Protein Complexes in Alginate Maturation

描述（由申请人提供）：胞外多糖在确定病原菌的毒力、影响其对表面的粘附以及保护其免受抗生素和宿主免疫系统的侵害方面发挥重要作用。在重要的人类病原体中，控制胞外多糖成熟和转运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。藻酸盐是一种无分支的杂聚碳水化合物，在细菌周围形成胶囊。聚合物合成的前体在细胞质中产生，并且通过整合的细胞质膜蛋白的作用聚合成聚甘露糖醛酸。我们描述了实验来表征聚合物形成和分泌之间发生的过程。聚甘露糖醛酸通过周质酶的作用在一些残基处经历差向异构化。我们已经表明，另一种蛋白质，AlgK，形成一个可分离的复合物与差向异构酶，复合物的形成需要第三种蛋白质AlgX。复合物的目的可能是保护藻酸盐免受藻酸盐裂解酶的降解，并促进藻酸盐递送至外膜上的孔蛋白。在其在周质中的时间期间，藻酸盐被部分乙酰化。已经证明乙酰化需要三种蛋白质：AlgI、AlgJ和AlgF。它们的作用仍然不明确，乙酰化过程是神秘的，因为在周质中没有乙酰辅酶A。我们建议表征与差向异构酶形成的蛋白质复合物，并确定它们对其功能特性的影响，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，并评估不同蛋白质在乙酰化过程中的作用。将采用动力学技术和生物物理方法表征蛋白质复合物，包括凝胶过滤色谱法、分析超离心和荧光各向异性。公共卫生相关性铜绿假单胞菌是一种机会性人类病原体，可在几种不同的患者人群中引起危及生命的感染。细菌的毒力和它们在宿主中感染的持续性的主要促成因素是胞外多糖藻酸盐。导致形成成熟藻酸盐的步骤的生物化学表征可以提供关于如何阻断或改善感染影响的见解。

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.