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

描述（由申请人提供）：本课题拟进行的研究将确定炭疽芽孢杆菌胶囊表达的分子机制。宿主体内聚d -谷氨酸胶囊的产生使细菌能够逃避免疫系统的检测，从而促进炭疽疾病的进展。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。胶囊生物合成酶由pXO2质粒上的操纵子capBCADE编码。操纵子的转录在升高的大气CO2和含有碳酸氢盐的培养基中被诱导。胶囊生物合成操纵子两侧有两个基因，acpA和acpB，它们编码积极调节操纵子转录的蛋白质。capBCADE的转录通读导致acpB的表达，形成一个正反馈循环。AtxA是炭疽芽孢杆菌的主要毒力调节因子，通过控制acpA正调控胶囊基因转录。当AtxA在高拷贝或AtxA活性增强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对胶囊操纵子转录的活性证明了这些蛋白在功能上的相似性。除了影响胶囊生物合成操纵子的表达外，AtxA在毒力质粒和染色体上都有一个广泛的由基因组成的调控子。AcpA和AcpB的调控规则不太明确，但被认为只包含少数基因。这三种调节因子在其氨基末端具有相似的假定dna结合域，但其靶特异性的基础尚不清楚。结构功能研究已经确定

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.