Isolation and physiological characterization of cell types from heterogeneous aggregates of Staphylococcus aureus using transcriptomic analysis

在这个SPP1617计划的第一阶段，我们展示了细菌病原体金黄色葡萄球菌的多细胞聚集体是由至少两种不同的细胞类型组成的异质群体，即生物膜生产者和毒素生产者。我们使用了一些生化技术来表征驱动这种细胞分化的分子机制。我们发现了一种复杂的遗传调控，由群体感应级联AGR的双峰行为触发，最终选择急性(较高浓度的毒素产生者)或生物膜相关的慢性感染(较高浓度的生物膜产生者)的进展。在这次更新中，我们将对我们在金黄色葡萄球菌生物膜中发现的这两个不同的亚群进行生理学表征。为了做到这一点，我们建立了一种新的细胞分选方法，允许分离这些亚群中的每一个，以便在分离后进一步分析它们的转录图谱。这种方法将使我们能够确定每种细胞类型的生理特征，以更好地了解它们在生物膜中的专门化。此外，为了测试我们方法的多功能性，我们将向该联盟中任何对表征细胞亚群感兴趣的实验室提供这种分类+RNA-SEQ技术。此外，我们将结合我们的细胞分选+RNA-seq技术和我们最先进的显微技术，对金黄色葡萄球菌聚集体中的亚群进行时空定位。这两种方法的结合将更好地理解特殊细胞类型在微生物群落中的作用，以及这是否与每种细胞类型的特定生理状态有关。该提案的第三个目标将研究生物被膜生产者和毒素生产者这两个亚群对常规用于消除金黄色葡萄球菌生物被膜的抗菌治疗的不同敏感性。具有不同转录谱和不同生理特征的细胞类型可能对抗生素表现出不同的敏感性，并可能发展为难以治疗的葡萄球菌感染。我们将根据每种细胞的生理特征来研究它们抵抗抗生素治疗的能力。总体而言，我们的项目将证明金黄色葡萄球菌群落是由生理上不同的细胞类型组成的，并将证明每种细胞类型的一般生理学与它们在生物膜中的作用和它们抵抗抗菌素处理的能力有关。我们的方法应该会引起该联盟的其他研究小组的兴趣。

During the first period of this SPP1617 program, we showed that multicellular aggregates of the bacterial pathogen Staphylococcus aureus are constituted by a heterogeneous population of, at least, two different cell types, biofilm producers and toxin producers. We used a number of biochemical techniques to characterize the molecular mechanism that drives to this cell differentiation. We found a complex genetic regulation triggered by the bimodal behavior of the quorum-sensing cascade agr, which ultimately selects for the progression of acute (higher concentration of toxin producers) or biofilm-associated chronic infections (higher concentration of biofilm producers). In this renewal, we will physiologically characterize these two distinct subpopulations that we find in S. aureus biofilms. To do this, we have setup a new cell-sorting methodology that permits the isolation of each one of these subpopulations to further analyze their transcriptomic profile after isolation. This approach will allow us determining the physiological characteristics of each cell type to better understand their specialization within the biofilm. Moreover, to test the versatility of our approach, we will provide this sorting + RNA-seq technology to any laboratory of this consortium that is interested in characterizing a subpopulation of cells. Furthermore, we will combine our cell sorting + RNA-seq technology with our state-of-the-art microscopic techniques to perform a spatio-temporal localization of the subpopulations within the S. aureus aggregates. The combination of these two approaches will provide a better understanding of the role of specialized cell types within microbial communities and whether this is connected to the specific physiological state of each cell type. The third goal of this proposal will study the differential sensitivity of the subpopulations, biofilm producers and toxin producers, to antimicrobial treatments that are conventionally used to eliminate S. aureus biofilms. Cell types with different transcriptomic profiles and different physiologies may display different sensitivities to antibiotics and may develop into difficult-to-treat staphylococcal infections. We will study the capacity of each cell type to resist an antibiotic treatment based on their physiological profile. Overall, our project will demonstrate that S. aureus communities are constituted by cell types that are physiologically different and will demonstrate that the general physiology of each cell type is related to their role within the biofilm and their capacity to resist antimicrobial treatments. Our approach should be of interest to other research groups of this consortium.