Bacillus anthracis Spores: Initiation of Germination

描述(由申请人提供)：在哺乳动物宿主中开始炭疽的一个重要的早期步骤是被巨噬细胞吞噬的炭疽芽胞的萌发。芽孢杆菌孢子萌发早期阶段的生物化学尚不清楚。目前炭疽感染周期中的治疗干预措施针对的是更晚的步骤。拟议的研究将把枯草芽孢杆菌的三蛋白GERB萌发受体的初步发现扩展到炭疽杆菌的GerX和Gerl同源物。GerX是由一个毒力质粒编码的，是完全毒力所必需的，而Gerl是染色体编码的受体之一，也可能有助于巨噬细胞的萌发。像GERB一样，GerX和Gerl是Gera型萌发受体的同源成员，芽胞对特定的营养发芽物质的反应是芽胞萌发所必需的。将寻求这些重要复合体的机制。初步证据表明，GERB是一种受营养发芽菌调控的离子通道。这一应用的假设是：(I)其他Gera型受体也是钾或钠通道；以及(Ii)当由萌发体触发时，通过受体通道的离子流动激活下游离子转运体和其他与萌发相关的蛋白质，从而导致一连串的萌发事件。具体目的1是进一步研究在初步研究中使用的异源系统(大肠杆菌膜囊泡和人胚胎肾细胞)中GERB通道的生物物理、抑制和调节特性。这三种GERB蛋白的作用将进一步确定。具体目标2是应用相同的方法来定义GerX和Gerl离子通道的属性、发芽物质、抑制物和对吞噬体体环境信号的响应。这些复合体中显示通道活性的单一蛋白质成分也将被表征。具体目标3是确定GerX和Gerl最近端的“下游”转运蛋白(S)的候选者，并表征它们的活性和特定的萌发受体激活特定的下游转运蛋白的基础。这种转运蛋白可能是新的毒力因子，即额外的潜在治疗靶点。

DESCRIPTION (provided by applicant): An essential early step in the initiation of anthrax in the mammalian host is the germination of the Bacillus anthracis (endo)spores that have been engulfed by macrophages. The biochemistry of early steps in Bacillus spore germination is not yet understood. Current therapeutic interventions in the anthrax infectious cycle target much later steps. The proposed studies will extend promising preliminary findings on the three-protein GerB germinant receptor of Bacillus subtilis to the GerX and Gerl homologues of B. anthracis. GerX is encoded by a virulence plasmid and is required for full virulence, and Gerl is one of the chromosomally encoded receptors that may also contribute to germination in the macrophage. Like GerB, GerX and Gerl are homologous members of the GerA-type germinant receptors required for germination of Bacillus spores in response to specific nutrient germinants. Mechanisms for these important complexes will be sought. Preliminary evidence indicates that the GerB is an ion channel that is regulated by its nutrient germinants. The hypothesis underlying the application is: (i) that other GerA-type receptors also are either potassium or sodium channels; and (ii) when triggered by a germinant, ion flux through the receptor channel activates downstream ion transporters and other germination-related proteins leading to a cascade of germination events. Specific Aim 1 is to further characterize the biophysical, inhibition and regulatory properties of the GerB channel in the heterologous systems (Escherichia coli membrane vesicles and human embryo kidney cells) used in preliminary studies. The roles of the three GerB proteins will be further defined. Specific Aim 2 is to apply the same approaches to define the GerX and Gerl ion channel properties, germinants, inhibitors, and response to signals of the phagolysosomal environment. Single protein components of these complexes that exhibit channel activity will also be characterized. Specific Aim 3 is to identify candidates for the most proximal, "downstream" transporter(s) of GerX and Gerl and to characterize their activities and the basis whereby specific germinant receptors may activate specific downstream transporters. Such transporters may be new virulence factors, i.e. additional potential therapeutic targets.