Protein Complexes in Alginate Maturation

描述（由申请人提供）：外多糖在确定致病菌的毒力，影响其对表面的粘附，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待了解。本申请描述了铜绿假单胞菌藻酸盐生物合成的最后步骤的研究。海藻酸盐是一种未支化的杂聚碳水化合物，在细菌周围形成一种胶囊。聚合物合成的前体在细胞质中产生，聚合到聚甘露聚糖是通过细胞质整体膜蛋白的作用发生的。我们描述的实验，以表征过程之间发生的聚合物形成和分泌。聚甘露聚糖在一些残基上通过质周酶的作用进行外聚化。我们已经证明，另一种蛋白质，AlgK，与外甲酶形成可分离的复合物，而这种复合物的形成需要第三种蛋白质AlgX。该复合物的目的可能是保护海藻酸盐不被海藻酸解酶降解，并促进海藻酸盐向外膜上的孔蛋白的传递。藻酸盐在周质中部分乙酰化。三种蛋白质已被证明是乙酰化所必需的，AlgI， AlgJ和AlgF。它们的作用仍然不明确，乙酰化过程是谜，因为没有乙酰辅酶a存在于周质中。我们建议表征与epimase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，并评估单独的蛋白质在乙酰化过程中的作用。动力学技术和生物物理方法表征蛋白质复合物，包括凝胶过滤层析，分析超离心和荧光各向异性，将被采用。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群体中引起危及生命的感染。细菌的毒力和它们在宿主体内持续感染的一个主要因素是藻酸盐的胞外多糖。对导致成熟海藻酸盐形成的步骤的生化表征可能为如何阻断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.