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

（2000会计年度）克隆了a.c rtinomycetemconutans （Aa）荚膜多糖和S.mutans细胞壁多糖的合成相关基因。对克隆基因序列的同源性研究表明，其中一些基因似乎编码糖基转移酶。（2001财政年度）用大肠杆菌中表达的基因产物详细分析了各基因产物的功能。结果表明，rgpA的基因产物为糖基转移酶，它位于rgp基因簇的最上游，催化鼠李糖第一残基的转移。此外，通过比较野生型菌株Xc及其突变株Xc41对抗生素的耐受性，可以明显看出血清型特异性细胞壁多糖对s.a mutans的耐药性起重要作用。（2002财政年度）从a型菌株SUNYaB75的染色体上克隆到了编码gdp -d -甘露糖4,6-脱水酶和gdp -酮-6-脱氧-d -甘露糖还原酶的基因。此外，通过插入失活，成功构建了缺失a、c、d血清型荚膜多糖抗原的变异突变株Aa。当检查这些变异对几种抗菌药物的耐受性时，对杆菌肽和万古霉素的敏感性在任何变异中也略有增加。（2003财政年度）抗体是针对在大肠杆菌中表达和纯化的s.a mutans的rgpC和rgpD基因产物而产生的。虽然在获得的抗体存在的情况下培养S.mutans，但不可能有效抑制细胞壁多糖的合成。将抗体置于脂质体中，加入培养基中，对细胞壁多糖合成有轻微的干扰倾向。对于Aa，制备了与多糖合成有关的ABC转运体抗体。各抗体对囊泡多糖的合成均有轻微的阻断作用。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