Virulence Determinants for Host Tropism in the Burkholderia cepacia complex

描述（由申请人提供）：洋葱伯克霍尔德菌复合体（Bcc）是一组革兰氏阴性菌，在环境中普遍存在，并在免疫功能低下患者中作为机会致病菌出现。这些细菌在两种看似不同的宿主中引起重大疾病：囊性纤维化（CF）和慢性肉芽肿病（CGD）。我们假设，在这两种缩短寿命的遗传疾病中，Bcc具有独特和共同的特征，使其成为机会性病原体，其中感染是导致死亡的主要原因。令人惊讶的是，目前还没有一项全面的研究来确定这些感染中常见或独特的细菌因素。在这里，我们建议使用两种不同的小鼠感染模型来鉴定CF和CGD中重要的基因。我们已经使用独特的mariner转座子系统构建了一个转座子突变文库，其中包含约60,000个随机突变，这些突变来自于已测序和注释的新洋葱伯克霍尔德菌J2315菌株。重要的是，该菌株已经在CF和CGD小鼠感染模型中显示出毒性。我们将使用高通量测序来鉴定CF和CGD小鼠模型系统感染后该文库中缺失的转座子突变体。这种负选择使我们能够集中突变体并使用相对较少的小鼠，而不是使用大量的小鼠来筛选单个Bcc突变体。在确定每种感染所需的因素之后，我们将重点关注在CF和CGD中重要的那些基因。我们预计，粘附所需的基因和体内代谢活动所需的基因将被认为是这两种感染的关键。我们将在这些途径中构建特定的基因突变，并确认这些因素在小鼠感染模型中的作用。我们还将在体外和离体毒力模型中确定这些突变的影响。彻底了解这些过程将有助于确定Bcc的潜在靶点，从而开发新的抗菌剂、抑制剂和免疫治疗干预措施，以保护这些脆弱的患者群体。

DESCRIPTION (provided by applicant): The Burkholderia cepacia complex (Bcc) is a group of Gram-negative bacteria that are ubiquitous in the environment and have emerged as opportunistic pathogens in immunocompromised patients. These bacteria cause significant disease in two seemingly disparate hosts: those with cystic fibrosis (CF) and those with chronic granulomatous disease (CGD). We hypothesize that there are unique and common features of Bcc that make it an opportunistic pathogen in these two life-shortening inherited diseases, in which infection is the primary cause of mortality. Surprisingly, there has not been a comprehensive study to identify the bacterial factors that are common or unique in these infections. Here we propose to identify genes important in both CF and CGD using two distinct mouse models of infection. We have already constructed a transposon mutant library encompassing approximately 60,000 random mutants of the sequenced and annotated Burkholderia cenocepacia strain J2315 using a unique mariner transposon system. Importantly, this strain has already been shown to be virulent in a CF and a CGD mouse model of infection. We will use high-throughput sequencing to identify transposon mutants missing from this library following infection in both a CF and a CGD mouse model system. This negative selection allows us to pool the mutants and use relatively few mice, rather than using large numbers of mice that would be required to screen individual Bcc mutants. Following the identification of factors required for each of these infections, we will focus on those genes important in both CF and CGD. We anticipate that genes needed for adherence and those necessary for metabolic activities in vivo will be recognized as critical for both these infections. We will construct specfic mutations in genes in these pathways and will confirm the role of these factors in the mouse models of infection. We will also determine the effect of these mutations in in vitro and ex vivo models of virulence. A thorough understanding of these processes will allow the identification of potential targets in Bcc for the development of novel antimicrobials, inhibitors, and immunotherapeutic intervention to protect these vulnerable patient populations.