Defining the molecular inventory of staphylococcal tissue abscesses and its effects on the host-pathogen interface

摘要： 耐多药细菌感染，特别是由金黄色葡萄球菌引起的细菌感染，被认为是21世纪最大的威胁之一。血液感染是葡萄球菌疾病最严重的表现形式，尽管我们采用了最好和最新的治疗方法，但它往往是致命的。器官脓肿是金黄色葡萄球菌全身性感染的主要因素，也是入侵病原体的初始储备库。脓肿形成本身经历了不同的发育阶段，受到宿主和细菌的积极促进，最终为金黄色葡萄球菌创造了有利的生态位。虽然过去的研究已经对脓肿的结构进行了概述，但我们缺乏关于脓肿的分子组成的信息，特别是在不同脓肿阶段的背景下。这种对脓肿形成过程中分子事件的有限了解尤其令人担忧，因为它阻碍了有意义的抗葡萄球菌策略设计的尝试。 我们的初步数据显示，脓肿形成的特点是宿主在脓肿附近的过渡金属广泛迁移，这一过程被称为营养免疫。因此，活体成像显示，脓肿内的细菌缺乏锌和铁。由于可用金属水平可以作为入侵病原体的生物标志物，我们假设元素分布的波动协调了与脓肿形成相关的细菌活动。沿着这些思路，我们表明锌饥饿为金黄色葡萄球菌随后与不同免疫细胞群的接触做好了准备。然而，除了这些发现之外，涉及金属重新定位、金黄色葡萄球菌对这些刺激的检测以及相应的细菌反应的年表和因素完全没有被探索。因此，我们计划在这项提案中解决这些问题。 目前对葡萄球菌组织脓肿的发展进行有意义的研究的一个障碍是脓肿异质性的显著程度，这可能是同一器官中单个病变的不同发展阶段的结果。为了解释组织脓肿的非同步性，我们已经确定了一组潜在的不同脓肿阶段的蛋白质标志物。这些蛋白质将作为分子时钟，这样我们就可以在整个发育过程中跟踪单个脓肿的进展。基于这些标记，我们将创建活体记者，并描述正在发展的脓肿的分子清单，重点是元素和蛋白质组成的变化。在这里，我们将通过微液萃取表面分析将各种体内成像方式，包括3D生物发光成像、MRI和成像质谱学与先进的蛋白质组学相关联。一旦我们确定了脓肿微环境在脓肿形成的不同阶段如何变化，我们将对细菌亚群进行转录组分析，以评估环境刺激如何影响葡萄球菌的病理生理学，进而影响脓肿的发展。结合起来，拟议的实验将检查宿主-病原体界面上的事件，并为抗击葡萄球菌感染的新颖和有针对性的治疗策略铺平道路。

Summary: Multidrug-resistant bacterial infections, particularly those caused by Staphylococcus aureus, are recognized as one of the greatest threats of the 21st century. Bloodstream infections are the most severe staphylococcal disease manifestation and are often fatal despite our best and most current therapies. Organ abscesses are primary contributors to S. aureus systemic infections and serve as initial reservoirs for the invading pathogen. Abscess formation itself follows distinct developmental stages, is actively facilitated by host and bacterium, and ultimately creates an advantageous niche for S. aureus. While past studies have generated an overview of abscess architecture, we lack information on the molecular composition of abscesses, particularly in the context of different abscess stages. This limited knowledge of the molecular events during abscess formation is especially alarming, for it hinders meaningful attempts at targeted design of anti-staphylococcal strategies. Our preliminary data show that abscess formation is characterized by the host's extensive relocation of transition metals in proximity to the abscess in a process known as nutritional immunity. Consequently, in vivo imaging reveals that bacteria within the abscess are starved for zinc and iron. Since available metal levels can serve as biomarkers for invading pathogens, we hypothesize that fluctuating elemental distributions orchestrate bacterial activities associated with abscess formation. Along these lines, we showed that zinc starvation primes S. aureus for subsequent contact with different immune cell populations. Beyond these findings, however, the chronology and factors involved in metal relocation, detection of these stimuli by S. aureus, and corresponding bacterial responses are entirely unexplored. We thus plan to address these questions in this proposal. One current barrier to the design of meaningful investigations into the development of staphylococcal tissue abscesses is a significant degree of abscess heterogeneity, likely a result of different developmental stages of individual lesions in the same organ. To account for the non-synchronous nature of tissue abscesses, we have identified a group of potential proteinaceous markers for different abscess stages. These proteins will serve as molecular clocks so we can follow the progression of individual abscesses through the developmental process. Based on these markers, we will create in vivo reporters and characterize the molecular inventory of developing abscesses, focusing on changes in elemental and proteinaceous compositions. Here, we will correlate various in vivo imaging modalities, including 3D-bioluminiscent imaging, MRI, and imaging mass spectrometry, with advanced proteomics via micro Liquid Extraction Surface Analysis. Once we have established how the abscess microenvironment changes during different phases of abscess formation, we will perform transcriptome analysis of bacterial subpopulations to assess how environmental stimuli affect staphylococcal pathophysiology and, in turn, abscess development. Combined, the proposed experiments will examine the events at the host-pathogen interface and pave the way for novel and targeted treatment strategies to combat staphylococcal infections.