Protein Complexes in Alginate Maturation

描述(由申请人提供)：胞外多糖在确定致病菌的毒力、影响它们在表面的粘附性以及保护它们免受抗生素和宿主免疫系统的伤害方面发挥着重要作用。关于在重要的人类病原体中调控胞外多糖成熟和运输的步骤，仍有许多有待了解。本申请描述了对铜绿假单胞菌中藻酸盐生物合成的最后步骤的研究。藻酸盐是一种未支化的杂多碳水化合物，在细菌周围形成一个胶囊。聚合物合成的前体是在细胞质中产生的，聚合成聚甘露糖是通过完整的细胞质膜蛋白的作用进行的。我们描述了描述聚合物形成和分泌之间的过程的实验。聚甘露糖在某些残基上通过周质酶的作用发生异构化。我们已经证明，另一种蛋白质ALGK与差向异构体酶形成可分离的复合体，而复合体的形成需要第三种蛋白质ALGX。该复合体的目的可能是保护藻酸盐不被海藻酸裂解酶降解，并促进海藻酸盐向外膜上的孔蛋白输送。在周质中，藻酸盐被部分乙酰化。已证明乙酰化需要三种蛋白质：海藻、海藻J和海藻F。它们的作用尚不明确，乙酰化过程也是个谜，因为周质中不存在乙酰辅酶A。我们建议表征与差向异构酶形成的蛋白质复合体，并确定它们对其功能性质的影响，并在分子细节上表征乙酰化过程。我们将确定乙酰基的来源，并评估单独的蛋白质在乙酰化过程中的作用。将使用动力学技术和生物物理方法来表征蛋白质复合体，包括凝胶过滤层析、分析超速离心法和荧光各向异性。公共卫生相关性铜绿假单胞菌是一种机会性的人类病原体，在几个不同的患者群体中引起危及生命的感染。细菌的毒力和其在宿主中的持续感染的一个主要因素是胞外多糖海藻酸盐。对导致成熟藻酸盐形成的步骤进行生化表征，可能会为如何阻止或改善感染的影响提供见解。

DESCRIPTION (provided by applicant): Exopolysaccharides play important roles in determining the virulence of pathogenic bacteria, affecting their adherence to surfaces, and protecting them from antibiotics and the host's immune system. Much remains to be learned about the steps that govern the maturation and transport of exopolysaccharides in important human pathogens. The present application describes investigations into the final steps of alginate biosynthesis in Pseudomonas aeruginosa. Alginate is an unbranched heteropolymeric carbohydrate that forms a capsule around the bacteria. The precursors for polymer synthesis are generated in the cytoplasm, and polymerization to polymannuronan occurs through the action of integral cytoplasmic membrane proteins. We describe experiments to characterize the processes that occur between polymer formation and secretion. Polymannuronan undergoes epimerization at some residues via the action of a periplasmic enzyme. We have shown that another protein, AlgK, forms an isolable complex with the epimerase, and that complex formation requires a third protein AlgX. It is possible that the purpose of the complex is to protect the alginate from degradation by alginate lyase, and to facilitate the delivery of alginate to a porin protein on the outer membrane. During its time in the periplasm, alginate is partially acetylated. Three proteins have been shown to be required for acetylation, AlgI, AlgJ, and AlgF. Their roles remain undefined, and the acetylation process is enigmatic, since there is no acetyl-CoA present in the periplasm. We propose to characterize the protein complexes formed with the epimerase and to determine their influence on its functional properties, and to characterize the acetylation process in molecular detail. We will determine the source of the acetyl groups, and evaluate the roles of the separate proteins in the acetylation process. Kinetics techniques and biophysical methods for characterizing the protein complexes, including gel filtration chromatography, analytical ultracentrifugation and fluorescence anisotropy, will be employed. PUBLIC HEALTH RELEVANCE Pseudomonas aeruginosa is an opportunistic human pathogen that causes life-threatening infections in several different patient populations. A major contributing factor to the virulence of the bacteria and the persistence of their infections in the host is the exopolysaccharide alginate. Biochemical characterization of the steps that lead to formation of mature alginate may provide insights into how to block or ameliorate the effects of infection.