天然形式的 BglA-2 晶体以及与不可水解类似物（硫代纤维二糖-6-磷酸）的复合物，并且已通过 X 射线（同步加速器）衍射和解析了该酶的结构。通过分子置换来解决相。未来的实验，包括定点突变和基因删除，将解决 BglA-2 对肺炎链球菌感染的致病作用。