Host-Microbial Control of Deoxycholate Producton

描述(由申请人提供)：脱氧胆酸盐生成增加是结肠癌和胆固醇结石发展的积极风险因素。然而，人们对调节脱氧胆酸盐产生的因素知之甚少。只有少数细菌菌株(真细菌和梭状芽孢杆菌属的特定成员)携带胆汁酸诱导(BAI)操纵子，该操纵子编码负责胆汁酸7a-脱羟基化的酶，并且这些微生物只占肠道菌群的一小部分。虽然脱氧胆酸盐的产生与含有白操纵子的细菌的肠道菌群含量直接相关，但人们对这些微生物的肠道内容物的控制因素知之甚少。我们最近建立了一种原发性胆汁酸吸收不良的小鼠模型，即回肠胆汁酸转运体(Slc10a2)基因敲除小鼠。在129S6/SvEv背景下，slcl0a2基因缺失的小鼠会出现严重的胆汁酸吸收不良。相反，将SLCl0a2零等位基因与C57BL/6J背景杂交，只会产生胆汁酸吸收不良，并伴随脱氧胆酸盐产生增加。这是宿主遗传背景明显调节产生脱氧胆酸细菌的结肠含量的第一个例子。此R21应用程序的目标是开发一种小鼠模型系统，以研究调节脱氧胆酸产生的遗传和环境因素。提出了三个具体目标。目的：验证肠道菌群差异是回肠胆汁酸转运蛋白(SIC10a2)缺失小鼠胆汁酸吸收差异的原因的假说。将使用抗生素和胆汁酸类似物来评估小鼠品系肠道菌群差异，以测量脱氧胆酸盐产生的肠道菌群差异。目的：建立直接定量检测含有白操纵子的肠道菌群的酶和聚合酶链式反应方法。许多负责将胆酸转化为脱氧胆酸盐的复杂途径的基因位于单个胆汁酸诱导(BAI)操纵子上。在人的结肠和粪便样本中，直接测定了胆酸7a-脱羟基酶的活性，并用聚合酶链式反应检测了BAI基因。这一目标的目的是将这些分析应用于小鼠，以量化宿主菌株在肠道菌群对胆汁酸7a脱羟基方面的差异。目的3：将SLCl0a2零等位基因与其他小鼠品系杂交，作为克隆负责控制脱氧胆酸产生(由白操纵子细菌定植)的基因的第一步。

DESCRIPTION (provided by applicant): Elevated deoxycholate production is a positive risk factor for the development of colon cancer as well as cholesterol gallstones. However, the factors that regulate deoxycholate production are poorly understood. Only a few bacterial strains (specific members of the Eubacterium and Clostridium genera) carry the bile acid inducible (bai) operon that encodes the enzymes responsible for bile acid 7a-dehydroxylation, and these organisms constitute only a tiny fraction of the intestinal flora. While deoxycholate production correlates directly with intestinal flora content of bai operon-containing bacteria, little is know about what factors control the intestinal content of these organisms. We have recently developed a mouse model of primary bile acid malabsorption, the ileal bile acid transporter (Slc10a2) knockout mouse. Slcl0a2 null mice in a 129S6/SvEv background develop severe bile acid malabsorption. In contrast, crossing the Slcl0a2 null allele into a C57BL/6J background produced only bile acid malabsorption accompanied by increased deoxycholate production. This is the first example of the host genetic background apparently regulating the colonic content of deoxycholate-producing bacteria. The goal of this R21 application is to develop a mouse model system to study the genetic and environmental factors that regulate deoxycholate production. Three specific aims are proposed. Aim 1: To test the hypothesis that differences in the gut flora deoxycholate production are responsible for bile acid absorption differences between mouse strains lacking the ileal bile acid transporter (Sic10a2). The mouse strain gut flora differences will be assessed using antibiotics and bile acid analogs to measure gut flora differences in deoxycholate production. Aim 2: To develop enzyme and PCR-based assays to directly quantify the gut flora bacterium harboring the bai operon. Many of the genes responsible for the complex pathway that converts cholate to deoxycholate are located on a single bile acid inducible (bai) operon. In human colonic and fecal samples, cholic acid 7a-dehydroxylation activity has been directly measured and the bai genes detected by PCR. The goal of this aim is to apply these assays to the mouse to quantify host strain differences in bile acid 7a-dehydroxylation by the enteric flora. Aim 3: To cross the Slcl0a2 null allele into additional mouse strains as a first step toward cloning the genes responsible controlling deoxycholate production (colonization by the bai operon bacteria).