Vanderbilt Institute of Chemical Biology



Discovery at the VICB







Visualizing Infection in Three Dimensions


By: Carol A. Rouzer, VICB Communications
Published: April 9, 2018


A new multimodal approach enables molecular-level imaging of the host-pathogen interaction.  


To truly understand the impact of any disease, we must be able to explore all of the metabolic changes that occur in response to that disease. Yet, our tools to accomplish this task are woefully inadequate. One approach is to use techniques such as immunohistochemistry to monitor levels of a particular protein of interest in an affected tissue. This approach retains tissue architecture but is limited to a small number of target species of known identity. Other methods enable the global evaluation of entire classes of molecules; however, these usually require destruction of the tissue of interest, so measured changes cannot be localized, and subtle regionalized changes are not captured. Now Vanderbilt Institute of Chemical Biology members Eric Skaar, James Cassat, and Richard Caprioli offer a new multi-modal approach to three-dimensionally image changes that occur in a complex tissue in response to infection by Staphylococcus aureus. Using this approach, they provide a new way to explore the interaction between host and pathogen spatially and at the molecular level [J. E. Cassat, et al., (2018) Sci. Transl. Med., 10, eaan6361].


Metal ions serve as critical co-factors in many proteins, so all organisms require nutrient metal for survival. Pathogenic bacteria have devised multiple capture and transport systems to obtain metals from the host, whereas the host produces proteins that sequester metals from invading bacteria, a process known as nutritional immunity. The importance of this competition for nutrient metal to bacterial survival led the investigators to focus their attention on changes in metal levels at the site of S. aureus infection. For these studies, they established a disseminated infection in mice using a genetically engineered strain of S. aureus that emits a luminescent signal under conditions of iron starvation. A characteristic of this infection model is the formation of abscesses – enclosed micro-colonies of bacteria that have been isolated by the innate immune system (Figure 1). The concentration of bacteria into abscesses enabled the investigators to use bioluminescence imaging (BLI) to obtain two- or three-dimensional images of the location of abscesses containing iron-starved S. aureus in the infected mice. They next used magnetic resonance imaging (MRI) to obtain a three-dimensional overview of the position of all abscesses. An overlay of the BLI and MRI results demonstrated the presence of many abscesses not detected by BLI. This implied that many abscesses did not contain iron-starved bacteria. In fact abscesses containing iron-starved bacteria often existed quite close to others in which metal starvation was not apparent by BLI. These findings suggested that either metal distribution within a tissue was not uniform or that the abscesses that did not exhibit a BLI signal contained few to no bacteria. To distinguish between these two possibilities, the researchers sectioned the mice transversely in the region of the kidneys and analyzed each section by digital photography (blockface imaging) and conventional microscopy. An overlay of these images with those from BLI demonstrated the presence of S. aureus in kidney abscesses that did not emit a BLI signal. Thus, the investigators concluded that the availability of iron likely varies in different regions of the tissue. Alternatively, bacteria in abscesses that did not emit a BLI signal may have been dead, or oxygen levels in those abscesses may have been too low to support luciferase activity.



FIGURE 1. Hematoxylin and eosin stain of kidney section from a mouse infected with S. aureus (left) and close-up (right). Abscesses are seen as finely demarcated circular lesions in the kidney parenchyma. Figure kindly provided by the Skaar lab. Copyright 2018.



To further explore the distribution of metal in infected tissues, the researchers used laser ablation inductively coupled mass spectrometry (LA-ICP-MS), which detects the presence of elements in a sample. Overlay of the results from LA-ICP-MS analysis of the slices from infected mice with MRI and blockface images provided a three-dimensional view of the localization of metal ions in normal tissue and the abscesses. The abscesses were rich in calcium, low in manganese, iron, and zinc, and devoid of copper. Magnesium and phosphorus were both present in abscesses, but distributed heterogeneously. Of particular interest was the finding that iron levels were particularly low in abscesses containing iron-starved bacteria as indicated by BLI.


The researchers next turned to matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) to investigate the distribution of proteins throughout their tissue slices (Figure 2). A particularly abundant signal associated with abscesses (m/z = 10,164) was identified as a subunit of calprotectin, a calcium-binding protein from neutrophils and other cells that plays a role in nutritional immunity by sequestering zinc and manganese. The investigators were not surprised to find that calprotectin co-localized with calcium; however, the discovery that the protein was not associated with zinc or manganese in abscesses was somewhat unexpected. Also of note was the presence of two bacterial proteins in the abscesses. These were a CsbD-like superfamily protein (m/z = 6888) and δ-hemolysin (m/z = 3006). Although the function of the CsbD-like protein is currently unclear, δ-hemolysin is a conserved virulence factor associated with severe S. aureus infections. A distinctive feature about these proteins was that they clearly did not co-localize with calprotectin. MALDI IMS also revealed changes in proteins that occurred with time during the course of the infection. For example, an as yet unidentified protein with m/z = 5047 was homogeneously distributed throughout the kidney at 4 and 7 days after the start of infection, but on the 10th day this protein was concentrated at the periphery of the abscesses.



FIGURE 2. MALDI-MS analysis of the kidney section shown in Figure 1. Each color represents a different protein. Figure kindly provided by the Skaar lab. Copyright 2018.



Together, this multi-modal high resolution imaging approach provides an unprecedented opportunity to gain important new insights regarding the interaction between host and pathogen during an infection (Figure 3). The current results demonstrate a surprising heterogeneity in the distribution of metals in S. aureus-infected tissues. Thus, bacteria may encounter quite different nutritional environments even within the same tissue, and BLI findings demonstrate that they respond appropriately to metal starvation. The results also yield interesting insights into the role of calprotectin. The absence of zinc or manganese in association with the protein found in abscesses suggests the possibility that metal binding leads to rapid degradation, that metal-bound calprotectin is removed from the abscess, or that other proteins may be sequestering these metals. Furthermore, the finding that calprotectin is completely absent in the immediate vicinity of the bacteria suggests the possibility that S. aureus has a mechanism to either destroy or exclude it from its environment. This could be an important survival mechanism, as calprotectin-mediated metal sequestration is an effective means to suppress S. aureus growth. These considerations are just the beginning of possible new information that can be obtained using this approach to imaging a disease process, which promises to be an important research tool in the future. The translation of this technology to the clinic, although not immediate, is also of great interest. For example, it opens the possibility that bacterial infections can be diagnosed by imaging of intact tissue and that the response to an infection and therapy can be monitored in a comprehensive fashion.




FIGURE 3. Blockface histology, ICP-MS, and MALDI-MS images of a single slice from a kidney of an infected mouse. The ability to overlay the three images enables investigators to simultaneously evaluate the presence of bacteria, metal ion content, and protein expression in the tissue. Figure kindly provided by the Skaar lab. Copyright 2018.




ViewScience Translational Medicine article: Integrated molecular imaging reveals tissue heterogeneity driving host-pathogen interactions








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