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感染有关，并极大地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抵抗力的增加，而且可能是由于突变体适应进化成不同的、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，能够长期选择机会致病菌Burkholderia cenocepacia的种群，这些种群经历了附着、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突变的基因座的突变，这表明生物膜适应概括了感染期间进化的各个方面。这一建议侧重于两个基因位点，它们协调分泌信号(BDSF)的感知和控制生物膜产生的开关(环二GMP)的内部调节。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大的生物膜。该项目的首要目标是准确地确定在不同的生物膜环境中具有不同信号、黏附和扩散策略的突变体的选择优势。为了解决这个复杂的问题，我们组建了一个多学科团队，拥有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的专业知识。我们的目标是1)量化在感知BDSF和产生环-diGMP方面发生变化的突变体如何适应生物被膜群落，2)确定BDSF如何机械地控制被选择的主要基因座的活性，以及适应性突变对这一过程的影响，以及3)建立一个生态模型，说明这些过程如何在混合突变和混合物种群落中运行。我们希望了解如何操纵这个系统来诱导生物膜的扩散并增加它们的敏感性，这可能对开发新的抗菌剂具有广泛的相关性。

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.