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）是一组革兰氏阴性细菌，在环境中普遍存在，并已成为免疫功能低下患者的机会病原体。这些细菌会在两个看似不同的宿主中引起严重的疾病：囊性纤维化（CF）患者和慢性肉芽肿病（CGD）患者。我们假设 Bcc 具有独特而共同的特征，使其成为这两种缩短寿命的遗传性疾病中的机会病原体，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识别 CF 和 CGD 中重要的基因。我们已经使用独特的水手转座子系统构建了一个转座子突变体库，其中包含已测序和注释的新洋葱伯克霍尔德菌 J2315 菌株的约 60,000 个随机突变体。重要的是，该菌株已被证明在 CF 和 CGD 小鼠感染模型中具有毒性。我们将使用高通量测序来识别 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汇集突变体并使用相对较少的小鼠，而不是使用筛选单个 Bcc 突变体所需的大量小鼠。在确定了每种感染所需的因素之后，我们将重点关注那些在 CF 和 CGD 中都很重要的基因。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对这两种感染至关重要。我们将在这些途径的基因中构建特定突变，并确认这些因素在小鼠感染模型中的作用。我们还将确定这些突变在体外和离体毒力模型中的影响。彻底了解这些过程将有助于识别 BCC 中的潜在靶点，用于开发新型抗菌药物、抑制剂和免疫治疗干预，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.