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.
Tissue abscesses formed by Staphylococcus aureus are important contributors to progression and persistence of infection, largely due to the physical protection they afford to bacteria from circulating immune cells. While general features of the abscess architecture are known, we lack a detailed understanding of the molecular processes that shape abscess formation, a shortcoming that complicates the development of novel anti-staphylococcal strategies. In this proposal, we outline experiments that are designed to overcome these limitations by i) identifying molecular markers of abscess formation, ii) characterizing the elemental and proteinaceous abscess composition throughout different developmental stages of the abscess, and iii) evaluating how S. aureus senses and responds to fluctuations in the abscess microenvironment.
