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

Summary

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.

Abstract

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.

Introduction

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 tissues^3,4,5. 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 tissues^{7,8,9,10,11,12,13}. 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 coagulase^14,18,19,20. 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 infection^25,26. 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 SarA^28,29, 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 activities^26,31. 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.

Protocol

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

1. Digest the genome integrative pRB4 vector sequentially with EcoRI and SmaI restriction enzymes. Following the manufacturer's protocol, set up an overnight digestion with SmaI using 1 µg of pRB4 at 25 °C, and then proceed with the second digestion for 1h at 37 °C by adding EcoRI to the reaction mixture. Inactivate the enzymes by incubating at 65 °C for 20 min.
2. 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' EcoRI restriction site and a 3' 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's recommendations with forward primer oRB015 (5'-atcattgaattctccaaagtaaattataagttatac-3') and reverse primer oRB016 (5'-gggcataactaacacctctttctttttag-3'). The annealing temperature was 53 °C. Purify the resulting fragment using a PCR clean-up kit following the manufacturer's instructions.
3. 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.
4. Electroporate the construct into S. aureus strain RN4220 as previously described³⁴. In brief, use 1 µg of plasmid with 70 µL of electro-competent cells (100 Ω, 25 µF, 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 (Em^r) at the permissive temperature (30 °C) on tryptic soy agar (TSA) supplemented with erythromycin (5 µg mL^-1) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal; 80 µg mL^-1). The resulting transformants are blue due to plasmid-encoded β-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.
5. Transfer plasmid to S. aureus USA300 strain of interest using electroporation or phage-mediated transduction³⁶ at the permissive temperature. In brief, propagate Φ₁₁ bacteriophage particles on the donor strain using TSA plates containing 5 mM CaCl₂ overnight at 30 °C, and then sterilize by filtration through a 0.45 µM cellulose acetate syringe filter. Use lysates to transduce S. aureus strain LAC to Em^r.
   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 Em^{r 37,38}.
6. Integrate the construct by two, successive homologous recombination events into the chromosome as described previously³². The recombinants are chloramphenicol-resistant (Cm^r) on TSA plates containing 5 μg mL^-1 of chloramphenicol, erythromycin-susceptible (Erm^s), 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.

2. Animal Infection: Preparation of the Inoculum, Systemic Infection, and Tissue Processing

1. Preparation of bacterial inoculum
   1. Streak S. aureus strains of interest for isolation on blood agar plates from a frozen glycerol stock. Incubate at 37 °C for 16–24 h to confirm hemolytic phenotypes based on their ability to lyse red blood cells (RBCs) and form clear transparent zones.
   2. 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 °C for 16–24 h in a tube roller set at approximately a 70° angle and 70 rotations per minute [RPM].
   3. 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 °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.
   4. 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.
   5. Harvest the cells during exponential phase (OD₆₀₀ ~0.6–0.8) by centrifuging at 3,000 x g for 10 min at room temperature.
   6. Wash the pellets twice in equivalent volume of sterile 1x phosphate-buffered saline (PBS; pH 7.4).
   7. 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.
2. Preparation of the animals and bacterial infection
   1. 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.
   2. Before infection, dilate mouse tail veins with lukewarm water.
   3. Infect animals by injecting 100 µL 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).
   4. Monitor animals daily and evaluate their health status using a monitoring system reviewed and approved by one's Institutional Animal Care and Use Committee.
   5. 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 cells^5,41.
3. Harvesting the organs and tissue processing
   1. Euthanize the animals by CO₂ inhalation and cervical dislocation as a secondary method and perform necropsy.
   2. 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.
   3. Transfer the left kidney to a sterile 2 mL impact-resistant tube containing ~500 µL of 2 mm silica beads and 1 mL sterile 1x PBS (pH 7.4). Proceed with this organ to step 4.2.
   4. 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.
   5. Embed organs in clear tissue freezing medium and store the tissues at -80 °C.
   6. Using a cryostat, section tissue into slices of 10 µm thickness.
   7. Dry the sections on a pre-cleaned, charged glass slide for 20 min in darkness, apply hard mounting medium with 4',6-diamidino-2-phenylindole (DAPI) stain, and apply coverslip. Cure mounted slides at room temperature overnight, and transfer to 4 °C for long-term storage.

3. Laser Scanning Confocal Microscopy and Image Processing

1. 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.
2. Acquire the image using the appropriate objective for visualizing individual cells (e.g., typically 20x or 40x objectives are used).
3. Measure the fluorescence intensities in the confocal images.
   1. Open the confocal image in Image J and adjust brightness/contrast to properly visualize the fluorescence signal of the lesion.
   2. Define the region of interest, and by using the Thresholding option, set the lower and upper fluorescence limits as necessary.
   3. Define the centroid by selecting Area → Mean gray value → Centroid → Limit to Threshold under the Analyze tab in Image J. 
   4. 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 µm² were used.
      NOTE: A blinded second analysis minimizes bias when the identity of the sample is known.
   5. Plot the data and perform the appropriate statistical analyses for each comparison.

4. Flow Cytometry Analysis

1. (Optional) Determine fusion activity of the inoculum on the day of the infection (from section 2.2.3)
   1. To 899 µL of 1x sterile PBS (pH 7.4) add 100 µL of each inoculum and 1 µL of membrane permeant nucleic acid stain (see Table of Materials). As a control, be sure to include a sample lacking nucleic acid stain.
   2. 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.
   3. To identify the bacterial population, plot events using nucleic acid stain (y-axis) and forward scatter (x-axis).
   4. 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 µm 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.
2. Analysis of tissue samples after sacrifice:
   1. 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 µL of 2 mm borosilicate beads.
   2. 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.
   3. Pellet the larger tissue debris by centrifuging at 250 x g for 3 min in a tabletop microcentrifuge cooled to 4 °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.
   4. Transfer 10 µL from the aqueous layer into a clean 1.5 mL microcentrifuge tube containing 1 µL of nucleic acid stain in 989 µL of PBS (total volume 1 mL).
   5. 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 "Live gate" 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.
   6. 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.

Results

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 (Ap^r) for maintenance and propagation in E. coli and...

Discussion

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

Materials

List of materials used in this article

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