Summary

High Throughput, in tempo reale, Dual-lettura Prove di intracellulare antimicrobica attività e eucariotica citotossicità cellulare

Published: November 16, 2016
doi:

Summary

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

Abstract

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

Introduction

Pathogens that grow or reside temporarily in intracellular compartments are difficult to therapeutically eradicate. Obligate or relatively obligate intracellular pathogens such as Legionella pneumophila, Coxiella burnetii, Brucella spp., Francisella tularensis, and Mycobacterium spp. often require prolonged courses of antimicrobial therapy for cure that may range from months to even years. Furthermore, extracellular pathogens may transiently occupy intracellular niches and in this way escape clearance by normal courses of antimicrobial therapy and later emerge to start new rounds of virulent infection. Staphylococcus aureus1 and uropathogenic Enterobacteriaceae2,3 infections are two increasingly recognized examples. Therefore, a fundamental drug discovery goal is to identify novel antimicrobials that penetrate into intracellular compartments. Optimal therapy to quickly eradicate intracellular organisms and prevent development of resistance through sub-inhibitory antimicrobial exposure is especially desirable.

To this end, we developed a high-throughput screening technology to identify intracellular-penetrant antimicrobials targeting the intracellular growth of the model pathogen, Legionella pneumophila.4 Previous clinical observations indicate that standard antimicrobial susceptibility testing did not accurately predict in vivo therapeutic efficacy against this organism.5 Specifically, this was because major classes of antimicrobials such as β-lactams and aminoglycosides, although highly effective against axenically grown Legionella, do not sufficiently penetrate into the intracellular compartments where Legionella resides.5,6 Later evidence suggested that technically more complex intracellular growth assays effectively predicted clinical efficacy.7 Unfortunately, these assays were extremely laborious endpoints assays, requiring infected macrophages, treated with antimicrobials, to be lysed at different times points for colony forming unit enumeration. Such assays are impractical to do on a large scale and are unsuitable for high-throughput drug discovery.

Therefore, we developed technology for real-time determination of intracellular bacterial growth.6 This was accomplished through use of a bacterial strain modified through integration of either a bacterial luciferase operon8 (first generation assay, described previously)4 or fluorescent protein9 reporters (second generation, orthogonal assay, described here) into the bacterial chromosome. In this way, luminescent or fluorescent signal provides a surrogate, real-time readout of bacterial number.

However, these attributes do not address a major confounder in intracellular infection assays, off-target effects on host cells. In particular, the death of the host cell inherently limits intracellular growth and leads to false positive identification of antimicrobial effect. As many compounds in screening libraries are eukaryotic cell toxic, such false positives would overwhelm true antimicrobials, necessitating a large number of follow-up, endpoint cytotoxicity assays for resolution.

Thus, it was of great interest to be able to assess eukaryotic cell viability and intracellular growth simultaneously. Notably, a characteristic of non-viable eukaryotic cells is loss of cell membrane integrity. Probes that test the permeability of the cell membrane may therefore be used to assess cell viability. We previously characterized the ability of a series of putatively cell membrane-impermeant, fluorescent, DNA-binding dyes to access and stain nuclear DNA of dead cells.4 On binding nuclear DNA, these dyes display a large increase in quantum fluorescent yield resulting in increased signal over background solution fluorescence. As such, these dyes provided a quantitative readout of eukaryotic cell death.4 Notably, we found that several were non-toxic themselves during prolonged co-incubation with J774 macrophages. When added during initial infection, they provided a real-time, fluorescent readout of eukaryotic cell death that can be measured by a microplate fluorimeter or observed microscopically.

Therefore, by combining use of a bacterial reporter and non-toxic, membrane-impermeant, DNA-binding dyes, we were able to develop a simple, non-destructive, real-time assay to measure both bacterial load and eukaryotic cell cytotoxicity simultaneously. This assay has allowed us to screen in 384-well plate format ~10,000 known bioactives including ~250 antimicrobials and >240,000 small molecules with functionally uncharacterized activity for the ability to inhibit intracellular growth of Legionella pneumophila, while at the same time generating eukaryotic cell cytotoxicity data for each compound.6 Our analysis of known antimicrobials against intracellular growth of Legionella was the most comprehensive exploration of this type to date.6

Based on the efficiency of our assay format, we also subsequently explored the potentially synergistic effects of known antimicrobials when used in combination. One of the most common synergy tests, the so-called checkerboard assay, is standardly performed by assessing combinatorial effects of two-fold serial dilutions of two or more antimicrobials.10 In these assays, synergy is defined by the observation of greater effect when two or more antimicrobials are applied together than the sum of the effects of each applied separately. Of note, heretofore, only focused and selective synergy testing was performed against intracellular Legionella pneumophila because of the great effort involved in traditional endpoint assays multiplied by the combinatorial permutations required.

To facilitate synergy testing, we made use of our real-time intracellular growth/eukaryotic cytotoxicity assay in combination with automated digital dispensing technology6. This automation permitted us to dispense serial dilutions of compounds dissolved in DMSO or aqueous solution alone or in combination in 384-well format.11 Furthermore, such robust liquid handling technology permitted us to easily perform higher resolution, square-root-of-two (rather than the standard, lower resolution, doubling) dilution combinations to achieve higher levels of specificity in our two-dimensional, checkerboard synergy analysis. This resolution was especially valuable in addressing concerns in the synergy field about reproducibility when using two-fold dilution series12. Lastly, our assay was quantitative and also therefore measured gradations of inhibition. As a result, the assay captured the entirety of inhibitory information, expressible in isocontour isobolograms in which isocontours connect combinatorial concentrations with similar levels of growth inhibition.6 This plotting strategy allowed visualization of combinatorial dose-response curves. To illustrate our methodology, we describe our protocol for performing these assays and show representative results.

Protocol

1. Real Time Intracellular Growth and Eukaryotic Cell Cytotoxicity Assay Preparing Host Cells (J774A.1 Cells) Culture J774A.1 Mus musculus macrophage-like cells in suspension in RPMI 1640 with 9% iron-supplemented calf serum. Initially passage in tissue culture flasks. After cells have become confluent in a 75 cm2 tissue culture flask in 15 ml of medium, split by scraping and dilution to 65 ml with the same type of medium, of which 15 ml is returned to the tissue culture flask…

Representative Results

Microplate intracellular growth assay Figure 1 diagrams the assay steps. The automated steps shown can be performed manually. However, throughput is greatly facilitated using liquid handling systems. Figure 2 shows representative results from a 384-well microplate, dual-readout, real-time intracellular growth and eukaryotic cell cytoto…

Discussion

We describe real-time assays for simultaneous detection of intracellular bacterial growth and host cell cytotoxicity. There are several critical steps in the protocol. First, for robust assay performance, there must be sufficient spectral separation between bacterial and cytotoxicity readouts. Such separation is intrinsic for combinations of luciferase operon reporters and fluorescent DNA-binding dyes. However, based on our experience (Table 1-3, Figure 2), use of dual, fluorescent reado…

Divulgations

The authors have nothing to disclose.

Acknowledgements

Research reported in this manuscript was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R01AI099122 to J.E.K. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We would like to thank Jennifer Smith, David Wrobel, Su Chiang, Doug Flood, Sean Johnston, Jennifer Nale, Stewart Rudnicki, Paul Yan, Richard Siu, and Rachel Warden from the ICCB-Longwood Screening Facility and/or the National Screening Laboratory for the New England Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases (supported by U54AI057159) for their assistance in development and performance of high-throughput screening assays. We also would like to thank Kenneth P. Smith for helpful comments on the manuscript.

Materials

J774A.1 cells American Type Culture Collection TIB-67 Host cell
ACES Sigma-Aldrich A9758  For making buffered charcoal yeast extract agar and buffered yeast extract medium
Yeast extract, ultrafiltered Becton-Dickinson/Difco 210929 For making buffered charcoal yeast extract agar and buffered yeast extract medium; lower grades may cause impaired growth and/or alter sensitivity of Legionella to growth inhibitors
Alpha-ketoglutaric acid, monopotassium salt Sigma-Aldrich K2000 For making buffered charcoal yeast extract agar and buffered yeast extract medium
Sodium pyruvate Sigma-Aldrich P5280 For making buffered charcoal yeast extract agar and buffered yeast extract medium
Potassium phosphate, dibasic Thermo Fisher Scientific P288-500 For making buffered charcoal yeast extract agar and buffered yeast extract medium
L-cysteine Sigma-Aldrich C-7755 For making buffered charcoal yeast extract agar and buffered yeast extract medium
Ammonium iron(III) citrate Sigma-Aldrich F5879 For making buffered charcoal yeast extract agar and buffered yeast extract medium; ferric pyrophosphate may be used instead but is more difficult to weigh accurately
Potassium hydroxide solution, concentrated Thermo Fisher Scientific SP236-500 For making buffered charcoal yeast extract agar and buffered yeast extract medium
Deonized water N/A N/A For making buffered charcoal yeast extract agar and buffered yeast extract medium
Thymidine (tissue culture grade) Sigma-Aldrich T1895 For supplementing both RPMI 1640 and buffered yeast extract agar/medium — lower grade thymidine may be used for the latter, but may cause impaired cell growth and/or cell death in RPMI 1640
RPMI 1640, standard formulation Corning via Thermo Fisher Scientific 10-040-CV For growing J774A.1 cells prior to plating; includes 2 mM L-glutamine
RPMI 1640 lacking phenol red Corning via Thermo Fisher Scientific 17-105-CV For plating J774A.1 cells in 384 well dishes (not suitable for growth prior to plating); also lacks L-glutamine — supplement to 2 mM before use
L-glutamine, 200 mM in 0.85% NaCl (tissue culture grade) HyClone via Thermo Fisher Scientific SH30034.02 For supplementing RPMI 1640 lacking L-glutamine, to 2 mM final concentration
Iron-supplemented calf serum Gemini Bioproducts 100-510 For supplementing RPMI 1640, to 9.1% final concentration
Trypan Blue solution Sigma-Aldrich T8154 For staining for J774A.1 cell death determination while counting cell density
SYTOX Green, 5 mM solution in DMSO Thermo Fisher Scientific S7020 For staining for J774A.1 cell death determination by fluorescence reading or epifluorescence microscopy (in conjunction with orange-red or far red fluorescent bacteria). Use at 125 nM final concentration.
GelRed, 10000X solution in water Biotium 41003 For staining for J774A.1 cell death determination by epifluorescence microscopy (in conjunction with green fluorescent bacteria). Use Gel Red  at 1X final concentration.  
Cell culture incubator Thermo Fisher Scientific 13-255-26 For incubation of J774A.1 cells (both before and after infection); can also be used for incubation of bacteria, or a standard atmosphere incubator can be used instead)
Orbital shaker BellCo Glass 7744-01010 For shaking incubation of J774A.1 cells before infection; fits inside cell culture incubator; includes shaker base 7744-01000 and tray 7740-01010 (these are also available separately)
Shaker flasks (250 ml) ChemGlass Life Sciences CLS-2038-04 For shaking incubation of J774A.1 cells before infection
Shaker clamps for flasks (250 ml) BellCo Glass 7744-16250 For shaking incubation of J774A.1 cells before infection
Shaker flasks (1000 ml) ChemGlass Life Sciences CLS-2038-07 For shaking incubation of J774A.1 cells before infection
Shaker clamps for flasks (1000 ml) BellCo Glass 7744-16100 For shaking incubation of J774A.1 cells before infection
Sponge foam caps for flasks (250 ml – 1000 ml) ChemGlass Life Sciences CLS-1490-038 For shaking incubation of J774A.1 cells before infection; reduces risk of contamination relative to standard metal caps
MultiDrop Combi programmable multichannel peristaltic pump Thermo Fisher Scientific 5840300 For dispensing J774A.1 cells, medium, and bacterial suspension containing fluorophores to large numbers of 384 well dishes
Combi standard bore manifold Thermo Fisher Scientific 24072670 Default predispense volume of 20 ml is insufficient to compensate for settling — increase to 80 ml
White 384 well dishes treated for tissue culture Corning 3570 For reading luminescence and fluorescence; Greiner catalog # 781080 also tested successfully
DMSO (tissue culture grade, in sealed ampoules) Sigma-Aldrich D2650 For dissolving positive control and test compounds
Azithromycin Sigma-Aldrich PHR1088 Antibiotic positive control
Saponin (from Quillaja bark) Sigma-Aldrich S-4521 Cytoxicity positive control
Multichannel pipettor Thermo Fisher Scientific Finnpipette For transfer of fixed amounts of positive control compounds; pipettor must have digital dispensing with detents to enable repetitive fixed volume dispensing
Epson pin transfer robot Epson/ICCB-L (Custom equipment) For transfer of fixed amounts of test compounds from library arrays
 D300 digital dispensing system Hewlett-Packard via Tecan D300 For transfer of variable amounts of test compounds ranging from 11 picoliters to 10 microliters
T8+ cartridges for D300 digital dispensing system Hewlett-Packard via Tecan T8+ For dispensing test compounds
EnVision multi-mode plate reader Perkin-Elmer (Contact manufacturer) For optimal detection of SYTOX Green fluorescence, use excitation filter 485/14, emission filter 535/25, and dichroic mirror 505 nm, with selection of minimum gain and transmittance, and “high concentration mode. For luminescence detection, use the "USLUM" protocol for high-sensitivity detection. For mNeptune2 detection, use excitation filter 600/8, emission filter 665/7.5, and dichroic mirror 658 nm, with selection of gain and transmittance to achieve the highest maximum signal possible without saturating the photomultiplier.
Epifluorescence microscope with computer-connected digital camera Nikon Ti For live cell imaging; any standard fluorescent microscope can substitute, with phase contrast or DIC optics, capable of imaging green (fluorescein), orange-red to red (Texas Red), and far-red (Cy5) fluorescence, with 100X oil objective for highest resolution
Glass-bottom tissue culture dishes MatTek Corporation P35G-1.5-20-C For live cell imaging. Dishes such as the MatTek allow microscopic visualization at 600X or 1000X magnification through use of an inverted epifluorescent or confocal microscope.  These specific dishes are 3.5 cm nominal diameter, 3.3 cm inside diameter, with 20 mm diameter #1.5 thickness cover slips inserted into the bottoms.
Photoshop CS6 Adobe Adobe photoshop or similar programs can be used to pseudocolor and merge light microscopic and fluorescent images.
Mathematica 10 Wolfam For generation of two-dimensioonal isocontour isobolograms and three-dimensional surface isobolograms.

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Chiaraviglio, L., Kang, Y., Kirby, J. E. High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity. J. Vis. Exp. (117), e54841, doi:10.3791/54841 (2016).

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