Char of Phospholipases C & the Twin Arginine Secretory System of B. pseudomallei

前言细胞内铁浓度的动态控制对所有生物系统来说都是至关重要的。 这个问题的一个方面是，对生物有用的铁(即Fe2“)非常有限，或者它是高度不溶的 (即Fe3)。因此，生物实体已经进化出有效的机制来从 不溶的形式，通常是大量的。另一方面，进一步收购铁以上 生物学上有用的浓度可能会对细胞产生可怕的后果。过多的游离铁将催化 产生高活性的氧和氮中间体，这将损害所有已知的生物 大分子。这种冲突在很大程度上是通过一系列不同的致病微生物来处理的，通过一种 经典的Fe2+依赖的抑制因子，称为Fur(铁摄取调节剂)，在控制铁的过程中起关键作用 动态平衡。虽然很明显，B类选择因子伯克霍尔德氏菌和马利氏杆菌表现出 与近缘生物(如铜绿假单胞菌)的毛皮高度同源的蛋白质 关于铁稳态在8.假鼻疽和马利氏杆菌毒力中的作用，人们知之甚少。更多 重要的是，目前还不知道铁稳态在这些细胞内的生活方式中所起的作用。 病原体。我们建议通过使用强大的分子和基因来补救这种情况 我们使用的方法来阐明毛皮在相关的 独特的机会致病菌铜绿假单胞菌。我们将鉴定毛皮调节基因6.假鼻孔菌和 编码调节因子(例如，Sigma因子、小调节RNA)的Mallei。然后我们将评估 这些选定的监管因素在这些代理人进入、生存、生长或贩运的能力中的作用 模型真核细胞(如巨噬细胞)，适用于发病机制的已知方面 这些潜在的生物恐怖分子。我们鉴定的基因产品最终可能会被用于 开发治疗和预防马蹄内翻足和扁桃体的药物。 除了与其他项目成员的明显互动之外的项目互动(例如，巴斯克斯博士- Torres、Holmes和Voskuil)，在本申请的正文和 项目II C简介，我们将特别与Robison博士(杨百翰大学选择代理档案馆)和Dr Robison互动。 Schweizer(微生物遗传学-CSU)在假鼻疽杆菌和马来杆菌的细菌学和遗传学方面。 我们还将对与Slayden博士(基因组学/蛋白质组学)的非常活跃的互动特别感兴趣 核心)，这将是至关重要的微阵列的发展和评估潜在的目标基因和 可能用于实验性预防和治疗药物的产品。此外，沿着这些思路，我们将 出于同样的目的，还与Brennan博士(Pdm Core)密切互动。

Introduction The dynamic control of intracellular iron concentrations is paramount to all biological systems. One aspect of this issue is that biologically useful iron (i.e. Fe2") is extremely limiting or it is highly insoluble (i.e.Fe3). Accordingly, biological entities have evolved efficient mechanisms to acquire this nutrient from the insoluble form, which is generally in plentiful quantities. On the other hand, further acquisition of iron above biologically useful concentrations can have dire consequences for a cell. Excess free iron will catalyze the generation of highly reactive oxygen and nitrogen intermediates that will damage all known biological macro-molecules. This conflict, in a major way is dealt with in a diverse array of pathogenic microbes, by a classic Fe2+-dependent represser called Fur (ferric uptake regulator) that plays the key role in controlling iron homeostasis. While it is clear that the Class B Select Agents Burkholderia pseudomallei & mallei express a protein that is highly homologous to Fur from closely related organisms (e.g. Pseudomonas aeruginosa) very little is known about the role of iron homeostasis in the virulence of 8. pseudomallei & mallei. More importantly, nothing is known about the role of iron homeostasis in the intracellular life style of these pathogens. We propose to remedy this situation through the use of powerful molecular and genetic approaches that we employed to elucidate the extensive and crucial role that Fur plays in the related, but distinct, opportunistic pathogen P. aeruginosa. We will identify the Fur regulated genes of 6. pseudomallei & mallei that encode regulatory factors (e.g. sigma factors, small regulatory RNAs). We will then evaluate the role of these selected regulatory factors in the ability of these agents to enter, survive in, grow in or traffick in model eukaryotic cells (e.g. macrophage), which are appropriate to the known aspects of the pathogenesis of these potential bioterrorist agents. The gene products that we identify may ultimately be used in developing therapeutic and prophylactic agents against Malleipdosis and Glanders. Project interactions In addition to the obvious interactions with other project members (e.g. Drs. Vazquez- Torres, Holmes, & Voskuil) that are described in more detail in the body of this application and in the Introduction to Project II.C, we will especially interact with Dr. Robison (BYU Select Agent Archive) and Dr. Schweizer (Microbial genetics - CSU) in terms of the bacteriology and genetics of B. pseudomallei & mallei. We will also be especially interested in very active interactions with Dr. Slayden (Genomics/Proteomics Core) that will be vital to development of microarrays and for the evaluation of potential target genes and products for possible use in experimental prophylactic and therapeutic agents. Also, along these lines we will also closely interact with Dr. Brennan (PDM Core) for the same purpose.