Host-Microbial Control of Deoxycholate Producton

描述（由申请方提供）：脱氧胆酸盐产生升高是结肠癌和胆固醇结石发生的积极风险因素。然而，调节脱氧胆酸盐生产的因素知之甚少。只有少数细菌菌株（真杆菌属和梭菌属的特定成员）携带编码负责胆汁酸7 α-脱羟基化的酶的胆汁酸诱导（bai）操纵子，并且这些生物体仅构成肠植物群的一小部分。虽然脱氧胆酸盐的产生与含bai操纵子的细菌的肠道植物群含量直接相关，但对控制这些生物体的肠道内容物的因素知之甚少。我们最近开发了一种原发性胆汁酸吸收不良的小鼠模型，即回肠胆汁酸转运蛋白（Slc 10a 2）敲除小鼠。在129 S6/SvEv背景中的Slcl 0a 2缺失小鼠发生严重的胆汁酸吸收不良。相反，将Slcl 0a 2无效等位基因杂交到C57 BL/6 J背景中仅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脱氧胆酸盐细菌的结肠内容物的第一个例子。这项R21应用的目标是开发一种小鼠模型系统，以研究调节脱氧胆酸盐产生的遗传和环境因素。提出了三个具体目标。目标1：为了检验以下假设：肠道植物群脱氧胆酸盐产生的差异是缺乏回肠胆汁酸转运蛋白（Sic 10 a2）的小鼠品系之间胆汁酸吸收差异的原因。将使用抗生素和胆汁酸类似物评估小鼠品系肠道植物群差异，以测量脱氧胆酸盐产生中的肠道植物群差异。目的2：建立基于酶和PCR的检测方法来直接定量含有bai操纵子的肠道植物群细菌。许多负责将胆酸盐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。在人结肠和粪便样品中，直接测量胆酸7 α-脱羟基活性，并通过PCR检测bai基因。本研究的目的是将这些试验应用于小鼠，以定量肠道植物群对胆汁酸7 α-脱羟基作用的宿主菌株差异。目标3：将Slcl 0a 2无效等位基因杂交到另外的小鼠品系中，作为克隆负责控制脱氧胆酸盐产生的基因（通过bai操纵子细菌定殖）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).