Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.
Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.
Type III secretion systems are specialized protein-export machines utilized by different genera of gram-negative bacteria to directly deliver bacterially encoded effector proteins into eukaryotic target cells. While the secretion machinery itself is highly conserved, specialized sets of effector proteins have evolved among the different bacterial species to manipulate cellular signaling pathways and facilitate specific bacterial virulence strategies1. In case of Yersinia, up to seven effector proteins, so called Yops (Yersinia outer proteins), are translocated upon host cell contact and act together to subvert immune cell responses such as phagocytosis and cytokine production, i.e., to permit extracellular survival of the bacteria2-4. The process of translocation is tightly controlled at different stages5. It is established that primary activation of the T3SS is triggered by its contact to the target cell6. However, the precise mechanism of this initiation is yet to be elucidated. In Yersinia a second level of so called fine-tuning of translocation is achieved by up- or down-regulating activity of the cellular Rho GTP-binding proteins Rac1 or RhoA. Activation of Rac1 e.g. by invasin or cytotoxic necrotizing factor Y (CNF-Y) leads to increased translocation7-9, while the GAP (GTPase activating protein) function of translocated YopE down-regulates Rac1 activity and accordingly decreases translocation by a negative feedback type of mechanism10,11.
Valid and precise methods are the prerequisite to investigate how translocation is regulated during Yersinia host cell interaction. Many different systems have been used for this purpose, each with specific advantages and drawbacks. Some approaches rely on lysis of infected cells but not bacteria by different detergents followed by western blot analysis. The common drawback of these methods is that minor but inevitable bacterial lysis potentially contaminates the cell lysate with bacteria-associated effector proteins. However, treatment of cells with proteinase K to degrade extracellular effector proteins and subsequent use of digitonin for selective lysis of the eukaryotic cells were proposed to minimize this problem12. Importantly, these assays crucially depend on high quality anti-effector antibodies, which are mostly not commercially available. Attempts to use translational fusions of effector proteins and fluorescent proteins like GFP to monitor translocation were not successful probably due to the globular tertiary structure of the fluorescent proteins and the inability of the secretion apparatus to unfold them before secretion13. However, several different reporter tags like the cya (calmodulin-dependent adenylate cyclase) domain of the Bordetella pertussis toxin cyclolysin14 or the Flash tag were successfully used to analyze translocation. In the former assay the enzymatic activity of cya is used to amplify the signal of the intracellular fusion protein, while the Flash tag, a very short tetracysteine (4Cys) motif tag, allows for labeling with the biarsenical dye Flash without disturbing the process of secretion15.
The approach applied here was reported for the first time by Charpentier et al. and is based on the intracellular conversion of the Förster resonance energy transfer (FRET) dye CCF4 by translocated effector TEM-1 beta-lactamase fusions16 (Figure 1A). CCF4/AM is a cell-permeant compound in which a coumarin derivate (donor) and a fluorescein moiety (acceptor) are linked by a cephalosporin core. Upon passive entry into the eukaryotic cell, the non-fluorescent esterified CCF4/AM compound is processed by cellular esterases to the charged and fluorescent CCF4 and thereby trapped within the cell. Excitation of the coumarin moiety at 405 nm results in FRET to the fluorescein moiety, which emits a green fluorescence signal at 530 nm. After cleavage of the cephalosporin core by the beta-lactamase FRET is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission at460 nm. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., the technique was used in a mouse infection model to identify the leukocyte populations targeted for translocation in vivo17-19. Readout of signals can be conducted using plate readers, FACS analysis or fluorescence microscopy. Of note, the method also provides the possibility to monitor translocation in real-time by live-cell microscopy during the infection process20,21. Here laser scanning fluorescence microscopy was applied for readout of fluorescence signals as it provides highest sensitivity and accuracy. In particular, the capability to adjust the emission window with nanometer precision in combination with high-sensitive detectors facilitates optimized fluorescence detection and minimized cross-talk. In addition this microscopy setup can be adapted for real-time monitoring of translocation and potentially permits for simultaneous analysis of host-pathogen interaction at the cellular level.
In this study translocation rates of a Y. enterocolitica wild type strain and a YopE deletion mutant exhibiting a hypertranslocator phenotype10,11 were exemplarily analyzed.
1. Peak Emission and Cross-talk Determination (See Also Figure 2)
2. Preparation of Y. enterocolitica Strains for Cell Culture Infection
3. Plating HeLa Cells
4. Infection of HeLa Cells and Loading with CCF4
5. Laser Scanning Microscopy and Analysis of Data
To demonstrate the capability of the described method to quantitatively analyze effector translocation into target cells, two Yersinia strains with different translocation kinetics were studied: Y. enterocolitica wild type strain WA-314 (serogroup O8, harboring the virulence plasmid pYVO822) and its derivative WA-314ΔYopE (WA-314 harboring pYVO8ΔYopE23). Earlier work showed that Y. enterocolitica mutants lacking functional YopE exhibit a significantly higher Yop translocation rate10. Both strains were transformed with vectors encoding for fusions of the n-terminal secretion signal of YopE and TEM-1 beta-lactamase (pMK-bla)17. WA-314 was additionally transformed with a vector encoding for a respective YopE ovalbumin fusion (pMK-ova17) to serve as a negative control. Incubation times of 30, 60 and 90 min were applied in order to further assess the method`s effective range and suitability for quantification. Two different readouts were used for data analysis: donor fluorescence (Ch1) and relative FRET coefficients (Ch3 = Ch2’ / (Ch1 + Ch2’)) (Figure 1B and 1C).
For the negative control strain WA-314-pMK-ova plotting of donor channel intensities (Ch1) over time shows no rise of fluorescence emission irrespective of the incubation time (Figure 1B). In contrast, in WA-314-pMK-bla infected cells an increase of fluorescence intensity over time was detected indicating the presence of YopE beta-lactamase fusion in the target cell cytoplasm. As expected the maximum slope of fluorescence intensity is positively correlated with the length of infection (30 min of infection: 0.10/min, 60 min infection: 0.33/min, 90 min infection: 0.76/min) indicating that different concentrations of translocated beta-lactamase can be distinguished. In accordance with previous studies WA-314ΔYopE-pMK-bla infected cells exhibit more rapid increase of fluorescence intensity compared to the wild type strain (30 min of infection: 1.28/min, 60 min infection: 1.53/min, 90 min infection: 1.41/min). Interestingly, expanding infection to 90 min did not further accelerate the rise of donor fluorescence compared to 60 min of infection (Figure 1B). This finding can presumably be explained by the progressive cytotoxic effect exerted by WA-314ΔYopE-pMK-bla, which could either inhibit efficient translocation beyond 60 min of infection or lead to loss of fluorescence dyes due to disruption of plasma membrane integrity. The results provided by using the relative FRET coefficient (Ch3) for analysis are in good accordance with the results obtained by the donor channel alone. However, in some conditions (WA-314ΔYopE-pMK-bla, 60 and 90 min infection) representing very large amounts of translocated beta-lactamase fusion, obviously the CCF4 substrate was instantly processed before imaging could be started. These conditions would not allow for calculating a reasonable maximum negative slope because the relevant part of the curve is missed in this data representation (Figure 1C). Images of the relative FRET coefficient channel provide the possibility to compare differences between individual cells (Figure 1D).
Figure 1. Quantitative analysis of effector translocation by TEM-1 YopE fusions in Y. enterocolitica. (A) Hydrolysis of CCF4 substrate by translocated TEM-1 beta-lactamase was monitored by laser scanning microscopy. Excitation at 405 nm resulted in green fluorescence emission (530 nm) of the intact substrate and in blue fluorescence emission (460 nm) of the cleaved hydrolysis product (coumarin). (B, C) Microscopy data were quantitatively analyzed by plotting the donor channel intensity (Ch1) or calculation and plotting of the relative FRET coefficients (Ch3 = Ch2’ / (Ch1 + Ch2’)). Data are representative of three independent experiments. (D) Depicted micrographs (FRET channel) were taken from representative movies at the indicated time points. WA-314ΔE-pMK-bla infected cells show more rapid decrease of the relative FRET coefficients compared to the wild type WA-314-pMK-bla (60 min infection). Scale bar, 20 µm. Please click here to view a larger version of this figure.
Figure 2. Cross-talk determination of the fluorophores V450 and FITC. The spectra of both individual fluorophores were measured with the confocal microscope. Bleed-through of the donor V450 into the acceptor channel within the 525-535 nm detector range was calculated from a linear regression (insert). The mean percentage of crosstalk calculated from two measurements equals 0.33. Bleed-through of the acceptor FITC into the donor channel (455-465 nm) was not detectable over the level of noise. Please click here to view a larger version of this figure.
We here successfully applied a TEM-1 beta-lactamase reporter based assay for quantitative analysis of effector translocation by Y. enterocolitica. Many different variations of this sensitive, specific and relatively straightforward technique have been described in the literature. In this study laser scanning microscopy was conducted for most sensitive and precise detection of fluorescence signals. Specifically the correction for cross-talk between the donor and acceptor dyes and the individually adjustable detection bandwidths allow for superior accuracy of measurement compared to other variations of the method. The results clearly show that the method is capable of quantitatively analyzing translocation. Different incubation times representing different concentrations of intracellular beta-lactamase were positively correlated with the slope of donor fluorescence intensity. Also a significantly higher translocation rate of the WA-314ΔYopE compared to the wild type strain WA-314 could be demonstrated.
Two alternative options for data analysis were applied: Donor channel intensities and relative FRET coefficients. The advantage of using the donor channel is that it directly represents the accumulation of a CCF-4 hydrolysis product (coumarin). This approach is possible in a microscopic setup, because other than in a plate reader correction for differences in cell densities by using a ratio of donor/acceptor is not necessary. However, export of the CCF4 dye or its hydrolysis products is still a possible confounder in this approach. This disadvantage can be overcome by calculating the relative FRET coefficient.
In the described setup experiments were conducted in 96 well plates. This format allows for testing of multiple conditions in a single experiment and helps to save expensive chemicals (specifically CCF4/AM loading kit). In the described workflow it is critical to start image acquisition at a defined time point shortly after addition of the CCF4/AM loading solution because hydrolysis of CCF4 by the translocated beta-lactamase starts immediately. In case of processing to many conditions at once, adjustment of focus and lateral positions (step 5.1.4) may take too long and hamper a timely start of image acquisition. Therefore, the maximum number of parallel samples needs to be determined individually.
One of the main problems encountered using this method was the considerable export of CCF4 substrate by the HeLa cells at 37 °C. This phenomenon could not be sufficiently counteracted by treating the cells with probenecid. We therefore decided, instead of monitoring translocation during the ongoing infection as described previously, to first complete infection and afterwards load the cells with the CCF4/AM dye. With this approach it was possible to monitor the processing of the CCF4 substrate at RT. Under these conditions reduced export of CCF4 was observed. The amount of export of CCF4 substrate seems to be cell line specific; the setup described here allows for reliable analysis of translocation also in cell lines, which strongly export CCF4 at 37 °C.
Export of CCF4 is not a problem in some other cell lines allowing to adopt the microscopy setup for monitoring the processes of infection and translocation simultaneously in real-time. This provides the possibility to analyze translocation into individual cells and could be used to correlate translocation with specific events of host cell interaction like bacterial adhesion or formation of micro-colonies20. Therefore, this assay is a valuable tool to further elucidate the mechanisms of how translocation is regulated during the interplay of bacteria and host cells.
The authors have nothing to disclose.
We thank Dr. Antonio Virgilio Failla for providing the FRET acquisition quantification algorithm and Erwin Bohn for providing the pMK-bla and pMK-ova constructs.
LB-Agar | Roth | X969.2 | |
LB-Medium | Roth | X968.2 | |
Dulbecco’s Phosphate Buffered Saline | Sigma-Aldrich | D8662-500ML | |
96-well plate (black, clear bottom) | Greiner Bio One | 655087 | |
DMEM, high glucose, GlutaMAX Supplement, pyruvate | Gibco/Life Technologies | 31966-047 | |
Probenecid | Sigma-Aldrich | P8761-25G | |
LiveBLAzer FRET-B/G Loading Kit with CCF4-AM | Life Technologies | K1095 | |
Lectin from Triticum vulgaris (wheat) FITC conjugate | Sigma-Aldrich | L4895 | Used for peak emission and cross-talk determination |
V450 Rat anti-Mouse CD8a | BD Bioscience | 560469 | Used for peak emission and cross-talk determination |
Immersol W immersion oil | Zeiss | 444969-0000-000 | Refractive index = 1.3339 @ 23 °C |
TCS SP5 II confocal laser scanning microscope | Leica microsystems | 2x GaAsP-Hybrid detectors, 4 channel spectrometer, acusto optical beam spliter, motorized XY stage, adjustable pinhole, objective 20x HC PL APO CS IMM/CORR, 405nm diode laser 50mW | |
Imaris 7.6 software | Bitplane | Plugins included ImarisXT and MeasurementPro | |
MatLab compiler runtime | MathWorks | ||
Prism 5 | GraphPad software |