Under-oil open microfluidic system (UOMS) for studying systemic fungal infection

项目概要/摘要 寄生真菌病原体是医疗保健相关血流感染的主要原因。念珠菌 具体地说，酵母引起80-90%的生物膜相关的侵袭性真菌感染， 接近50%。此外，念珠菌感染的发病率随着导管使用的增加而上升， 其他基于器械的干预措施。到目前为止，大多数工作与真菌感染和潜在的 治疗集中在生物膜及其预防上。然而，最近的证据表明， 酵母细胞从生物膜（进入血液）和持久性，这些分散的细胞是 可能是更重要的毒力因子，代表重要但未充分利用的治疗靶点。 此外，现有的测定和仪器不适合测量分散和表型 分散的细胞它们也不能概括体内条件（例如流动、与宿主细胞的相互作用）。因此，在这里，我们 将开发一种新型的油下开放微流控系统（UOMS），以量化分散能力， 生物膜，并评估念珠菌突变株和临床分离株中分散细胞的表型。UOMS 该平台是建立在一个新观察到的现象称为排他性液体排斥（ELR）的基础上。 ELR提供了一个独特的环境，其中液体完全从固体表面排斥，以消除 生物污染此外，ELR扩展了简单开放微流体设备的功能，使我们能够 克服了现有方法的局限性，并提供了一种能够定量研究真菌的系统， 分散体我们将首先（目标1）开发一种油下微通道装置来测量分散能力， 念珠菌生物膜和毒力表型。第二（目标2），我们将使UOMS自动化，并开发一个单一的- 细胞分布测定以测量单个分散细胞的表型。最后（目标3），我们将使用 UOMS平台开发念珠菌突变库和临床分离株的分散表型谱。我们 将测量来自数百株临床白念珠菌的分散能力和分散细胞的表型 分离株和突变体在现有的图书馆，提供线索的遗传决定因素的分散。

Project Summary/Abstract Opportunistic fungal pathogens are a leading cause of healthcare associated bloodstream infections. Candida yeasts, specifically, cause 80-90% of biofilm-associated invasive fungal infections and mortality rates can approach 50%. Furthermore, the incidence of Candida infections is rising with the increased use of catheter and other device-based interventions. To date, the majority of work related to fungal infections and potential treatments has focused on biofilms and their prevention. However, recent evidence suggests that the dispersion of yeast cells from the biofilm (into the bloodstream) and the persistence of these dispersed cells are perhaps more important virulence factors and represent significant but underutilized treatment targets. Further, existing assays and instrumentation are not amenable to measuring dispersion and phenotyping dispersed cells. Nor do they recapitulate in vivo conditions (e.g. flow, interaction with host cells). Thus, here we will develop a new type of under-oil open microfluidic system (UOMS) to quantify the dispersive capacity of biofilms and assess the phenotype of dispersed cells in Candida mutants and clinical isolates. The UOMS platform is built on the foundation of a newly observed phenomena called Exclusive Liquid Repellency (ELR). ELR provides a unique environment where liquid is completely repelled from a solid surface to eliminate biofouling. Additionally, ELR expands the capabilities of simple open microfluidic devices allowing us to overcome the limitations of current methods and provide a system capable of quantitatively studying fungal dispersion. We will first (Aim 1) develop an under-oil microchannel device to measure the dispersive capacity of Candida biofilms and virulence phenotypes. Second (Aim 2) we will automate the UOMS and develop a single- cell distribution assay to measure the phenotype of individual dispersed cells. And finally (Aim 3), we will use the UOMS platform to develop dispersion phenotype profiles for Candida mutant libraries and clinical isolates. We will measure the dispersive capacity and phenotype of dispersed cells from hundreds of Candida albicans clinical isolates and mutants available in existing libraries, providing clues to the genetic determinants of dispersion.