Molecular mechanisms of adaptive diversity in Burkholderia biofilms

描述（由申请人提供）：生物膜与每年数十万例感染有关，并大大增加了治疗成本和死亡率。这些不利影响不仅是由生活在生物膜基质中的细菌的抗性增加引起的，而且还可能是由突变体适应性进化成不同的、不稳定的形式引起的。为了研究生物膜中的这种进化过程，我们设计了一种方法，该方法能够长期选择经历附着，生物膜组装和分散循环的机会致病菌新洋葱伯克霍尔德氏菌的种群。在慢性感染过程中通常发生突变的基因座中，选择反复倾向于突变，这表明生物膜适应重现了感染过程中的进化过程。该建议集中在两个遗传基因座，其协调分泌信号（BDSF）的感测与控制生物膜产生的开关（cyclic-di-GMP）的内部调节。不同蛋白质结构域的突变导致不同的生态策略，不同的突变体一起产生了更强大的生物膜。该项目的总体目标是精确定义在不同生物膜环境中具有不同信号传导，粘附和分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，他们拥有分子遗传学和进化生物学，生物化学和质谱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）量化突变体在生物膜群落中的适应性，2）确定BDSF如何机械地控制选择下的主要位点的活性，以及适应性突变对该过程的影响，以及3）开发这些过程如何在混合突变体和混合物种群落中运作的生态模型。我们希望了解如何操纵该系统以诱导生物膜的分散并增加其敏感性，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.