Extracellular Proteins in Virulence of B. (pseudo)mallei

描述(申请人提供)：假鼻疽伯克霍尔德氏菌(BP)及其缺失克隆伯克霍尔德氏菌(BM)可引起致命的人类疾病。它们固有的抗生素耐药性、通过气雾剂感染的能力以及缺乏保护性疫苗，导致它们被归类为主要的生物制剂和选择性制剂。尽管关于这些细菌自然感染的报道可以追溯到100多年前，每年仍有数十例新病例出现，但人们对它们的毒力和致病机制知之甚少。然而，最近获得了多株BP、BM及其非致病亲缘株的完整基因组序列，以及新的鸟枪式蛋白质组学技术的发展，为快速鉴定这些病原体和其他病原体的数十种新的胞外毒力因子开辟了新的途径。我们建议使用原位离子陷阱串联质谱仪方法来快速识别编码&gt；100分泌和细胞表面暴露蛋白的BM和BP基因。接下来，为了找到这些基因中最有可能参与疾病的15-20个，我们将搜索这些基因：1)我们的微阵列(表达谱)数据库，包含BM基因在小鼠脾和肝脏中的表达变化；以及2)电子产生的数据库，包含390个基因，BP和BM共享，但从5个密切相关但非致病性的伯克霍尔德氏菌中缺失。这样选择的基因将被定点灭活，并在我们的Galleria mellonella(蜡蛾幼虫)代理疾病模型中对产生的突变进行毒力评估，在该模型中，BM及其一些毒力突变的行为与在受感染的仓鼠中观察到的行为很好地相关。我们还将评估这些突变体入侵和存活于人、鼠巨噬细胞和肺上皮细胞的能力。在不止一种检测中显示出毒力显著降低的突变株将在叙利亚仓鼠动物模型中进行测试。这些实验将发现并部分定义BM和BP的新的分泌和细胞表面蛋白，它们在疾病期间以不同的方式与动物宿主相互作用。所获得的结果不仅可能为微生物发病机制带来新的范式和见解，而且还可能为BM和BP以及其他重要病原体和生物制剂提供新的诊断、疫苗和治疗方法。

DESCRIPTION (provided by applicant): Burkholderia pseudomallei (Bp), and its diminutive deletion clone Burkholderia mallei (Bm) cause fatal human diseases. Their inherent antibiotic resistance, ability to infect via aerosol, and lack of protective vaccines has led to their classification as major biothreats and select agents. Although reports of natural infections by these bacteria date back over 100 years, and dozens of new cases still arise each year, relatively little is known about their virulence and pathogenicity mechanisms. However, the recent availability of complete genome sequences for multiple strains of Bp, Bm, and their nonpathogenic relatives, as well as the development of new shotgun proteomic technologies, has opened up new avenues to quickly identify dozens of novel extracellular virulence factors of these and other pathogens. We propose to use in-place ion-trap tandem mass spectrometry methods to rapidly identify the genes encoding >100 secreted and cell surface-exposed proteins of Bm and Bp. Next, to find the 15-20 of these genes with the highest probability of involvement in disease, we will search these genes against: 1) our microarray (expression profiling) databases containing Bm genes showing altered expression in mouse spleen and liver; and 2) against an in silico-generated database of 390 genes shared by Bp and Bm, but missing from 5 closely-related, but nonpathogenic Burkholderia. Genes thus selected will be site-specifically inactivated and the resultant mutants assessed for virulence in our Galleria mellonella (wax moth larvae) surrogate disease model, in which behavior of Bm and some of its virulence mutants correlates well with what is observed in infected hamsters. We will also assess the ability of these mutants to invade and survive in human and mouse macrophages and lung epithelial cells. Mutants showing significantly reduced virulence in more than one of these assays will then be tested in a Syrian hamster animal model. These experiments will discover and partially define new secreted and cell surface proteins of Bm and Bp that interact in diverse ways with animal hosts during disease. The results obtained may not only lead to new paradigms and insights into microbial pathogenesis, but also to new diagnostics, vaccines and therapeutics for Bm and Bp and other important pathogens and biothreat agents.