Development of new antibiotics targeting cell wall biosynthesis of oral pathogenic bacteria

(2000财政年度)克隆了与放线菌衣壳多糖和变形链球菌胞壁多糖合成相关的基因。对克隆的基因序列进行同源性研究，发现其中一些基因似乎编码糖基转移酶。(2001财年)利用在大肠杆菌中表达的基因产物对每个基因产物的详细功能进行分析。结果表明，rgpA基因产物为糖基转移酶，位于rgp基因簇最上游，催化第一鼠李糖残基的转移。此外，通过比较野生型菌株Xc和突变菌株Xc41对抗生素的耐受性，发现血清型特异性细胞壁多糖对变形链球菌的耐药性有重要作用。(2002年财政年度)编码GDPD-甘露糖4，6-脱水酶和GDP酮-6-脱氧-D-甘露糖还原酶的基因是…从AA型AA型SUNYaB75株的染色体上克隆了更多。另外，通过插入失活，成功构建了缺失a、c、d三种衣壳多糖抗原的突变株AA。当检测这些变异体对几种抗菌药的耐受性时，所有变异体对杆菌肽和万古霉素的敏感性也略有增加。(2003财年)针对变形链球菌在大肠杆菌中表达和纯化的rgpC和rgpD基因产物，抗体被提高。虽然变形链球菌在获得的抗体存在下培养，但不可能有效地抑制细胞壁多糖的合成。当抗体进入脂质体并添加到培养基中时，对细胞壁多糖合成的干扰倾向被轻微认识到。在AA的情况下，制备了与多糖合成有关的抗ABC转运蛋白的抗体。各抗体对囊多糖的合成均有轻微的阻断作用。较少

(The 2000 fiscal year)The genes which were related to the synthesis of the capsule polysaccharide of A.actinomycetemconutans(Aa) and cell wall polysaccharide of S.mutans was cloned. Homologous research for the determined sequence of the cloned genes revealed that some of them seemed to encode glycosyltransferases.(The 2001 fiscal year)The detailed function of each gene product was analyzed with the expressed gene products in Escherichia coli. The result indicated that the gene product of rgpA which is located in the uppermost stream of rgp gene cluster was glycosyltransferase which catalyzes the transfer of the first rhamnose residue. Furthermore, it became to be obvious that the serotype specific cell wall polysaccharide was important for the drug tolerance of S.mutans by comparing the drug tolerance for antibiotics of wild type strain Xc and its mutant strain Xc41.(The 2002 fiscal year)The genes which encode GDP-D-mannose 4,6-dehydratase and GDP-keto-6-deoxy-D-mannose reductase were … More cloned from the chromosome of the serotype a Aa SUNYaB75 strain. In addition, construction of the variant mutant strains Aa which lost serotype a,c, and d capsular polysaccharide antigens was succeeded by the insertional inactivation. When the tolerance of these variants for several antimicrobial drugs was examined, the sensitivity for bacitracin and vancomycin also slightly increased in any variants.(The 2003 fiscal year)The antibodies were raised against the rgpC and rgpD gene products of S.mutans expressed in and purified from E.coli. Though S.mutans was cultivated in the presence of the obtained antibodies, it was not possible to effectively inhibit the cell wall polysaccharide synthesis. When the antibody was taken in liposome and added to the culture medium, the interference tendency in the cell wall polysaccharide synthesis was slightly recognized. In case of Aa, antibodies against ABC transporters concerning the synthesis of the polysaccharide were prepared. Every antibodies showed slight blocked effect on capusular polysaccharide synthesis. Less