Functional similarity of PRD-containing virulence regulators in B. anthracis

描述(由申请人提供)：本提案中将要进行的研究将定义炭疽芽孢杆菌中胶囊表达的分子机制。宿主体内产生的聚D-谷氨酸胶囊使细菌能够逃避免疫系统的检测，从而促进了炭疽病的发展。促进胶囊表达的环境和培养条件已经确定，但导致胶囊生物合成操纵子表达的机制尚不清楚。衣壳生物合成酶由质粒pXO2上的操纵子capBCADE编码。操纵子的转录是在大气中高浓度的二氧化碳和含有碳酸氢盐的介质中生长过程中诱导的。胶囊生物合成操纵子的两侧有两个基因，ACPA和AcPB，它们编码正向调节操纵子转录的蛋白质。CapBCADE的转录通读导致AcPb的表达，从而产生一个正反馈环。AtxA是炭疽杆菌的主要毒力调节因子，通过控制ACPA正向调控被膜基因的转录。当在高拷贝或在增强atxA活性的生长条件下表达时，atxa可以在没有ACPA和AcPb的情况下控制胶囊操纵子的表达。ACPA、AcPb和Atxa对衣壳操纵子转录的活性表明这些蛋白质在功能上是相似的。除了影响衣壳生物合成操纵子的表达外，AtxA还具有广泛的调控基因，包括毒力质粒和染色体上的基因。ACPA和AcPb的调控基因没有那么明确，但被认为只包含几个基因。这三个调节器在其氨基末端具有相似的假定DNA结合域，但其靶标特异性的基础尚不清楚。结构-功能研究已经确定 Atxa的活性受到二聚化和磷酸化的影响。除了表型研究旨在评估在ACPA-和Ac铅缺失突变体中的被膜表达外，关于ACPA和AcPb活性的机制尚不清楚。这项研究将(1)确定与靶基因特异性相关的AtxA、ACPA和AcPb结构域；(2)评估ACPA和AcPb的多聚化状态和磷酸化位点及其对调控活性的影响。这项研究旨在确定三种蛋白质在炭疽杆菌中负责表达荚膜合成基因的不同角色。确定ATXA、ACPA和AcPb活性所需的离散功能结构域和蛋白质-蛋白质相互作用的作用将突出抑制包膜表达和炭疽发病机制的新靶点。

DESCRIPTION (provided by applicant): The research to be performed in this proposal will define a molecular mechanism for capsule expression in Bacillus anthracis. Production of the poly-D-glutamic acid capsule within the host allows the bacterium to evade detection by the immune system facilitating anthrax disease progression. Environmental and culture conditions that promote capsule expression have been defined, however the mechanism leading to expression of the capsule biosynthetic operon are not well understood. The capsule biosynthetic enzymes are encoded by an operon, capBCADE, on plasmid pXO2. Transcription of the operon is induced during growth in elevated atmospheric CO2 and medium containing bicarbonate. The capsule biosynthetic operon is flanked by two genes, acpA and acpB, that encode proteins that positively regulate transcription of the operon. Transcriptional read-through of capBCADE results in expression of acpB, resulting in a positive feedback loop. AtxA, the master virulence regulator in B. anthracis, positively regulates capsule gene transcription via control of acpA. When expressed in high copy or in growth conditions that enhance AtxA activity, AtxA can control capsule operon expression in the absence of acpA and acpB. The activity of AcpA, AcpB, and AtxA on transcription of the capsule operon demonstrates functional similarity between these proteins. In addition to influencing expression of the capsule biosynthetic operon, AtxA has a broad regulon consisting of genes on both virulence plasmids and the chromosome. The regulons of AcpA and AcpB are less well-defined, but are thought to contain only a few genes. The three regulators have similar putative DNA-binding domains at their amino termini, but the basis for their target specificities is unknown. Structure-function studies have identified that AtxA activity is influenced by dimerization and phosphorylation. Aside from phenotypic studies designed to assess capsule expression in acpA- and acpB-null mutants, nothing is known regarding the mechanism of AcpA and AcpB activity. The research proposed here will (1) determine AtxA, AcpA, and AcpB domains associated with target gene specificity and (2) assess multimerization states and phosphorylation sites of AcpA and AcpB and the effects on regulator activity. This study is designed to define discrete roles for three proteins responsible for expression of capsule synthesis genes in B. anthracis. Identifying the role of discrete functional domains and protein-protein interactions required for activity of AtxA, AcpA, and AcpB will highlight novel targets for the inhibition of capsule expression and anthrax pathogenesis.