Molecular mechanisms of adaptive diversity in Burkholderia biofilms

描述（由申请人提供）：生物膜每年与数十万例感染有关，并大大增加了治疗成本和死亡率。这些不利影响不仅是由于生活在生物膜基质中的细菌的抗性增加，而且可能是由于突变体的适应性进化成不同的，顽固的形式。为了研究生物膜中的这一进化过程，我们设计了一种方法，可以长期选择机会性病原体新绿伯克氏菌的种群，使其经历附着、生物膜组装和扩散的循环。选择反复倾向于在慢性感染期间通常发生突变的位点突变，这表明生物膜适应概括了感染期间进化的各个方面。本研究的重点是两个基因位点，它们协调分泌信号的感知（BDSF）和控制生物膜产生的开关的内部调节（cyclic-di-GMP）。不同蛋白质结构域的突变导致了不同的生态策略，不同的突变体共同产生了更强健的生物膜。本项目的总体目标是精确定义突变体在不同生物膜环境中具有不同的信号、粘附和扩散策略的选择优势。为了解决这个复杂的问题，我们组建了一个多学科团队，他们在分子遗传学和进化生物学、生物化学和质谱学以及生物物理结构-功能分析方面具有专业知识。我们的目标是1)量化在感知BDSF和产生循环二gmp方面变化的突变体如何在生物膜群落中适应；2)确定BDSF如何在选择中机制控制主要位点的活性，以及适应性突变对这一过程的影响；3)建立一个生态模型，说明这些过程如何在混合突变体和混合物种群落中运作。我们希望了解如何操纵该系统来诱导生物膜的扩散并增加其敏感性，这可能与开发新型抗菌剂广泛相关。

DESCRIPTION (provided by applicant): Biofilms are associated with hundreds of thousands of infections each year and greatly increase treatment costs and mortality. These adverse effects are caused not only by increased resistance of bacteria living in the biofilm matrix but likely also by adaptive evolution of mutants into different, recalcitrant forms. To study this evolutionary process in biofilms we devised a method enabling long-term selection of populations of the opportunistic pathogen Burkholderia cenocepacia that undergo a cycle of attachment, biofilm assembly, and dispersal. Selection repeatedly favored mutations in loci commonly mutated during chronic infections, suggesting that biofilm adaptation recapitulates aspects of evolution during infections. This proposal focuses on two genetic loci that coordinate the sensing of a secreted signal (BDSF) with internal regulation of a switch governing biofilm production (cyclic-di-GMP). Mutations in different protein domains led to different ecological strategies and together different mutants produced a more robust biofilm. The overarching goal of this project is to precisely define the selective advantages of mutants with different strategie of signaling, adhesion, and dispersal in diverse biofilm environments. To tackle this complex problem we have assembled a multidisciplinary team with expertise in molecular genetics and evolutionary biology, biochemistry and mass spectrometry, and biophysical structure-function analysis. Our objectives are to 1) quantify how mutants that vary in sensing BDSF and producing cyclic-di-GMP are adaptive in biofilm communities, 2) determine how BDSF mechanistically controls the activity of the primary locus under selection, and the effects of adaptive mutations on this process, and 3) to develop an ecological model of how these processes operate in mixed-mutant and mixed-species communities. We expect to learn how this system may be manipulated to induce dispersal from biofilms and increase their susceptibility, which could be broadly relevant for developing novel antimicrobials.