基于CcpA蛋白调控山羊瘤胃牛链球菌对乳酸代谢的分子机制

The mechanism of lactic acid production and metabolic regulation in the rumen is one of a key scientific issue, which is closely related to the rumen acidosis caused by the high-grain feeding in modern intensive ruminant production. Previous studies have shown that the catabolite control protein (CcpA) is a regulator that has regulatory effect on the production of lactic acid metabolism in the rumen; however its regulatory mechanism through CcpA is unclear at present. This proposal study focuses on CcpA protein manipulation on lactate production and metabolism in the rumen. In vitro culture and In vivo experiment, chromatin coprecipitation tandem ChIP detection, Immunoprecipitation technology and high throughput sequencing technology were used to study the transcriptional regulation of lactate dehydrogenase (LDH) and pyruvate formate-lyase (PFL) in the lactate pathway of ruminal Streptococcus bovis. Studies will focus on In vitro cultivation substrates and dietary carbohydrate structure, rumen lactic acid produced by Streptococcus bovis and metabolic pathways and targets of lactic acid metabolism modulated by CcpA protein and its mechanism, and the ruminal lactic acid production of Streptococcus bovis, differences in metabolic pathways and molecular mechanism regulated by the CcpA protein are planned to investigate. Furthermore, the transcriptional regulation of lactate dehydrogenase and pyruvate formate-lyase in the ruminal lactate production pathway will be conducted and verifed by yeast bihybridization and overexpression and RNAi technology. Finally, as a whole to clarify regulation mechanism of ruminal lactate production by Streptococcus bovis and lactate metabolism through CcpA protein under the condition of different dietary structure of carbohydrate. This study will provide a scientific basis for rumen acidosis prevention and control policy measures.

瘤胃牛链球菌的乳酸产生和代谢调控机制是重要的科学问题，它与集约化反刍动物生产中因饲喂高精饲料而产生的瘤胃酸中毒的关系密切。前期研究表明CcpA蛋白具有对瘤胃中牛链球菌产乳酸代谢的调控作用，但其调控机制不清楚。本研究围绕CcpA对瘤胃内牛链球菌乳酸产生和代谢调控，采用体外与体内试验，应用基因敲除、染色质免疫共沉淀和高通量测序等技术研究CcpA对瘤胃牛链球菌产生乳酸通路中乳酸脱氢酶和丙酮酸甲酸裂解酶转录调控；从体外培养底物和饲喂动物日粮的碳水化合物组成结构、牛链球菌内乳酸产生与代谢通路及其CcpA调控乳酸代谢的靶点出发，研究瘤胃牛链球菌对乳酸产生、代谢通路的差异，利用超表达及RNAi技术验证研究CcpA对瘤胃牛链球菌产生乳酸通路中乳酸产生和代谢调控的影响；从整体上阐明CcpA对瘤胃牛链球菌产生乳酸和代谢调控的分子机制，为今后制定反刍动物瘤胃酸中毒防控策措提供科学依据。

亚急性瘤胃酸中毒（SARA）是反刍动物因大量饲喂谷物日粮而诱发的一种常见营养代谢病，瘤胃中的牛链球菌（Streptococcus bovis）快速增殖并产生过量的乳酸是诱发瘤胃酸中毒的重要因子。本项目通过ccpA基因敲除试验研究揭示了分解代谢性控制蛋白（CcpA）对牛链球菌产乳酸起关键调控作用，即CcpA通过调控S. bovis的乳酸脱氢酶（LDH）和丙酮酸甲酸裂解酶（PFL）的转录水平而影响其有机酸发酵模式，CcpA调控S. bovis S1产生的关键靶点是丙酮酸下游关键代谢酶和碳代谢方向，使其由乳酸发酵向混合酸发酵转变，而且在高葡萄糖浓度下的调控作用更有效。ccpA基因敲除导致牛链球菌的生长减慢。证实了CcpA可调控瘤胃中牛链球菌的增殖及其产酸模式，ccpA基因缺失后导致S. bovis S1产酸模式发生改变，即由乳酸发酵向混合酸发酵转变，其中乳酸比例显著降低（15.13%），甲酸和乙酸比例显著升高。ccpA基因敲除后，参与编码丙酮酸下游代谢关键酶的基因和蛋白表达发生变化，其中向乳酸发酵方向的关键酶LDH（ldh）下调，而向混合酸发酵方向的关键酶PFL（pfl）和ACKA（acka）上调；细菌糖代谢产酸的关键酶活性发生改变，其中LDH活性下降，ACKA活性上升。同时，研究阐明了在不同碳水化合物底物（麦芽糖、纤维二糖和蔗糖）下CcpA 对牛链球菌S1 产酸模式的调控作用的变化规律。对牛链球菌菌株S1进行了全基因组及生物信息学分析，发现S1菌株基因组是一个全长为1.88 Mb的环状染色体，基因数为1802，GC含量为37.89%。同时S1菌株在泛核基因组、碳水化合物活性酶方面均与JB1菌株有较近的进化关系，具有较多的CE1、GT2、GH51、GH127等碳水化合物活性酶。构建了CcpA蛋白三级结构并预测碳水化合物代谢关键酶基因pflD、pck、ldh、ackA、amy、hpr具有与CcpA蛋白结合的cre位点。从蛋白水平、代谢水平上揭示了CcpA蛋白对牛链球菌产生乳酸代谢的调控机制。总体上阐明了瘤胃中牛链球菌在不同底物类型和不同pH酸度下CcpA对牛链球菌产有机酸的代谢位点、关键代谢酶的功能以及CcpA蛋白对乳酸代谢调控通路，为今后制定反刍动物瘤胃酸中毒防控措施提供科学依据。