Rotation 1: Investigating the role(s) of the putative FANCJ helicase in the malaria parasite Plasmodium falciparum

囊性纤维化（CF）患者的呼吸道经常被各种各样的微生物“动物园”定植，特别是在他们的早期和青少年时期。历史证明，这些多微生物群落很难在实验室环境中重现，因此，到目前为止，大多数研究都涉及关键病原体的单一培养，如铜绿假单胞菌。然而，越来越清楚的是，病原体在多物种系统中的表现往往与在纯无菌培养物中生长时截然不同，因此，近年来人们对在体外重现复杂的CF微生物群的努力越来越感兴趣。Welch实验室开发了一种体外实验室模型，可以准确捕获CF气道中存在的多微生物群落的长期稳定性。简而言之，该装置由一个连续流动的生物反应器组成，其中液体（特别是人工痰介质）的置换率反映了气道中的情况。目前的模型包含三种主要的cf相关病原体：铜绿假单胞菌、金黄色葡萄球菌和白色念珠菌。该系统为探究cf相关的多微生物气道感染的生物学问题开辟了新的可能性，并且已经产生了一些有趣的新见解。在这个项目中，我将在体外模型系统中引入其他常见的cf相关病原体，以及目前的三种病原体，例如：流感嗜血杆菌、粘液罗氏菌和米勒链球菌。然后，我将使用这个“改进”的模型系统来检查铜绿假单胞菌突变体在多微生物群落中的存在的影响。在CF气道感染中观察到的铜绿假单胞菌群体不是均匀的，经常观察到突变变体。某些基因的功能丧失突变在CF中通常被过度代表，这表明它们可能赋予这种环境下更高的适应性；然而，我们仍然不知道为什么会这样。关键的“致病适应性”基因包括mutS（导致高易变性的突变）、lasR（影响毒力群体感应）、mucA（粘液样变性）、next（环丙沙星耐药性）和metF（蛋氨酸营养不良）。为了研究这些致病适应性突变对铜绿假单胞菌适应度的影响，我将生成每个靶基因的功能丧失突变体，然后研究这些突变体（或野生型和突变型的混合物）的引入如何影响多微生物培养的多样性和物种轨迹。这将进一步加深我们对铜绿假单胞菌进入CF气道如何影响现有微生物组的理解，以及是否一些突变体比其他突变体更成功地定殖了这个生态位。韦尔奇小组的初步数据表明，与野生型相比，lasR突变体确实显示出物种间相互作用的主要改变，我希望在其他病理适应性突变体中看到类似的有趣结果。我还将研究模型系统中病理适应性突变体的存在如何影响临床相关因素，如相关病原体对抗菌剂的反应。这个项目将帮助我们理解为什么一些突变体在CF中出现如此高的频率，这些突变体可能赋予铜绿假单胞菌的优势，以及它们对治疗干预的影响：因此它具有生物学和临床的兴趣和重要性。

Bioscience for an integrated understanding of healthThe airways of people with cystic fibrosis (CF) are often colonised by a diverse "zoo" of microbes, especially during their early and teenage years. These polymicrobial communities have historically proven difficult to recapitulate in the laboratory environment, so, until now, most research has involved monoculture of key pathogens such as Pseudomonas aeruginosa. It is becoming increasingly clear, however, that pathogens in multi-species systems often behave radically differently than when grown in pure axenic cultures, and, as such, recent years have seen a growing interest in efforts to recapitulate the complex CF microbiota in vitro. The Welch Lab has developed an in vitro laboratory model that accurately captures the long-term stability of the polymicrobial community present in CF airways. In brief, the setup consists of a continuous flow bioreactor in which the rate of fluid - specifically, artificial sputum media - replacement reflects that seen in the airways. The current model incorporates three major CF-associated pathogens: P. aeruginosa, Staphylococcus aureus, and Candida albicans. This system has opened new possibilities for interrogation of the biology of CF-associated polymicrobial airway infections, and has already yielded some intriguing new insights. In this project, I will introduce other common CF-associated pathogens into the in vitro model system, alongside the current three, for example: Haemophilus influenzae, Rothia mucilaginosa, and Streptococcus milleri. I will then use this "improved" model system to examine the impacts of the presence of P. aeruginosa mutants in the polymicrobial community. The P. aeruginosa population observed in CF airway infections is not homogenous, with mutant variants frequently observed. Loss-of-function mutations in certain genes are commonly over-represented in CF, indicating they may confer increased fitness in this environment; however, we still have very little idea as to why this is. Key "pathoadaptive" genes include mutS (mutations leading to hypermutability), lasR (affecting virulence quorum sensing), mucA (mucoidy), mexT (ciprofloxacin resistance), and metF (methionine auxotrophy). To investigate the impact of these pathoadaptive mutations on P. aeruginosa fitness, I will generate loss-of-function mutants for each target gene, then examine how introduction of these mutants (or mixtures of both wild type and mutant(s)) influences the diversity and species trajectory of the polymicrobial culture. This will further our understanding of how the arrival of P. aeruginosa into the CF airway influences the existing microbiome, and whether some mutants are more successful at colonising this niche than others. Preliminary data from the Welch Group indicate that lasR mutants do indeed show majorly altered inter-species interactions as compared to the wild type, and I hope to see similarly interesting results for other pathoadaptive mutants. I will also examine how the presence of pathoadaptive mutants in the model system impacts clinically relevant factors, such as response of the involved pathogens to antimicrobials. This project will help us understand why some mutants arise with such high frequency in CF, the advantages these mutants potentially confer on P. aeruginosa, and their impact on therapeutic interventions: it therefore has both biological and clinical interest and importance.