Name: investigation of microbial cooperation via imaging mass spectrometry analysis of bacterial colonies grown on agar and in tissue during infection
Uploaded: 2022-11-18T14:00:00
Description: Watch this Scientific Journal Video about Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection at JoVE.com

This work was supported by the National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) under award GM138660. J.T.S. was supported by the Charles and Monica Burkett Family Summer Fellowship from the University of Florida. J.P.Z. was supported by NIH grants K22AI7220 (NIAID) and R35GM138369 (NIGMS). A.B.S. was supported by the Cell and Molecular Biology Training Grant at the University of Pennsylvania (T32GM07229).

NOTE: Animal experiments were approved by the Animal Care and Use Committees of the Children&#39;s Hospital of Philadelphia and the University of Pennsylvania Perelman School of Medicine (protocols IAC 18-001316 and 806279).
CAUTION: Clostridium difficile (C. difficile) and Enterococcus faecalis (E. faecalis) are BSLII pathogens and should be handled with extreme caution. Utilize proper decontamination protocols when necessary.
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During MALDI imaging mass spectrometry, it is important to have a flat sample surface to provide for a consistent focal diameter of the incident MALDI laser on the sample substrate. Deviations in sample height can cause the MALDI laser beam to shift out of focus, causing alterations in beam diameter and intensity, which can affect MALDI ionization efficiency. These alterations in ionization efficiency can result in differences in analyte intensity across the tissue surface that are not reflective of the underlying tissue...

The human microbiome is a highly dynamic ecosystem involving molecular interactions of bacteria, viruses, archaea, and other microbial eukaryotes. While microbial relationships have been intensely studied in recent years, much remains to be understood about microbial processes at the chemical level1,2. This is in part due to the unavailability of tools capable of accurately measuring complex microbial environments. Advances in the field of imaging mass spectrometry (IMS) over the past decade have enabled in situ and label-free spatial mapping of many metabolites, lipids, and proteins in biological substrates3,4. Matrix-assisted laser desorption/ionization (MALDI) has emerged as the most common ionization technique used in imaging mass spectrometry, involving the use of a UV laser to ablate material from the surface of a thin tissue section for measurement by mass spectrometry4. This process is facilitated by the application of a chemical matrix applied homogeneously to the surface of the sample, allowing for sequential measurements to be made in a raster pattern across the sample surface. Heat maps of analyte ion intensities are then generated following data acquisition. Recent advances in ionization sources and sampling techniques have enabled the analysis of non-traditional substrates such as bacterial5 and mammalian6,7,8 cellular specimens grown on nutrient agar. The molecular spatial information afforded by IMS can provide unique insight into the biochemical communication of microbe-microbe and host-microbe interactions during infection9,10,11,12,13,14.
Upon Clostridioides difficile infection (CDI), C. difficile is exposed to a rapidly changing microbial environment in the gastrointestinal tract, where polymicrobial interactions are likely to impact infection outcomes15,16. Surprisingly, little is known about the molecular mechanisms of interactions between C. difficile and resident microbiota during infection. For example, enterococci are a class of opportunistic commensal pathogens in the gut-microbiome and have been associated with increased susceptibility to and severity of CDI17,18,19,20. However, little is known about the molecular mechanisms of the interactions between these pathogens. To visualize small-molecule communication between these members of the gut microbiome, bacterial macrocolonies were grown herein on agar to simulate microbe-microbe interactions and&#160;bacterial biofilm formation in a controlled environment. However, obtaining representative metabolic distributions upon MALDI imaging mass spectrometry analysis of bacterial culture specimens is challenging due to the high water content and uneven surface topography of these samples. This is largely caused by the highly hydrophilic nature of agar and the non-uniform agar surface response during moisture removal.
The high water content of agar can also make it challenging to achieve homogeneous MALDI matrix coating and can interfere with subsequent MALDI analysis performed in vacuo21. For example, many MALDI sources operate at pressures of 0.1-10 Torr, which is a sufficient vacuum to remove moisture from the agar and can cause deformation of the sample. These morphological changes in the agar induced by the vacuum environment cause bubbling and cracking in the dried agar material. These artifacts reduce the adherence of the agar to the slide and can cause dismounting or flaking of the sample into the instrument vacuum system. The thickness of the agar samples can be up to 5 mm off the slide, which can create insufficient clearance from ion optics inside the instrument, causing contamination and/or damage to instrument ion optics. These cumulative effects can result in reductions of ion signal reflective of the surface topography, rather than the underlying microbial biochemical interactions. Agar samples must be homogeneously dried and strongly adhered to a microscope slide prior to analysis in vacuo.
This paper demonstrates a sample preparation workflow for the controlled drying of bacterial culture macrocolonies grown on agar media. This multi-step, slower drying process (relative to those previously reported) ensures that the agar will dehydrate uniformly while minimizing the effects of bubbling or cracking of agar samples mounted on microscope slides. By using this gradual drying method, samples are strongly adhered to the microscope slide and amenable for subsequent matrix application and MALDI analysis. This is exemplified using model bacterial colonies of C. difficile grown on agar and murine tissue models harboring CDI with and without the presence of commensal and opportunistic pathogen,&#160;Enterococcus faecalis. MALDI imaging mass spectrometry analyses of both bacterial and tissue models allow for the spatial mapping of amino acid metabolite profiles, providing novel insight into bioenergetic microbial metabolism and communication.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#956;m Titan3 nylon syringe filters</td>
			<td>Thermo Scientific</td>
			<td>42225-NN</td>
			<td></td>
		</tr>
		<tr>
			<td>1,5-diaminonaphthalene MALDI matrix</td>
			<td>Sigma Aldrich</td>
			<td>2243-62-1</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL Henke Ject luer lock syringes</td>
			<td>Henke Sass Wolf</td>
			<td>4200.000V0&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>275i series convection vacuum gauge</td>
			<td>Kurt J. Lesker company</td>
			<td>KJL275807LL</td>
			<td></td>
		</tr>
		<tr>
			<td>7T solariX FTICR mass spectrometer equipped with a Smartbeam II Nd:YAG MALDI laser system (2 kHz, 355 nm)&#160;</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid solution, suitable for HPLC</td>
			<td>Sigma Aldrich</td>
			<td>64-19-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile, suitable for HPLC, gradient grade, &#8805;99.9%</td>
			<td>Sigma Aldrich</td>
			<td>75-05-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution, 28% NH3 in H2O, &#8805;99.99% trace metals basis</td>
			<td>Sigma Aldrich</td>
			<td>1336-21-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclavable biohazard bags: 55 gal</td>
			<td>Grainger</td>
			<td>45TV10</td>
			<td></td>
		</tr>
		<tr>
			<td>Biohazard specimen transport bags (8 x 8 in.)</td>
			<td>Fisher Scientific</td>
			<td>01-800-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain heart infusion broth</td>
			<td>BD Biosciences</td>
			<td>90003-040</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 male mice&#160;</td>
			<td>Jackson Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CanoScan 9000F Mark II photo and document scanner</td>
			<td>Canon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CM 3050S research cryomicrotome</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator cabinet</td>
			<td>Sigma Aldrich</td>
			<td>Z268135</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond tip scriber, Electron Microscopy Sciences&#160;</td>
			<td>Fisher Scientific</td>
			<td>50-254-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Drierite desiccant pellets</td>
			<td>Drierite</td>
			<td>21005</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 200 Proof</td>
			<td>Decon Labs</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>flexImaging software</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ftmsControl software</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vacuum trap</td>
			<td>Sigma Aldrich</td>
			<td>Z549460</td>
			<td></td>
		</tr>
		<tr>
			<td>HTX M5 TM robotic sprayer</td>
			<td>HTX Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Indium Tin Oxide (ITO)-coated microscope slides</td>
			<td>Delta Technologies</td>
			<td>CG-81IN-S115</td>
			<td></td>
		</tr>
		<tr>
			<td>In-line HEPA filter to vacuum pump</td>
			<td>LABCONCO</td>
			<td>7386500</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol, HPLC Grade</td>
			<td>Fisher Chemical</td>
			<td>&#160; 67-56-1</td>
			<td></td>
		</tr>
		<tr>
			<td>MTP slide-adapter II</td>
			<td>Bruker Daltonics</td>
			<td>235380</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal cutting temperature (OCT) compound</td>
			<td>Fischer Scientific</td>
			<td>23-730-571</td>
			<td></td>
		</tr>
		<tr>
			<td>Peridox RTU Sporicide, Disinfectant and Cleaner</td>
			<td>CONTEC</td>
			<td>CR85335&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE (Teflon) printed slides, Electron Microscopy Sciences</td>
			<td>VWR</td>
			<td>100488-874</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary vane vacuum pump RV8</td>
			<td>Edwards</td>
			<td>A65401903</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Accu-Edge Disposable High Profile Microtome Blades</td>
			<td>Electron Microscopy Sciences</td>
			<td>63068-HP</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent vacuum tubing</td>
			<td>Cole Palmer</td>
			<td>EW-06414-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultragrade 19 vacuum pump oil</td>
			<td>Edwards</td>
			<td>H11025011</td>
			<td></td>
		</tr>
		<tr>
			<td>Variable voltage transformer</td>
			<td>Powerstat</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water, suitable for HPLC</td>
			<td>Sigma Aldrich</td>
			<td>7732-18-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-mouth dewar flask</td>
			<td>Sigma Aldrich</td>
			<td>Z120790</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigation of microbial cooperation via imaging mass spectrometry analysis of bacterial colonies grown on agar and in tissue during infection

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical imaging. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry represents a label-free, in situ imaging modality capable of generating spatial maps for a wide variety of metabolites. While thinly sectioned tissue samples are now routinely analyzed via this technology, imaging mass spectrometry analyses of non-traditional substrates, such as bacterial colonies commonly grown on agar in microbiology research, remain challenging due to the high water content and uneven topography of these samples. This paper demonstrates a sample preparation workflow to allow for imaging mass spectrometry analyses of these sample types. This process is exemplified using bacterial co-culture macrocolonies of two gastrointestinal pathogens: Clostridioides difficile and Enterococcus faecalis. Studying microbial interactions in this well-defined agar environment is also shown to complement tissue studies aimed at understanding microbial metabolic cooperation between these two pathogenic organisms in mouse models of infection. Imaging mass spectrometry analyses of the amino acid metabolites arginine and ornithine are presented as representative data. This method is broadly applicable to other analytes, microbial pathogens or diseases, and tissue types where a spatial measure of cellular or tissue biochemistry is desired.

We have performed metabolite MALDI imaging mass spectrometry of model bacterial colonies and mice co-colonized with E. faecalis and C. difficile to study the role of amino acids in microbe-microbe interactions. Bacterial macrocolonies grown on agar serve as a well-defined model to analyze distinct biochemical changes in bacterial biofilm formation. It is important to ensure a controlled drying process for bacterial culture macrocolonies grown on agar media to minimize deformations and cracking in the ag...

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Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical ...

Our protocol enables the visualization of spatial metabolism of microbial interactions that occur during infection. We have developed this imaging mass spectrometry workflow for bacterial co-cultures grown on agar and tissue models. Analyzing non-traditional substrates such as bacterial cultures grown on agar presents many challenges.Using our protocol, bacterial specimens can be appropriately dried to facilitate MALDI matrix application in subsequent imaging mass spectrometry analysis. This method is broadly applicable to a variety of metabolites, microbial pathogens or diseases, and sample types where a spatial measure of cellular or tissue biochemistry is desired, particularly when studying sample types that require dehydration prior to analysis. After preparing the bacterial culture macrocolonies, remove the agarose bacterial colonies mounted on ITO-coated microscope slides from the packaging material.Place the samples in a dry box with dessicant for 48 to 72 hours at room temperature. Visually inspect the colonies for deformations in the agar surface. Next, close the vacuum line to the sample chamber, and turn on the power switch to the rotary vane pump to allow the vacuum pump to warm up and achieve proper vacuum pressure.Turn on the variable voltage transformer to heat the wire filament wrapped around the chamber. Adjust the variable power supply until the internal temperature of the vacuum chamber reaches approximately 50 degrees Celsius. While the pump is warming up, place a slurry of dry ice and 100%ethanol into the cold trap condenser.Open the vacuum chamber using a wrench to loosen the 16-millimeter double cloth flange clamps. Insert the agarose samples into the chamber, and seal the chamber tightly with the 16-millimeter double cloth flange clamps. Then open the pump valve to evacuate the chamber, and allow the samples to dry for 60 to 120 minutes.When complete, slowly vent the vacuum chamber to ambient pressure by closing the valve on the rotary vane pump, and opening the vacuum flange to ambient air. Open the chamber as demonstrated previously, and remove the dried agarose sample from the chamber. Use 1, 5-Diaminonaphthalene MALDI matrix for its favorable desorption and ionization of amino acids and negative ion mode.Attach the sample to the sprayer tray, and load the prepared solutions into the sprayer line. Using the computer software, specify the necessary parameters for a uniform coating of the matrix compound. Monitor the spraying sequence until it is finished.Remove the sample from the sprayer tray, and store it in a desiccation cabinet until analysis. Insert the matrix-coated sample into the MALDI target plate microscope slide adapter. Inscribe at least three fiducial markers that encompass the sample area using permanent markers.Use a flatbed scanner to acquire an optical image of the microscope slide including the fiduciary markers. Now define a mass spectrometry instrument method as described in the manuscript. In negative ion mode, select the 100 dalton mass window from mass by charge 100 to 200 for gas phase signal enrichment using a continuous accumulation of selected ions approach which encompasses the mass by charge values of the metabolites of interest.Open the instrument's image acquisition software, and use the setup wizard to define the file name and location, the MS acquisition mode, regions of interest to be sampled, and the spatial resolution of the image. Teach the sample by using the fiducial markers from the optical image to synchronize with the instrument sample carrier. Then define the regions of interest on the optical image using the software's regions of interest selection tool.Once all the parameters have been defined, start the acquisition sequence to serially acquire a mass spectrum at each pixel across the defined region of interest. Following image acquisition, save the data with a MIS file extension which is a vendor-specific file format for imaging mass spectrometry platforms. Open the data file in flexImaging, or SCiLS software packages, or export to a non-proprietary data format such as mzML, and visualize using vendor neutral software.Using the reference software, define mass windows for peaks of interest to generate false color heat maps of ion distributions across the sampled regions. On the average mass spectrum display, zoom in to the peak of interest, and select an appropriate mass window that encompasses the area of the peak profile. Now, right-click on the highlighted mass window, and select Add Mass Filter.Label the selected mass by charge value using the tentative identification of the suspected metabolite as determined by the high-resolution accurate mass measurement. Select the intensity threshold to adjust the dynamic range of the analyte image displayed by the false color heat map. Further adjust the mass-filtering parameters so that the mass window encompasses the area of the peak profile, and then add the mass filter.Clean all cryosectioning equipment by rinsing it with 100%ethanol, and allow to dry before placing samples into the cryostat chamber. Prepare ITO-coated microscope slides using an ohmmeter to identify the side of the slide with the conductive coating. Using a diamond-tipped scribe, etch and label the ITO-coated side of the microscope slide.Then perform cryosectioning on a research cryomicrotome. Transfer the OCT-embedded mouse ceca tissues from a 80 degree Celsius freezer to the cryomicrotome chamber on dry ice. Inside the cryomicrotome chamber, mount the OCT-embedded tissue on a sample chuck using an additional OCT solution.After the OCT solution has solidified, fix the chuck to the specimen head and begin cryosectioning at 10 to 50 micrometers increments to reach the desired tissue depth or plane of the organ. Once an optimal cross-section of the tissue is reached, begin collecting sections at 12 micrometers thickness. Gently manipulate the slice using artist paintbrushes, and place it onto a Teflon-coated microscope slide.To pick up the tissue from the Teflon-coated slide, roll the ITO-coated microscope slide on top of the tissue section. Thaw-mount the tissue section to the microscope slide by pressing the palm to the back of the ITO-coated slide. Continue to thaw-mount until the tissue section transitions from translucent to an opaque or matte texture.For same day analysis, transfer the tissue-mounted microscope slides on dry ice to a dry box with dessicant for storage. Perform the MALDI matrix application and MALDI imaging mass spectrometry as demonstrated earlier. Analyze the ion images from multiple biological replicates, and compare the results for a significance via intensity box plot comparisons using the SCiLS statistical software.Negative ion mode MALDI analysis of bacterial culture samples allows for detecting dozens of metabolites. High-resolution accurate mass measurements allow for the tentative identification of ornithine and arginine. The measurement regions for imaging mass spectrometry analysis are shown.Imaging mass spectrometry of these samples allows for the spatial mapping of both ornithine and arginine amino acid metabolites which are found to be altered and differentially localized in the presence of Enterococcus faecalis. The tissue morphology of infected mouse ceca was visualized using hematoxylin and eosin staining. Optical images of mouse ceca tissues after applying a 1, 5-Diaminonaphthalene MALDI matrix layer are shown.MALDI ion images for ornithine and arginine display unique spatial distributions across the tissue samples. The most important consideration for this protocol is to maintain gentle drying and handling of bacterial culture specimens to minimize bubbling, cracking, and other deformation of the sample. Following imaging mass spectrometry, bright-field and fluorescence microscopy analyses of tissue samples can be performed to evaluate tissue morphology.This can also enable targeted validation of molecular pathways using antibody-based imaging methods.

Research

JoVE Journal

Biochemistry

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