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ability to cause a broad range of life-threatening illnesses in a vertebrate host, ranging from soft tissue infections that present with abscess formation to systemic conditions such as sepsis. Abscess architecture consists of staphylococcal communities
surrounded by layers of healthy and necrotic innate immune cells. Upon recognition
of a pathogen, a vertebrate host will deploy mechanisms of innate immunity. These
mechanisms vary from pathogen killing by oxidative stress to more specialized
responses, such as nutrient metal sequestration in processes known as nutritional
immunity [76–79]. Characterization of the host-pathogen interface in these infections by spatially-targeted molecular analysis technologies will enable a deeper
understanding of bacterial pathogenesis and host defense mechanisms. IMS has
been employed to investigate this interface, identifying both host and pathogen factors [80–84].
Advances in IMS instrumentation using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) allow for the measurement of isotopically
resolved protein species directly from tissue, Fig. 7.6a, b [82]. Previous research
of protein distributions at the infectious interface focused on the heterodimer
calprotectin (CP) constructed by S100A8 and S100A9 subunits [82, 83]. CP can
bind manganese, zinc, calcium, and iron [83, 85, 86]. By decreasing the availability of necessary metal species, metalloproteins inhibit proliferation of pathogens by processes of nutritional immunity [79]. The resolving power provided by
a 15 T FT-ICR MS results in the identification of post-translational modifications
(PTMs) to S100A8 and S100A9. Modified S100A8 and S100A9 species were
observed to form a spatial gradient from singly to triply oxidized species as distributions approached bacterial foci, Fig. 7.6d [83]. This observation can be
explained by reactive oxygen species (ROS) used by a host to kill pathogens.
Application of IMS in tandem with multiple other imaging modalities such as
laser ablation inductively coupled plasma imaging mass spectrometry (LA-ICP
IMS), bioluminescent imaging, magnetic resonance imaging (MRI), and blockface imaging provided 3-dimensional (3D) distributions of protein and metal
abundances at 50 μm spatial resolution [84]. Multimodal analysis revealed heterogeneous bacterial transcriptional responses and host inflammatory responses.
Specifically, IMS allowed visualization of CP throughout a 3D volume of an
S. aureus infected murine kidney, Fig. 7.6e. Use of genetically modified bacteria
that produce a bioluminescent response to iron starvation allowed visualization of
transcriptional response in vivo, Fig. 7.6e. LA-ICP IMS provided elemental distributions translocated by processes of nutritionally immunity, Fig. 7.6e. MRI provided a scaffold for 3D reconstruction of image data file. Finally, blockface image
analysis provided visual context to imaging data as well as verification of signal
localizations within tissue. A major discovery from this study is that abscesses
within the same organ have heterogeneous molecular architectures despite having
a homogeneous appearance by traditional microscopy. Consistent with this observation, bacterial pathogens exhibited heterogeneous responses both within and
between tissue lesions. Collectively, IMS is a promising research tool for the
label-free identification of tissue pathology characterized by altered protein or element abundance.
7 Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry…
ability to cause a broad range of life-threatening illnesses in a vertebrate host, ranging from soft tissue infections that present with abscess formation to systemic conditions such as sepsis. Abscess architecture consists of staphylococcal communities
surrounded by layers of healthy and necrotic innate immune cells. Upon recognition
of a pathogen, a vertebrate host will deploy mechanisms of innate immunity. These
mechanisms vary from pathogen killing by oxidative stress to more specialized
responses, such as nutrient metal sequestration in processes known as nutritional
immunity [76–79]. Characterization of the host-pathogen interface in these infections by spatially-targeted molecular analysis technologies will enable a deeper
understanding of bacterial pathogenesis and host defense mechanisms. IMS has
been employed to investigate this interface, identifying both host and pathogen factors [80–84].
Advances in IMS instrumentation using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) allow for the measurement of isotopically
resolved protein species directly from tissue, Fig. 7.6a, b [82]. Previous research
of protein distributions at the infectious interface focused on the heterodimer
calprotectin (CP) constructed by S100A8 and S100A9 subunits [82, 83]. CP can
bind manganese, zinc, calcium, and iron [83, 85, 86]. By decreasing the availability of necessary metal species, metalloproteins inhibit proliferation of pathogens by processes of nutritional immunity [79]. The resolving power provided by
a 15 T FT-ICR MS results in the identification of post-translational modifications
(PTMs) to S100A8 and S100A9. Modified S100A8 and S100A9 species were
observed to form a spatial gradient from singly to triply oxidized species as distributions approached bacterial foci, Fig. 7.6d [83]. This observation can be
explained by reactive oxygen species (ROS) used by a host to kill pathogens.
Application of IMS in tandem with multiple other imaging modalities such as
laser ablation inductively coupled plasma imaging mass spectrometry (LA-ICP
IMS), bioluminescent imaging, magnetic resonance imaging (MRI), and blockface imaging provided 3-dimensional (3D) distributions of protein and metal
abundances at 50 μm spatial resolution [84]. Multimodal analysis revealed heterogeneous bacterial transcriptional responses and host inflammatory responses.
Specifically, IMS allowed visualization of CP throughout a 3D volume of an
S. aureus infected murine kidney, Fig. 7.6e. Use of genetically modified bacteria
that produce a bioluminescent response to iron starvation allowed visualization of
transcriptional response in vivo, Fig. 7.6e. LA-ICP IMS provided elemental distributions translocated by processes of nutritionally immunity, Fig. 7.6e. MRI provided a scaffold for 3D reconstruction of image data file. Finally, blockface image
analysis provided visual context to imaging data as well as verification of signal
localizations within tissue. A major discovery from this study is that abscesses
within the same organ have heterogeneous molecular architectures despite having
a homogeneous appearance by traditional microscopy. Consistent with this observation, bacterial pathogens exhibited heterogeneous responses both within and
between tissue lesions. Collectively, IMS is a promising research tool for the
label-free identification of tissue pathology characterized by altered protein or element abundance.
7 Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry…