Extracellular Proteins in Virulence of B. (pseudo)mallei

描述（由申请方提供）：类鼻疽伯克霍尔德氏菌（Bp）及其小型缺失克隆鼻疽伯克霍尔德氏菌（Bm）可引起致命的人类疾病。它们固有的抗生素耐药性、通过气溶胶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威胁和选择性病原体。虽然这些细菌的自然感染报告可以追溯到100多年前，每年仍有数十例新病例出现，但对它们的毒力和致病机制知之甚少。然而，最近获得的多株Bp、Bm及其非致病性亲属的全基因组序列，以及新的鸟枪蛋白质组学技术的发展，为快速鉴定这些和其他病原体的数十种新型细胞外毒力因子开辟了新的途径。我们建议使用原位离子阱串联质谱方法来快速鉴定编码Bm和Bp的> 100个分泌和细胞表面暴露蛋白的基因。接下来，为了找到这些基因中参与疾病的可能性最高的15 - 20个基因，我们将针对以下基因搜索这些基因：1）我们的微阵列（表达谱）数据库，其包含在小鼠脾脏和肝脏中显示改变表达的Bm基因;和2）针对计算机生成的由Bp和Bm共享的390个基因的数据库，但从5个密切相关但非致病性的伯克霍尔德氏菌中缺失。这样选择的基因将位点特异性失活，并在我们的大蜡螟（蜡螟幼虫）替代疾病模型中评估所得突变体的毒力，其中Bm的行为和它的一些毒力突变体与在感染的仓鼠中观察到的行为密切相关。我们还将评估这些突变体在人类和小鼠巨噬细胞和肺上皮细胞中侵入和存活的能力。然后在叙利亚仓鼠动物模型中检测在一种以上试验中显示毒力显著降低的突变体。这些实验将发现并部分定义Bm和Bp的新分泌蛋白和细胞表面蛋白，这些蛋白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导致对微生物发病机理的新范式和见解，而且可能导致对Bm和Bp以及其他重要病原体和生物威胁剂的新诊断、疫苗和治疗。

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.