The work was supported by Swiss National Science Foundation 310030_156818, 310030_182315, and NCCR_ 180541 AntiResist (to DB).

Whole&#45;Organ Pathogen Tracking via Fluorescent Reporter Quantification

<ol>
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The authors have nothing to disclose.

The local tissue context of bacterial pathogens is crucial for determining local host attacks, bacterial adaptations, the local outcome of the host pathogen interactions and antimicrobial chemotherapy, and the individual contributions to overall disease outcome. Imaging micrometer sized bacteria in centimeters size organs has been challenging. Serial two-photon (STP) tomography provides sufficient spatial resolution to detect individual bacterial cells in entire organs, automatized sectioning and imaging, and sufficient throughput (~1 organ per day)11. While host antigens can be stained in vivo, pathogen cells should express suitable fluorescent proteins to ensure comprehensive detection of intracellular pathogen cells. The resulting data sets (0.5-1.5 TeraByte per organ) pose substantial challenges for IT infrastructures for data analysis and storage.
There are several critical steps in this method. First, a pathogen strain with detectable and homogeneous expression of fluorescent protein GFP or YFP is required. Ideally, a chromosomal expression cassette25 is used to minimize fluorescence heterogeneity due to plasmid copy-number variation. Sufficient fluorescence intensity is required but excessive levels of fluorescent protein should be avoided to avoid fitness impairments of the pathogen23. Appropriate expression levels can be obtained by selection of an appropriate promoter and fine-tuning of the ribosomal binding site25 or the entire 5&#39; untranslated region (UTR)26. Second, the perfusion fixation should involve an initial wash with buffer to remove as many erythrocytes as possible from the blood circulation. This is particularly critical for spleen and liver (although complete erythrocyte removal from these organs is difficult). Remaining erythrocytes absorb light in the visible part of the spectrum compromising imaging quality27. Third, storage of the fixed tissues in the cryoprotectant is critical to reduce tissue autofluorescence, which is particularly high in inflamed tissues and can overshadow the comparatively weak fluorescence of the pathogen cells11. Fourth, the effective cross-linking of the tissue to the surrounding agarose block is critical for smooth vibratome-cutting without the tissue jumping out of the agarose block. Fifth, fluorescent signals and their identification as pathogen cells must be independently verified using orthogonal approaches such as staining with antibodies to pathogen components (such as lipopolysaccharide for Gram-negative bacteria) and confocal microscopy of the sections retrieved from the tomograph11. Some infected tissues contain auto-fluorescent particles with similar shape and overlapping fluorescence spectra that can be easily misinterpreted as pathogen cells. Sixth, the quantity of pathogen cells within microcolonies should be compared to orthogonal approaches such as confocal microscopy to assess the accuracy. The overall bacterial loads based on these calculations should be verified by comparison to orthogonal approaches such as flow cytometry and plating.
Important modifications of the widely used STP protocol include placing a narrow bandpass filter 510/20 nm in front of photomultiplier 211, to reduce interference of green-yellow autofluorescence that is particularly strong in infected and inflamed liver, spleen, and Peyer&#39;s patches. The strong autofluorescence and increased light scattering of such organs compared to brain (which dominates other applications of STP) also generates a need for more effective correction for uneven illumination. As another modification, this protocol employs the CIDRE approach22 for this purpose (Figure 3) and AI-based segmentation of bacteria. Finally, tissue preprocessing was altered by including an incubation step in cryoprotectant at -20 &#176;C which reduces tissue autofluorescence and thus facilitates detection of small pathogen cells with relatively weak fluorescence11.
Troubleshooting might be necessary if no pathogen signals can be detected, or segmentation yields insufficient sensitivity (too many pathogen cells are missed) or insufficient precision (too many background particles are segmented as pathogen cells). If background tissue autofluorescence is detectable but there are too few pathogen signals, the pathogens might contain insufficient amounts of fluorescent proteins. This can be tested using confocal microscopy of tissue sections from the same infected tissue or flow cytometry of tissue homogenates19,28. Underlying reasons could be insufficient expression levels or instability of the expression cassette. Mitigation strategies could include alternative promoters to drive expression, codon-adaptation of the genes encoding the fluorescent protein for the pathogen species, employment of episomal constructs with higher copy number, or stabilization of expression cassettes by chromosomal integration or balanced-lethal complementation29. The choice of fluorescent protein is also important, but detection is possible with GFP.mut2, mWasabi, YPet, and TIMERbac. If segmentation is inaccurate, this might be caused by too weak pathogen fluorescence which could be addressed as described above, or too high tissue autofluorescence background. Extensive perfusion of wash solution or prolonged incubation in storage buffer immediately before embedding in the agarose block and tomography might resolve these issues. Finally, sufficient training of the neural network is required for precise classification, but excessive training can lead to overfitting that impairs performance for new samples.
Currently, no other method can image entire organs at sufficient spatial resolution in 3D for detecting individual bacteria. Future improvements in tissue clearing and light-sheet microscopy might achieve similar resolution. This might enable imaging at higher speed and with more fluorescent channels.
An important limitation of STP is the in-plane pixel resolution of ~0.5 &#181;m and vertical resolution of 5 to 10 &#181;m, which is insufficient for resolving closely located bacteria, e.g., within a densely packed microcolony. However, it is possible to retrieve tissue sections after tomography for secondary high-resolution confocal microscopy of selected tissue parts. Another limitation of STP is the availability of only three fluorescence channels, which restricts the number of fluorophores that can be imaged simultaneously. Again, secondary analysis of retrieved tissue sections with multiplexing methods can reveal the location and intensity of many more markers for selected tissue parts. This information could be integrated into the overall 3D structure of the surrounding tissue as determined with STP.
In conclusion, this protocol enables detailed investigations of host-pathogen interactions at the local and whole-organ level. The protocol should be easily adaptable to other pathogens (provided they can be obtained as fluorescent strains), other organs, and different host species.

Infections occur in tissues with complex anatomy and compartmentalized physiology. The diverse microenvironments that co-exist in the infected tissue can determine the fates of local pathogen subsets and their contributions to overall disease outcome1,2,3. However, comprehensive 3D mapping of microbial pathogens in cm-sized tissues remains challenging4. Imaging of the brain and other organs is a highly active research field with constantly improving experimental strategies5, but many methods still lack the sub-&#181;m resolution that would be required to identify &#181;m-sized bacterial pathogens confidently. In contrast, serial two-photon (STP) tomography6 enables automated multi-color, deformation-free imaging of entire tissues with sub-&#181;m in-plane resolution, yielding full volumetric datasets. This method combines repeated physical sectioning of the tissue using a vibratome with intermittent two-photon imaging of the emerging block faces with infrared light. STP tomography has been widely used for mapping thin axons in the brain to establish connectivity maps7,8,9,10.
STP tomography also enables 3D mapping of individual microbial pathogen cells (Salmonella, Toxoplasma) in the entire infected tissues11,12 using a tomograph. Second-harmonic generation reveals collagen sheaths around arteries and in fibrous bands such as the trabeculae of spleen, thus providing anatomical context. In vivo injected fluorescent antibodies can be used to stain host cells to reveal interactions between individual pathogen cells and infiltrating immune cells such as neutrophils. Here, the pipeline involving processing of the tissue, imaging, stitching of imaging tiles with illumination correction, stacking of images in three dimensions, and segmentation using machine-learning tools, is described. This pipeline yields 3D positions of individual pathogens cells and microcolonies within their host context. Counting the number of individual cells within microcolonies remains difficult because of resolution limits, but such numbers can be estimated based on the integrated brightness of the microcolony. The pipeline can be readily adapted to other infection models if recombinant GFP- or YFP-expressing pathogens are available.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose Low Melt</td>
			<td>Roth</td>
			<td>Art. 6351.5 25g</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma-Aldrich</td>
			<td>6768-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Instant adhesive Loctite 435</td>
			<td>Henkel</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene glycol)</td>
			<td>Sigma-Aldrich</td>
			<td>P5413-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone</td>
			<td>Sigma-Aldrich</td>
			<td>PVP-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium borohydride</td>
			<td>Sigma-Aldrich</td>
			<td>71321-25g</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Merck</td>
			<td>106453</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium periodate</td>
			<td>Sigma-Aldrich</td>
			<td>311448-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>71640-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>71500-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium tetraborate</td>
			<td>Sigma-Aldrich</td>
			<td>221732-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>AppliChem</td>
			<td>A4734,1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-buffered saline (TBS)</td>
			<td>Merck</td>
			<td>T5912-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum filtration 500</td>
			<td>TPP</td>
			<td>TPP99250</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blade</td>
			<td>Campden Instruments Limited&#160;</td>
			<td>01-01-4692</td>
			<td></td>
		</tr>
		<tr>
			<td>MAITAI Laser</td>
			<td>Spectra-Physics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peel away plastic mold</td>
			<td>Sigma-Aldrich</td>
			<td>E6032-1CS</td>
			<td></td>
		</tr>
		<tr>
			<td>TissueCyte 1000 tomograph</td>
			<td>TissueVision</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody/dyes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Merck</td>
			<td>D9542-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-LPS Salmonella, rabbit</td>
			<td>Sifin</td>
			<td>REF TS 1624</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD169-PE, clone 3D6.112</td>
			<td>Biolegend&#160;</td>
			<td>142403</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Ly-6G-PE, clone 1A8</td>
			<td>Biolegend&#160;</td>
			<td>127608</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>chicken anti-rabbit Alexa 647</td>
			<td>Invitrogen</td>
			<td>A-21443</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td>Company</td>
			<td>Version</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>Image J</td>
			<td>1.54g or later</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>2017b/2018b or later</td>
			<td></td>
		</tr>
		<tr>
			<td>Orchestrator (tomograph)</td>
			<td>TissueVision</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Visualization software Imaris</td>
			<td>Oxford Instruments</td>
			<td>9.9.0 or later</td>
			<td></td>
		</tr>
	</tbody>
</table>

high&#45;resolution three&#45;dimensional whole&#45;organ tomography of microbial infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

The described procedure enables detection of individual Salmonella cells in entire mouse organs such as spleen, liver, mesenteric lymph nodes, and Peyer&#39;s patches11 (Figure 5 and Figure 6). It also detects Toxoplasma gondii parasites in mouse brain12. Some infected tissues including liver, Peyer&#39;s patches, and spleen emit substantial autofluorescence in the green-yellow range. Autofluorescence is further enhanced by the fixation with paraformaldehyde, which is necessary to preserve the tissue structure. Detection of green fluorescence from GFP, mWasabi, and the green component of TIMERbac against this autofluorescence background is improved by putting a narrow bandpass filter 510/20 nm (transmitting most GFP emissions but blocking a large part of the autofluorescence spectrum) in front of photomultiplier 2 (which collects green emissions) and by reducing tissue autofluorescence by storing fixed tissues for 3 or more days in cryoprotectant11. However, bacteria should still express at least a few thousand copies per cell of GFP or other fluorescent proteins. On the other hand, excessive fluorescent protein levels should be avoided to minimize fitness costs that could lead to attenuated virulence23.
Correct segmentation of fluorescent bacteria in the tissues can be confirmed by immunohistochemistry of retrieved tissue sections after imaging. Specifically, objects stained with an antibody to bacterial surface components such as lipopolysaccharide can be aligned with the fluorescence image obtained by STP tomography (Figure 5). It is important to note that some stained Salmonella cells lack fluorescent proteins and thus detectable fluorescence in both confocal microscopy and STP tomography. These cells are Salmonella that have been killed by the host immune system as demonstrated by flow cytometric sorting and growth cultures from single sorted cells as well as the close correlation between the number of colony-forming units on agar plates and the number of fluorescent Salmonella cells as determined by flow cytometry24. In addition, plasmid loss can result in non-fluorescent viable cells and this needs to be tested by plating on media with and without appropriate antibiotics corresponding to the selection marker on the plasmid. For pSC101-derived plasmids, plasmid loss in vivo is rare19. For most chromosomally integrated expression cassettes such as sifB::gfp used in11, loss of expression is undetectable in vivo. If the segmentation is inconsistent with immunohistochemistry data, the segmentation pipeline needs to be modified.
The resolution of STP tomography is insufficient to resolve individual bacterial cells within densely packed microcolonies. However, the total fluorescence intensity of the microcolony enables estimation of the number of Salmonella cells. This requires a fluorescent strain with highly homogenous fluorescence levels such as Salmonella sifB::gfp11. Combining the estimated number of Salmonella cells for all microcolonies and single cells yields total bacterial tissue loads that are consistent with alternative methods such as plating or flow cytometry of tissue homogenates11. Plating and flow cytometry cannot be done directly from the same tissues because they need to be perfusion-fixed for STP tomography. Instead, they have to be done with additional animals that are not fixed. If the median bacterial loads as determined by the various approaches differ by more than 3-fold, the viability of fluorescent bacteria might be compromised (in case of lower colony-forming units) or some bacteria might have lost the fluorescent reporter construct (in case of higher colony-forming units). Control experiment will be required to identify the source of such discrepancies.
STP tomography provides the localization of bacterial cells within the 3D structure of the infected tissues. The second harmonic signals of collagen provide anatomical landmarks such as arteries and trabeculae. In addition, host cells can be stained in vivo by injecting an antibody to surface markers prior to perfusion (Figure 5 and Figure 6). This staining provides additional landmarks for tissue compartments and specific microenvironments including inflammation foci (intracellular markers and some compartments with diffusional barriers such as the splenic white pulp or the brain are not easily accessible suitable for this in vivo staining). This approach revealed the splenic white pulp as a tissue compartment that enables long-term survival of Salmonella during antimicrobial chemotherapy11.
Finally, Salmonella replicating at moderate or slow rates can be identified and localized within the 3D structure of tissues using strains that express timerbac, a single cell reporter for replication rate11,15.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66469/66469fig01.jpg" /> 
Figure 1: Procedure for whole-organ imaging using serial two-photon (STP) tomography. (A-B) The organ is harvested after transcardial perfusion and stored overnight in 4% paraformaldehyde (PFA) at 4 &#176;C. (C-E) The organ is embedded in oxidized agarose and cross-linked, then the tissue is scanned and sliced using STP tomography. The slices are collected for subsequent immunohistochemistry. (F-J) Computational analysis pipelines for quantifying bacterial numbers, confirmation of fluorescent bacteria, and 3D reconstruction of bacterial positions. <a href="https://www.jove.com/files/ftp_upload/66469/66469fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66469/66469fig02.jpg" /> 
Figure 2: Serial two-photon (STP) tomography setup. (A) Tomograph which incorporates (B) 2-photon imaging with (C) automated serial tissue sectioning. (D) Collected tissue slices for follow-up investigations. <a href="https://www.jove.com/files/ftp_upload/66469/66469fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66469/66469fig03.jpg" /> 
Figure 3: Tile stitching and illumination correction. Tiles are stitched and uneven illumination is corrected for using corrected intensity distributions using regularized energy minimization (CIDRE). <a href="https://www.jove.com/files/ftp_upload/66469/66469fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66469/66469fig04.jpg" /> 
Figure 4: Segmentation of Salmonella expressing the green fluorescent protein (GFP) using a Support Vector Machine (SVM) and a Convolutional Neural Network (CNN). (A) Representative images of GFP objects segmented by SVM (left) and corresponding images (right) with regions segmented by SVM (Scale bar: 10 &#181;m). (B) Clustered red-green-blue (RGB) values distribution for the segmented regions. (C)&#160;Representative images of non-GFP objects falsely identified by the SVM as bacteria (left). CNN correctly dismisses them as background (right, scale bar: 10 &#181;m). <a href="https://www.jove.com/files/ftp_upload/66469/66469fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66469/66469fig05.jpg" /> 
Figure 5: Detection of Salmonella expressing the green fluorescent protein (GFP) by tomography and confirmation by immunohistochemistry. Images of the same section acquired by tomography (left) or confocal microscopy after staining with an antibody to Salmonella lipopolysaccharide (right). Neutrophils (red) were stained by in vivo injection of a PE-labeled anti-Ly-6G antibody prior to perfusion. <a href="https://www.jove.com/files/ftp_upload/66469/66469fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66469/66469fig06.jpg" /> 
Figure 6: 3D reconstruction and localization of Salmonella in infected mouse spleen. (A) Three-dimensional (3D) reconstruction of a 5 mm thick spleen slice and stained in vivo before perfusion with anti-CD169 antibody (red). The blue signal represents collagen detected by second harmonics. (B) One optical plane of the 3D stack shown in (A). The positions of Salmonella cells or microcolonies are indicated by stars. (C) 3D reconstruction of Salmonella positions&#160;(stars) in infected liver. The arteries are visible based on their collagen sheaths (blue). Scale bar: 1 mm. <a href="https://www.jove.com/files/ftp_upload/66469/66469fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Read this Scientific Article Protocol about Author Spotlight: Unraveling Bacterial Responses to Antibiotics and Immune System in Tissues at JoVE.com

Author Spotlight: Unraveling Bacterial Responses to Antibiotics and Immune System in Tissues

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...

high-resolution-three-dimensional-whole-organ-tomography-microbial

Research

JoVE Journal

Immunology and Infection

752 Views.  University of Basel. Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

Video: Author Spotlight: Unraveling Bacterial Responses to Antibiotics and Immune System in Tissues

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and sectioning of intact microbial colony biofilms using perfused paraffin wax. To adapt this method for use on colony biofilms, we developed techniques for maintaining each sample on its growth substrate and laminating it with an agar overlayer, and added lysine to the fixative solution. These optimizations improve sample retention and preservation of micromorphological features. Samples prepared in this manner are amenable to thin sectioning and imaging by light, fluorescence, and transmission electron microscopy. We have applied this technique to colony biofilms of Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilis, and Vibrio cholerae. The high level of detail visible in samples generated by this method, combined with reporter strain engineering or the use of specific dyes, can provide exciting insights into the physiology and development of microbial communities.

We describe fixation, paraffin embedding, and thin sectioning techniques for microbial colony biofilms. In prepared samples, biofilm substructure and reporter expression patterns can be visualized by microscopy.

Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.