Name: a fluorescence-based method to study bacterial gene regulation in infected tissues
Uploaded: 2019-02-19T15:00:00.000+00:00
Duration: 7 min 10 s
Description: Watch this Scientific Journal Video about A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues at JoVE.com

We thank Alexander Horswill for the gift of the PsarAP1-tdTomato fusion, and Karen Creswell for help with flow cytometry analysis. We also thank Alyssa King for advice on statistical analysis. This work was funded in part by an NIH Exploratory/Developmental Research Award (grant AI123708) and faculty startup funds to SRB. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Georgetown University.
1. Generation of the Fluorescent Reporter Strain
<ol>
	<li>Digest the genome integrative pRB4 vector sequentially with EcoRI and SmaI restriction enzymes. Following the manufacturer&#39;s protocol, set up an overnight digestion with SmaI using 1 &#181;g of pRB4 at 25 &#176;C, and then proceed with the second digestion for 1h at 37 &#176;C by adding EcoRI to the reaction mixture. Inactivate the enzymes by incubating at 65 &#176;C for 20 min.</li>
	<li>PCR amplify the regulatory region of interest from genomic DNA (in this case, a ~350 base pair DNA fragment containing the thermonuclease [nuc] promoter region), incorporating a 5&#39; EcoRI restriction site and a 3&#39; SmaI restriction site. The SmaI recognition sequence must be in-frame with the translational start codon. In this study, the PCR was performed using a high-fidelity DNA Polymerase according to the manufacturer&#39;s recommendations with forward primer oRB015 (5&#39;-atcattgaattctccaaagtaaattataagttatac-3&#39;) and reverse primer oRB016 (5&#39;-gggcataactaacacctctttctttttag-3&#39;). The annealing temperature was 53 &#176;C. Purify the resulting fragment using a PCR clean-up kit following the manufacturer&#39;s instructions.</li>
	<li>Digest the resulting PCR product with EcoRI and SmaI as performed in step 1.1 and ligate the digested DNA fragment to the same sites of pRB4 using T4 DNA ligase as recommended by the manufacturer to generate the integrative construct. Introduce the ligation mixture into E. coli and confirm construct sequence.</li>
	<li>Electroporate the construct into S. aureus strain RN4220 as previously described34. In brief, use 1 &#181;g of plasmid with 70 &#181;L of electro-competent cells (100 &#937;, 25 &#181;F, and 2.3 kV) in B2 broth (1% [w/v] Casein hydrolysate, 2.5% [w/v] Yeast extract, 2.5% [w/v] NaCl, 0.1% [w/v] K2PO4, 0.5% [w/v] Glucose). Select for erythromycin resistance (Emr) at the permissive temperature (30 &#176;C) on tryptic soy agar (TSA) supplemented with erythromycin (5 &#181;g mL-1) and 5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-Gal; 80 &#181;g mL-1). The resulting transformants are blue due to plasmid-encoded &#946;-galactosidase activity. 
	NOTE: Transfer of DNA into clinical isolates is extremely difficult due to a strong restriction-modification barrier35. Thus, S. aureus RN4220 was used as an intermediate recipient of the fusion construct because it is restriction deficient and highly transformable.</li>
	<li>Transfer plasmid to S. aureus USA300 strain of interest using electroporation or phage-mediated transduction36 at the permissive temperature. In brief, propagate &#934;11 bacteriophage particles on the donor strain using TSA plates containing 5 mM CaCl2 overnight at 30 &#176;C, and then sterilize by filtration through a 0.45 &#181;M cellulose acetate syringe filter. Use lysates to transduce S. aureus strain LAC to Emr. 
	NOTE: LAC is a widely used clinical isolate that was previously made Em sensitive by serial passage in TSB medium to cure the strain of the native plasmid pUSA03 that confers Emr 37,38.</li>
	<li>Integrate the construct by two, successive homologous recombination events into the chromosome as described previously32. The recombinants are chloramphenicol-resistant (Cmr) on TSA plates containing 5 &#956;g mL-1 of chloramphenicol, erythromycin-susceptible (Erms), and no longer blue. 
	NOTE: When possible, re-introduce the marked fusion into USA300 via phage-mediated transduction to avoid the accumulation of mutations associated with in vitro strain passage39 and temperature shifts.</li>
</ol>
2. Animal Infection: Preparation of the Inoculum, Systemic Infection, and Tissue Processing
<ol>
	<li>Preparation of bacterial inoculum
	<ol>
		<li>Streak S. aureus strains of interest for isolation on blood agar plates from a frozen glycerol stock. Incubate at 37 &#176;C for 16&#8211;24 h to confirm hemolytic phenotypes based on their ability to lyse red blood cells (RBCs) and form clear transparent zones.</li>
		<li>Initiate overnight cultures by inoculating single colonies of each strain to 4 mL of tryptic soy broth (TSB) in sterile glass tubes. Rotate the tubes at 37 &#176;C for 16&#8211;24 h in a tube roller set at approximately a 70&#176; angle and 70 rotations per minute [RPM].</li>
		<li>Use a spectrophotometer to measure the optical density at 600 nm (OD600) of the cultures from step 2.1.2. Use sterile medium as an optical reference (blank). Dilute these cells to an OD600 of 0.05 in 25 mL of sterile Tryptic Soy Broth (TSB) in separate, 125 mL DeLong flasks (5:1 flask to volume ratio) and incubate cultures in a water bath (for proper heat transfer to the cultures), shaking at 280 RPM and set to 37 &#176;C. 
		NOTE: Growth of the inoculum can be performed in various ways; one method for growing cells to exponential phase is presented. Regardless, it is important to consistently use the same method for preparing the cells for inoculation.</li>
		<li>At an OD600 of ~1, re-dilute the cells into fresh medium to a starting OD600 of 0.05 to ensure the cells achieve exponential phase and that factors that accumulate during overnight incubation have been reduced to exponential phase levels.</li>
		<li>Harvest the cells during exponential phase (OD600 ~0.6&#8211;0.8) by centrifuging at 3,000 x g for 10 min at room temperature.</li>
		<li>Wash the pellets twice in equivalent volume of sterile 1x phosphate-buffered saline (PBS; pH 7.4).</li>
		<li>Suspend the cells in 1x PBS (pH 7.4) to a concentration of 1 x 108 colony forming units (CFUs) mL-1 or as appropriate for the desired experiment. 
		NOTE: it is important to determine the relationship between OD600 and CFU mL-1 for each strain of interest because strain-dependent alterations of cell size and shape can affect light scattering properties and ultimately, the infection dose.</li>
	</ol>
	</li>
	<li>Preparation of the animals and bacterial infection
	<ol>
		<li>Acclimate mice for 7 days on purified diet AIN-93. The diet is formulated to provide adequate nutrition while reducing tissue auto-fluorescence associated with the consumption of plant-based ingredients used in standard mouse chow40. 
		NOTE: Female C57/BL6 mice (6-8 weeks old) are used here, but the mouse strain and sex will depend on the study.</li>
		<li>Before infection, dilate mouse tail veins with lukewarm water.</li>
		<li>Infect animals by injecting 100 &#181;L of the bacterial inoculum via tail-veins to produce a systemic infection. Save an aliquot of the initial inoculum if performing flow cytometry (see Section 4).</li>
		<li>Monitor animals daily and evaluate their health status using a monitoring system reviewed and approved by one&#39;s Institutional Animal Care and Use Committee.</li>
		<li>Allow the infection to progress for the desired duration. Here, the experiments are terminated 3 days post-infection. 
		NOTE: Under these conditions, abscesses consist of a staphylococcal abscess community of bacteria, enclosed by fibrin deposits, and surrounded by concentric layers of immune cells5,41.</li>
	</ol>
	</li>
	<li>Harvesting the organs and tissue processing
	<ol>
		<li>Euthanize the animals by CO2 inhalation and cervical dislocation as a secondary method and perform necropsy.</li>
		<li>Harvest kidney (right) and other vital organs (heart, liver, lungs, spleen) and transfer into 15 mL polypropylene tubes containing 10% [v/v] buffered formalin. Proceed with these organs to step 2.3.4 below.</li>
		<li>Transfer the left kidney to a sterile 2 mL impact-resistant tube containing ~500 &#181;L of 2 mm silica beads and 1 mL sterile 1x PBS (pH 7.4). Proceed with this organ to step 4.2.</li>
		<li>Allow organs from step 2.3.2 to fix in the dark at room temperature with gentle shaking or rotation for at least 24 hours but no more than 48 hours.</li>
		<li>Embed organs in clear tissue freezing medium and store the tissues at -80 &#176;C.</li>
		<li>Using a cryostat, section tissue into slices of 10 &#181;m thickness.</li>
		<li>Dry the sections on a pre-cleaned, charged glass slide for 20 min in darkness, apply hard mounting medium with 4&#39;,6-diamidino-2-phenylindole (DAPI) stain, and apply coverslip. Cure mounted slides at room temperature overnight, and transfer to 4 &#176;C for long-term storage.</li>
	</ol>
	</li>
</ol>
3. Laser Scanning Confocal Microscopy and Image Processing
<ol>
	<li>Examine mounted slides to locate lesions using lasers appropriate for the fluorescent reporter being used. In this case, the green (GFP), red (tdTomato), and blue (DAPI) fluorescence signals are used.</li>
	<li>Acquire the image using the appropriate objective for visualizing individual cells (e.g., typically 20x or 40x objectives are used).</li>
	<li>Measure the fluorescence intensities in the confocal images.
	<ol>
		<li>Open the confocal image in Image J and adjust brightness/contrast to properly visualize the fluorescence signal of the lesion.</li>
		<li>Define the region of interest, and by using the Thresholding option, set the lower and upper fluorescence limits as necessary.</li>
		<li>Define the centroid by selecting Area &#8594; Mean gray value &#8594; Centroid &#8594; Limit to Threshold under the Analyze tab in Image J.&#160;</li>
		<li>Extract centroid fluorescence intensity value or measure the mean fluorescence intensity in a given lesion per unit area (mean fluorescence intensity, MFI) for GFP and tdTomato. Here, areas of one &#181;m2 were used. 
		NOTE: A blinded second analysis minimizes bias when the identity of the sample is known.</li>
		<li>Plot the data and perform the appropriate statistical analyses for each comparison.</li>
	</ol>
	</li>
</ol>
4. Flow Cytometry Analysis
<ol>
	<li>(Optional) Determine fusion activity of the inoculum on the day of the infection (from section 2.2.3)
	<ol>
		<li>To 899 &#181;L of 1x sterile PBS (pH 7.4) add 100 &#181;L of each inoculum and 1 &#181;L of membrane permeant nucleic acid stain (see Table of Materials). As a control, be sure to include a sample lacking nucleic acid stain.</li>
		<li>Perform flow cytometry on all samples. 
		NOTE: For the very first experiment, it is useful to include a vector only control for the fusion of interest to determine the proper voltage to use. A clear separation between positive and negative samples is essential for data analysis, indicating the reporter is well above background signal.</li>
		<li>To identify the bacterial population, plot events using nucleic acid stain (y-axis) and forward scatter (x-axis).</li>
		<li>Draw an inclusion gate around events that are both nucleic acid stain positive and the correct size of the bacterium based on the forward scatter (between 0.5 to 2.0 &#181;m for S. aureus). 
		NOTE: Be rigorous when identifying this population as it is critical to include events that are clearly within these parameters. Be sure this gate excludes all events for the negative controls under other conditions. In this study, roughly 70% the cell population met these requirements in the analysis.</li>
	</ol>
	</li>
	<li>Analysis of tissue samples after sacrifice:
	<ol>
		<li>Euthanize the animals, harvest the left kidneys (see step 2.3), and transfer into bead-beating tubes containing 1 mL of 1x sterile PBS (pH 7.4) and 250 &#181;L of 2 mm borosilicate beads.</li>
		<li>Disrupt tissues to release bacterial cells. For kidneys, use a homogenizer to disrupt cells. Here, three 30 s bursts at 6800 rpm with 1 min cooling periods on wet ice between cycles were used to minimize heating samples.</li>
		<li>Pellet the larger tissue debris by centrifuging at 250 x g for 3 min in a tabletop microcentrifuge cooled to 4 &#176;C. 
		NOTE: Typically, there is a cell pellet at the bottom of the tube and a layer of floating debris on the top. The middle aqueous layer contains the bacterial cells.</li>
		<li>Transfer 10 &#181;L from the aqueous layer into a clean 1.5 mL microcentrifuge tube containing 1 &#181;L of nucleic acid stain in 989 &#181;L of PBS (total volume 1 mL).</li>
		<li>Perform flow cytometry using the same data acquisition parameters and gating as for the initial inocula. 
		NOTE: Bacterial cell counts will be very low because the sample mainly contains tissue debris. The &#34;Live gate&#34; option can also be used if the cells are nucleic acid stain positive and size-gated when data are loaded into the application. This will also help to analyze more of the target events and reduce file size. Nearly one million events per sample are counted, but more event counts may be necessary depending on the severity of the infection and size of tissue analyzed.</li>
		<li>Data Analysis: Determine mean fluorescent intensity (MFI) for each fluorophore in a given sample using the inclusion gates determined in section 4.1.4. Normalize samples in flow analysis software to event counts. 10,000 counts are usually sufficient in the analysis. 
		NOTE: It is not uncommon to find samples that contain less than this number of events since this is dependent on abscess formation and infection severity. Using this approach, 500-40,000 events are typically found in infected tissue.</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing financial interests.

Bacterial infectious diseases are an increasing health problem worldwide due to the acquisition of antibiotic resistance determinants46. Because adaptation to host environments is essential for growth and survival during infection, strategies targeting gene expression programs that increase pathogen fitness may prove useful therapeutically. One such program is the set of genes controlled by the SaeR/S two component system (TCS), shown previously to play an essential role in immune evasion<sup clas...

Bacteria respond to changing physiological conditions and alterations in the nutritional state of their environment by differentially expressing genes required for adaptation and survival. For instance, opportunistic pathogens colonize body surfaces at relatively low densities, and are often harmless. However, once the bacterium has penetrated physical and chemical barriers, it must contend with host immune cell counter-defenses and restricted nutrient availability1. As an example, Staphylococcus aureus colonizes approximately one third of the population asymptomatically but is also the cause of devastating skin and soft tissue infections, osteomyelitis, endocarditis, and bacteremia2. The success of S. aureus as a pathogen is often attributed to its metabolic flexibility as well as an arsenal of surface-associated and secreted virulence factors that enable the bacterium to escape the bloodstream and replicate in peripheral tissues3,4,5. Because host death due to staphylococcal disease is an evolutionary dead end and limits transmission to new hosts6, the commitment to virulence factor production must be carefully controlled.
A complex regulatory network of proteins and non-coding RNAs responds to a variety of environmental stimuli, including cell density, growth phase, neutrophil-associated factors, and nutrient availability, to ensure that virulence genes are expressed at the precise time and location within host tissues7,8,9,10,11,12,13. For instance, the SaeR/S two component system (TCS) regulates expression of several virulence factors via the sensor kinase (SaeS) and the response regulator (SaeR)14. SaeS is autophosphorylated on a conserved histidine residue in response to host signals (e.g., human neutrophil peptides [HNPs], calprotectin)8,15,16. The phosphoryl group is then transferred to an aspartate residue on SaeR, activating it as a DNA-binding protein (SaeR~P)17. The SaeR/S TCS regulates over 20 genes that contribute to pathogenesis including fibronectin binding proteins (FnBPs), leukocidins, and coagulase14,18,19,20. Targets can be classified into high-affinity and low-affinity gene targets, which are likely induced as the level of SaeR~P rises when exposed to its cues21. The SaeR/S activity is controlled by other regulators of gene expression such as the Agr quorum sensing system, repressor of toxins protein (Rot), and the alternative sigma factor B (SigB)22,23,24.
nuc is an Sae-dependent virulence gene in Staphylococcus aureus and encodes thermonuclease (Nuc), which is essential for escaping from neutrophilic extracellular traps (NETs) and for dissemination during the course of infection25,26. The expression of nuc is also strongly indirectly repressed by CodY in the presence of branched-chain amino acids and GTP27, and directly repressed by the staphylococcal accessory regulator protein SarA28,29, whose activity is influenced by oxygen (redox state) and pH30. Given that sae and nuc mutants are attenuated in mouse models of infection, there is interest in developing chemical interventions that inhibit their corresponding activities26,31. Despite this, there is no information regarding their regulation during infection.
Fluorescent reporters have been used to monitor and quantify gene expression on the single cell level. Herein, we present a method for quantifying S. aureus gene expression during infection that, when paired with in vitro transcriptome analysis and powerful imaging techniques like magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS), can reveal how bacterial physiology is regulated in vivo and the relative abundances of nutrients in certain niches. The method can be applied to any bacterial pathogen with a tractable genetic system.
Overview of the genome integrative vector. 
The genome integrative vector pRB4 contains 500 base pairs each from the upstream and downstream regions of the S. aureus USA300 SAUSA300_0087 pseudogene to facilitate homologous recombination. pRB4 is derived from the temperature-sensitive pMAD vector backbone containing the erythromycin resistance cassette (ermC) and thermostable beta-galactosidase gene bgaB for blue/white screening of recombinants32. The engineered reporter construct also contains a chloramphenicol resistance marker (cat) for selection after genome integration and plasmid elimination, as well as EcoRI and SmaI sites to fuse the regulatory region of interest to superfolder green fluorescent protein (sGFP) (Figure 1). It is known that the choice of ribosome binding site (RBS) influences the activity of the reporter, and often requires empirical optimization33. Thus, an RBS is not supplied. Here, the native ribosome binding site is used to provide for a more natural pattern of gene expression, but other sites may be used.

<table><tbody><tr><td>5% sheep blood</td><td>Hardy Diagnostics (Santa Maria, CA)</td><td>A10</td><td></td></tr><tr><td>5-bromo-4-chloro-3-indolyl-&amp;beta;-D-galactopyranoside (X-Gal)</td><td>ThermoScientific</td><td>R0402</td><td></td></tr><tr><td>Ampicillin</td><td>Fisher Scientific</td><td>BP1760-25</td><td></td></tr><tr><td>C57Bl/6 Mice</td><td>Charles River</td><td>NA</td><td></td></tr><tr><td>Chloramphenicol</td><td>Sigma-Aldrich</td><td>C-0378</td><td></td></tr><tr><td>confocal laser scanning microscope</td><td>Zeiss</td><td>NA</td><td></td></tr><tr><td>cryostat microtome</td><td>Thermo Scientific</td><td>NA</td><td></td></tr><tr><td>Culture, Cap</td><td>VWR International</td><td>2005-02512</td><td></td></tr><tr><td>D+2:25NA Oligo</td><td>Integrated DNA Technologies (Coralville, Iowa)</td><td>NA</td><td></td></tr><tr><td>DNA Ligase</td><td>New England Biolabs</td><td>M0202S</td><td></td></tr><tr><td>Erythromycin</td><td>Sigma-Aldrich</td><td>E5389</td><td></td></tr><tr><td>Flow Analyzer</td><td>Becton Dickinson</td><td>NA</td><td></td></tr><tr><td>glass beads</td><td>Sigma</td><td>Z273627</td><td></td></tr><tr><td>Miniprep, plasmid</td><td>Promega</td><td>A1220</td><td></td></tr><tr><td>orbital shaking water bath</td><td>New Brunswick Innova</td><td>NA</td><td></td></tr><tr><td>PCR purification</td><td>QIagen</td><td>28106</td><td></td></tr><tr><td>Phosphate Buffer Saline (PBS)</td><td>Cellgrow</td><td>46-013-CM</td><td></td></tr><tr><td>Plate reader</td><td>Tecan</td><td>NA</td><td></td></tr><tr><td>Precellys 24 homogenizer</td><td>Bertin Laboratories</td><td>NA</td><td></td></tr><tr><td>pUC57-Kan</td><td>GenScript (Piscataway, NJ)</td><td>NA</td><td></td></tr><tr><td>Q5 Taq DNA Polymerase</td><td>New England Biolabs (Ipswich, MA)</td><td>M0491S</td><td></td></tr><tr><td>Restriction Enzymes</td><td>New England Biolabs (Ipswich, MA)</td><td>R0150S (PvuI), R3136S (BamHI), R0144S (BglII), R3131S (NheI), R0101S (EcoRI), R0141S (SmaI).</td><td></td></tr><tr><td>Reverse transcriptase</td><td>New England BioLabs</td><td>E6300L</td><td></td></tr><tr><td>Sanger Sequencing</td><td>Genewiz (Germantown, MD)</td><td>NA</td><td></td></tr><tr><td>Sub Xero clear tissue freezing medium</td><td>Mercedes Medical</td><td>MER5000</td><td></td></tr><tr><td>Superfrost Plus microscope slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>superloop</td><td>GE Lifesciences</td><td>18111382</td><td></td></tr><tr><td>Syringe, Filter</td><td>VWR International</td><td>28145-481</td><td></td></tr><tr><td>Syto 40</td><td>Thermo Fisher Scientific</td><td>S11351</td><td>Membrane permeant nucleic acid stain</td></tr><tr><td>Tetracycline</td><td>Sigma-Aldrich</td><td>T7660</td><td></td></tr><tr><td>Tryptic Soy Broth</td><td>VWR</td><td>90000-376</td><td></td></tr><tr><td>UV-visible spectrophotometer</td><td>Beckman Coulter-DU350</td><td>NA</td><td></td></tr><tr><td>Vectashield Antifade Mounting medium with DAPI</td><td>Vector Laboratories</td><td>H-1500</td><td></td></tr></tbody></table>

a fluorescence-based method to study bacterial gene regulation in infected tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

We developed a plasmid derived from pMAD32 that can deliver any reporter fusion construct into the chromosome by double crossover homologous recombination (Figure 1). This construct allows for quantitative analysis of any regulatory region that supports the production of GFP protein and fluorescent signal above background. The plasmid confers ampicillin resistance (Apr) for maintenance and propagation in E. coli and...

Watch this Scientific Journal Video about A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues at JoVE.com

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

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Genetics

8.8K Views.  Georgetown University. Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Video: A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

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 ...

Immunology and Infection

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

NF-&#954;B-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of various cytokines and chemokines. NF-&#954;B is constitutively expressed and is sequestered in the cytosol by the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (I&#954;B&#945;). Activation of NF-&#954;B requires the degradation of I&#954;B&#945;, which then exposes a nuclear localization signal on NF-&#954;B and promotes its trafficking to the nucleus. Once in the nucleus, NF-&#954;B binds to the promotor region of NF-&#954;B target genes such as interleukin 6 (IL-6) and IL-23, to promote their expression.
The activation of NF-&#954;B occurs independently of transcription or translation. Therefore, the activation state of NF-&#954;B must be measured either by quantifying NF-&#954;B specifically in the nucleus, or by quantifying expression of NF-&#954;B target genes. In this protocol, cells stably transfected with an NF-&#954;B::luciferase reporter construct are assayed for NF-&#954;B activation using in vitro tissue culture techniques. These cells are infected with Salmonella Typhimurium to activate NF-&#954;B, which traffics to the nucleus and binds to &#954;B sites in the promoter region of luciferase, inducing its expression. Cells are lysed and analyzed with the luciferase assay system. The amount of luciferase produced by the cells correlates with the intensity of the luminescence signal, which is detected by a plate reader. The luminescence signal generated by this procedure provides a quick and highly sensitive method by which to assess NF-&#954;B activation under a range of conditions. This protocol also utilizes quantitative reverse transcription PCR (RT-qPCR) to detect relative mRNA levels that are indicative of gene expression.

Here, we present a protocol to quickly and easily measure nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&#954;B) activation in cell lines expressing NF-&#954;B::luciferase reporter constructs, via measurements of luminescence in the cell lysate. Additionally, gene expression is determined via RT-qPCR isolated from cells infected with Salmonella Typhimurium.

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

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

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.

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.

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

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

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