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 r...

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 border="0" cellpadding="0" cellspacing="0" style="width:1015px;" width="1014">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">5% sheep blood</td>
			<td style="width:343px;">Hardy Diagnostics (Santa Maria, CA)</td>
			<td style="width:139px;">A10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-Gal)</td>
			<td>ThermoScientific</td>
			<td>R0402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Ampicillin</td>
			<td style="width:343px;">Fisher Scientific</td>
			<td style="width:139px;">BP1760-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">C57Bl/6 Mice</td>
			<td style="width:343px;">Charles River</td>
			<td style="width:139px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Chloramphenicol</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td style="width:139px;">C-0378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">confocal laser scanning microscope</td>
			<td>Zeiss</td>
			<td style="width:139px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cryostat microtome</td>
			<td>Thermo Scientific</td>
			<td style="width:139px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture, Cap</td>
			<td>VWR International</td>
			<td>2005-02512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">D+2:25NA Oligo</td>
			<td>Integrated DNA Technologies (Coralville, Iowa)</td>
			<td style="width:139px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA Ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Erythromycin</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td style="width:139px;">E5389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow Analyzer</td>
			<td>Becton Dickinson</td>
			<td style="width:139px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glass beads</td>
			<td>Sigma</td>
			<td>Z273627</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Miniprep, plasmid</td>
			<td>Promega</td>
			<td>A1220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">orbital shaking water bath</td>
			<td>New Brunswick Innova</td>
			<td style="width:139px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR purification</td>
			<td>QIagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Phosphate Buffer Saline (PBS)</td>
			<td style="width:343px;">Cellgrow</td>
			<td style="width:139px;">46-013-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate reader</td>
			<td style="width:343px;">Tecan</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Precellys 24 homogenizer</td>
			<td>Bertin Laboratories</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pUC57-Kan</td>
			<td>GenScript (Piscataway, NJ)</td>
			<td style="width:139px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Q5 Taq DNA Polymerase</td>
			<td>New England Biolabs (Ipswich, MA)</td>
			<td style="width:139px;">M0491S</td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:367px;">Restriction Enzymes</td>
			<td>New England Biolabs (Ipswich, MA)</td>
			<td style="width:139px;">R0150S (PvuI), R3136S (BamHI), R0144S (BglII), R3131S (NheI), R0101S (EcoRI), R0141S (SmaI).</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reverse transcriptase</td>
			<td>New England BioLabs</td>
			<td>E6300L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Sanger Sequencing</td>
			<td>Genewiz (Germantown, MD)</td>
			<td style="width:139px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sub Xero clear tissue freezing medium</td>
			<td>Mercedes Medical</td>
			<td style="width:139px;">MER5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost Plus microscope slides</td>
			<td>Fisher Scientific</td>
			<td style="width:139px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">superloop</td>
			<td>GE Lifesciences</td>
			<td>18111382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe, Filter</td>
			<td>VWR International</td>
			<td>28145-481</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syto 40</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:139px;">S11351</td>
			<td>Membrane permeant nucleic acid stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Tetracycline</td>
			<td style="width:343px;">Sigma-Aldrich</td>
			<td style="width:139px;">T7660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Tryptic Soy Broth</td>
			<td style="width:343px;">VWR</td>
			<td style="width:139px;">90000-376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV-visible spectrophotometer</td>
			<td>Beckman Coulter-DU350</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield Antifade Mounting medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td style="width:139px;">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 ...

This method can reveal spatial patterns of bacterial gene expression and strategies for physiological adaptation to host tissue micro-environments that are lost when averaged across the entire population in tissues. With this technique potential heterogeneities can be detected that must be considered when identifying new bacterial pathways and proteins to be targeted by antivirulence or antibacterial compounds. Begin by streaking S.aureus strains of interest on blood agar plates from a frozen glycerol stock and incubating the plates at 37 degrees Celsius for 16 to 24 hours to confirm their hemolytic phenotypes based on their ability to lyse red blood cells and to form clear transparent zones.Inoculate single colony isolates of each strain in four milliliters of tryptic soy broth, or TSB, in sterile glass tubes and rotate the tubes at 37 degrees Celsius for 16 to 24 hours in a tube roller at an approximately 70 degree angle and 70 rotations per minute. The next morning measure the optical density at 600 nanometers, or OD 600, of the cultures on a spectrophotometer and dilute the cells to an OD 600 of 0.05 in 25 milliliters of sterile TSB at a five to one flask to volume ratio in 125 milliliter Delong flasks. Incubate the cultures in a 37 degree Celsius water bath at 280 rotations per minute and harvest the cells during the exponential phase.Pellet the cells by centrifugation and wash the cells two times in an equal volume of sterile PBS. Then resuspend the cells in fresh PBS to a one times 10 to the eighth colony forming units per milliliter concentration for their subsequent injection into a recipient animal. At the appropriate experimental end point, harvest the kidney, heart, liver, lungs, and spleen into 15 milliliter polypropylene tubes containing 10%buffered formalin for a 24 to 48 hour incubation in the dark at room temperature with gentle shaking or rotation.At the end of the fixation, embed the organs in clear tissue freezing medium for storage at minus 80 degrees Celsius. Use a cryostat to acquire 10 micrometer thick tissue sections and dry the sections on individual pre-cleaned charged glass slides for 20 minutes in the dark before applying hard mounting medium supplemented with DAPI. Then apply cover slips and cure the slides at room temperature overnight before transferring the samples to long term storage at four degrees Celsius.To identify lesions within the samples, place a slide on the stage of a laser scanning confocal microscope and use an appropriate objective for visualizing individual cells to acquire images of the lesions. To measure the fluorescence intensities within the confocal images, open an image in ImageJ and adjust the brightness and contrast to properly visualize the fluorescence signal of the lesion. Define the region of interest by using the polygon selections tool and add the ROI to the ROI manager under the Edit tab in ImageJ.Select Edit, then Selection, and finally Add to Manager. Next, rename the ROI and label the respective image. To define the centroid, open the Analyze tab and select Area, Mean Gray Value, Centroid, and Limit to Threshold.Extract the centroid fluorescence intensity value or measure the mean fluorescence intensity in a given lesion per unit area. This can be achieved by using the thresholding function to set the lower and upper fluorescence limits as necessary. Export the data to an Excel file and prepare a column indicating the mean fluorescence intensity per unit area for the periphery and core.To calculate the mean fluorescence intensities per unit area in the periphery and core, manually define the upper and lower limits of thresholding. Staphylococcus aureus thermonuclease GFP fusion fluorescence is on average nearly ninefold higher in cells carrying the fusion gene than in cells not carrying the reporter fusion. Similarly, tandem dimeric Tomato fusion fluorescence is about sixfold higher than the null reporter control.The pattern of reporter activities can be confirmed by flow cytometry with tissue homogenates. Although the fold differences in fluorescence are typically lower. Variations in the fluorescence measurements can be due to heterogeneous expression of the reporters.For example, in these representative samples it was observed that some lesions expressed either one or both of the reporters within approximately 100 micrometers of distance in the same tissue sample. Examining the reporter activity to a single cell resolution in staphylococcal abcess communities reveals a spatial regulation of staphylococcus aureus thermonuclease expression in abscesses circumscribed with strong DAPI staining, likely associated with the formation and release of neutrophil extracellular traps. For example, significantly higher staphylococcus aureus thermonuclease GFP fusion fluorescence is observed in the interior core of the staphylococcal abcess community compared to localized to the periphery.In the same abcess, the pattern for the tandem dimeric Tomato fusion fluorescence appears to be inverted. However, the pattern in this experiment was not statistically significant. Obtaining intact cryo-embedded dissections is vital for the fluorescence image analysis and should be done carefully.Sorting of the bacterial cells to capture sub-populations expressing the fusion to varying extents, coupled with RNA-Seq analysis could illuminate genome wide changes in the transcriptome. Take care when working with formalin as it is a carcinogen and may cause skin irritation, allergic reactions, or eye damage upon direct exposure. Generating mutant derivatives of strains carrying fluorescent reporters of interest can help reveal the genetic basis for the observed spatial regulatory patterns.

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Research

JoVE Journal

Genetics

8.7K Görüntüleme.  Georgetown University. Bu yöntem, bakterigen ekspresyonunun uzamsal desenlerini ve dokularda ortalama olarak tüm popülasyonda kaybolan doku mikro ortamlarına fizyolojik adaptasyon stratejilerini ortaya çıkarabilir. Bu teknik ile antivirülans veya antibakteriyel bileşikler tarafından hedef alınacak yeni bakteriyel yollar ve proteinler belirlenirken göz önünde bulundurulması gereken potansiyel heterojeniteler tespit edilebilir. Dondurulmuş bir gliserol stokundan kan agar plakaları üzerinde ilgi S.aur...