Dispersion and the Biofilm Matrix of Pseudomonas aeruginosa

项目摘要 抗生素耐药性慢性感染是当今社会面临的最大挑战之一。这 抗生素耐药性问题如此严重，以至于世界卫生组织将其列为十大问题之一 2019年全球健康威胁这些感染中有80%以上是由于 细菌形成生物膜的能力，包裹在保护性基质中的微生物群落， 细胞外聚合物，对抗菌剂无反应。铜绿假单胞菌是第2位 这些感染的主要原因，通常是医院感染患者死亡的最常见原因。 感染.分散被认为是生物膜控制的一个有前途的途径，通过帮助解决生物膜- 相关感染。这是因为分散是一个主动的、受调节的过程，细菌通过这个过程逃逸 生物膜作为自由生活的细胞，再次对抗菌剂敏感，因此分散体能够 增强抗生素的功效。然而，虽然铜绿假单胞菌的分散提示感测以及这些 提示被翻译成c-di-GMP库的调节已经被详细阐明，但知之甚少 关于使铜绿假单胞菌从生物膜中排出的下游事件，c-di-GMP的贡献，或 通过分散的细胞驱动毒力转换的因素。使用RNA-seq，我们已经确定了独特的基因， 分散的细胞（称为“分散调节子”），并有助于分散细胞的表型 包括药物敏感性、毒性和基质降解。我们确定了基质中的相关因素 降解和强化生物膜基质以有助于分散。然而，没有明显的矩阵 到目前为止，已经鉴定了靶向基质EPS Psl的降解酶，这表明了 基质降解和分散的不同类别的基质生产铜绿假单胞菌菌株。而且我们 研究结果表明，c-di-GMP的调节不足以诱导分散， 分散细胞的表型。基于这些前提，我们假设色散和色散- 相关的表型需要c-di-GMP-（in）依赖性途径，包括参与 基质降解可能在基质利用的类别之间不同。该项目的目标是 阐明导致分散的信号传导和调节事件，包括基质降解和分散 采用急性毒性表型的细胞。因此，我们将在目标1中阐明导致分散的途径 和分散相关的表型在体外和体内，并时空评估的命运基质 在目标2中分散的组分。目标3的目的是确定是否分散和所需的因素 在代表4种不同基质类别的非粘液样临床分离株中，分散度相似或不同。 我们希望我们的发现不仅有助于理解感染，而且有助于指导治疗。 分散体朝着生物膜控制的连续治疗发展。

PROJECT SUMMARY Antimicrobial resistant, chronic infections are one of the greatest challenges facing our society today. This problem is so significant that the World Health Organization named antibiotic resistance as one of the top ten threats to global health in 2019. More than 80% of these infections have been attributed to the remarkable capabilities of bacteria to form biofilms, communities of microorganisms encased in a protective matrix of extracellular polymeric substance, that are irresponsive to antimicrobials. Pseudomonas aeruginosa is the 2nd leading cause of these infections, and routinely the most common cause of fatalities in patients with nosocomial infections. Dispersion is considered a promising avenue open for biofilm control, by assisting in resolving biofilm- associated infections. This is because dispersion is an active, regulated process by which bacteria escape biofilms as free-living cells that are once again susceptible to antimicrobials, with dispersion thus capable of enhancing the efficacy of antibiotics. However, while dispersion cue sensing by P. aeruginosa and how these cues are translated into the modulation of the c-di-GMP pool has been elucidated in great detail, little is known about downstream events enabling P. aeruginosa to egress from the biofilm, the contribution of c-di-GMP, or factors driving the virulence switch by dispersed cells. Using RNA-seq, we have identified genes that are unique to dispersed cells (termed “dispersion regulon”) and contribute to the phenotype ascribed to dispersed cells including drug susceptibility, virulence, and matrix degradation. We confirmed factors involved in matrix degradation and the reinforcement of the biofilm matrix to contribute to dispersion. However, no obvious matrix degrading enzymes have been thus far identified which target the matrix EPS Psl, suggesting differences in matrix degradation and dispersion by different classes of matrix-producing P. aeruginosa strains. Moreover, our findings suggest that the modulation of c-di-GMP is not sufficient to induce dispersion and only explains some of the phenotypes of dispersed cells. Based on these premises, we hypothesize that dispersion and dispersion- related phenotypes require c-di-GMP-(in)dependent pathways including genes and gene functions involved in matrix degradation that likely differ amongst the classes of matrix utilization. The goal of this project is to elucidate the signaling and regulatory events leading to dispersion including matrix-degradation, and dispersed cells adopting an acute virulence phenotype. We will therefore elucidate in Aim 1 pathways leading to dispersion and dispersion-related phenotypes in vitro and in vivo, and spatio-temporally assess the fate of matrix components during dispersion in Aim 2. Aim 3 aims at determining whether the dispersion and factors required for dispersion are similar or are different in non-mucoid clinical isolates representing 4 different matrix classes. We anticipate our findings to not only contribute to understanding infection but also assist in guiding the development of dispersion towards an adjunctive therapy for biofilm control.