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Development of a Coxiella burnetii culture method for high-throughput assay to identify host-directed therapeutics
Journal of Microbiological Methods 169 (2020) 105813
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Development of a Coxiella burnetii culture method for high-throughput assay to identify host-directed therapeutics
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Cheryl N. Millera, , Maisha Khana,b, S. Ashraf Ahmedc, Krishna Kotaa, Rekha G. Panchala, Martha L. Halea a
Countermeasures Division, United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, United States Department of Chemistry and Physics, Hood College, 401 Rosemont Ave, Frederick, MD 21701, United States c Systems and Structural Biology, United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, United States b
A R T I C LE I N FO
A B S T R A C T
Keywords: Coxiella burnetii High-content imaging Host-directed therapeutics Axenic culture method
The intracellular Gram-negative bacterium, Coxiella burnetii, is a worldwide zoonotic pathogen and the causative agent of Q fever. The standard of care for C. burnetii infections involves extended periods of antibiotic treatment and the development of doxycycline-resistant strains stress the need for new treatment strategies. A previously developed axenic medium has facilitated in vitro growth of the organism. In this study, we have developed a simple culture method that is inexpensive, reliable and utilizes a modular hypoxic chamber system for either small or large scale production of bacteria without the need of a tri-gas incubator. This method provides consistent growth and yields sufficient viable bacteria within four days of culture and can be used for highthroughput screening. The viable bacteria were quantified by counting colony forming units and total bacteria were enumerated using a genomic equivalent method. The characterized bacterial inoculum was then used to optimize cell-based high-throughput immunofluorescence assays with a goal to quantify intracellular bacteria and then screen and identify compounds that inhibit early stages of C. burnetii infection in macrophages.
1. Introduction Coxiella burnetii is a wide-spread zoonotic pathogen and the causative agent of Q fever. The bacterium has a diverse animal reservoir that includes cattle, sheep, and goats as well as rodents, birds, and fish (Gurtler et al., 2014). Inhaled soil and dust that is contaminated with feces, urine, milk, placenta, and amniotic fluids from infected animals provide the primary mechanism for human infection (Gurtler et al., 2014). At least 50% of human C. burnetii infections are asymptomatic or result in nonspecific flu-like symptoms that are often undiagnosed (Dupuis et al., 1985). The acute infection may result in a self-limited fever and flu-like symptoms, and, if diagnosed in time, may be treated with antibiotics such as doxycycline (Kersh, 2013). Complications with the Q fever can be life-threatening and include endocarditis, vasculitis, osteomyelitis, hepatitis, interstitial lung fibrosis, and recurring fever (Gurtler et al., 2014; Dupuis et al., 1985; Melenotte et al., 2018; Harris et al., 2000). Treatment of Q fever endocarditis is among the longest for bacterial diseases. Initial combination therapies with antibiotics such as doxycycline and chloroquine have had some success (Harris et al., 2000; Calza et al., 2002). More recent recommendations for treatment of Q fever endocarditis of native ⁎
valves include antibiotic combinations of doxycycline and hydroxychloroquine for 18 months (Million et al., 2010). With the extended treatment regimes, the risk of development of antibiotic resistance increases. Also with the advancement in genetic manipulation of C. burnetii, there presents a potential risk for engineered antibiotic resistant strains to be developed. Additionally, C. burnetii infections may initiate development of B cell lymphomas, which further emphasize the need for novel medical interventions (Melenotte et al., 2016). As an obligate intracellular pathogen, C. burnetii has been difficult to culture, making the bacterium difficult to study. In mammalian cells, the bacterium replicates in highly acidic phagolysosomal compartments where oxygen concentration is sub-atmospheric (Omsland et al., 2008; Omsland et al., 2009). Development of an axenic culture medium that resembles the acidic environment of the bacterium's intracellular niche has drastically improved the ability to grow and study the microorganism. The low oxygen concentration critical for axenic culture can be difficult to maintain in a conventional CO2-based incubator that requires purging with N2 gas in order to maintain the low oxygen level (Omsland et al., 2008; Omsland et al., 2009; Omsland et al., 2011). In this study, we report the development of a simple culture system that utilizes a modular hypoxic chamber for small or large scale
Corresponding author at: 1425 Porter Street, Fort Detrick, Frederick, MD 21702, United States. E-mail address: [email protected] (C.N. Miller).
https://doi.org/10.1016/j.mimet.2019.105813 Received 6 November 2019; Received in revised form 16 December 2019; Accepted 16 December 2019 Available online 17 December 2019 0167-7012/ © 2019 Published by Elsevier B.V.
Journal of Microbiological Methods 169 (2020) 105813
C.N. Miller, et al.
production of the bacteria and results in reliable and consistent bacterial growth. This method is inexpensive as it does not require the purchase of a special incubator and can be set up in space constrained laboratories as it does not require two separate gas cylinders or a nitrogen generator. The bacteria generated using this method were characterized using the genomic equivalent and colony forming unit (CFU) methods. Infection conditions of macrophage cells were optimized with a goal to develop a quantitative, high-throughput, imagebased immunofluorescence screening assay that can identify compounds which effectively inhibit early stages of C. burnetii infection in macrophage cells.
chamber at a flowrate of 60 l per minute for four minutes. The chamber was then placed on a rotating platform (30 rpm) in a non-humidified incubator (37 °C). The chamber was gassed every one to two days. After 4 days, the culture was harvested by centrifugation at 1000 xg (4 °C) for 45 min. Culture fluid was removed and the bacterial pellet re-suspended to an OD600 = 0.350 in Dulbecco's modified minimal essential medium (DMEM) containing 20% fetal bovine serum and 20% DMSO and stored at −80 °C. For experimental studies, CBMNIIC4 was cultured as described at the beginning of this section except that CBNMIIC4 stock suspensions were used instead of the original seed suspension by adding 3 × 106 bacteria/ml to ACCM-2 medium.
2. Material and methods
2.3. Bacterial enumeration
2.1. Axenic medium and reagents Acidified cysteine citric medium (ACCM-2) was prepared using the formula developed by Omsland and coworkers (Omsland et al., 2008; Omsland et al., 2009) with minor modification: concentrated stock solutions of most chemicals were prepared and stored at 4 °C until medium preparation. Unless otherwise stated, all chemicals were purchased from Sigma Aldrich (St. Louis MO). When medium was needed, stock solutions were mixed together to yield the final concentrations: 13.4 mM citric acid, 16.1 mM sodium citrate, 3.67 mM potassium phosphate, 1 mM magnesium chloride, 0.02 mM calcium chloride, 0.01 mM iron sulfate (Fisher Scientific, Hampton, NH), 125.4 mM sodium chloride, L-cysteine (1.5 mM), 0.1 g/l Bacto Neopeptone (BD, Franklin Lakes, NJ), 2.5 g/l casamino acids (Fisher Scientific), methyl beta cyclodextrin (1 g/l), and 125 ml/l RPMI containing glutamax (Thermo Fisher Scientific, Waltham, MA). A 2× solution was prepared for the CFU assays. The pH of both preparations was adjusted to 4.75 with 6 N sodium hydroxide. Media was filter-sterilized, and stored at 4 °C for 1–2 weeks.
Viable bacteria were enumerated with colony forming units (CFU) (Omsland et al., 2011). Equal volumes of the 2× ACCM-2 were mixed with a 2% agarose solution and poured into sterile plastic petri dishes. Dilutions of the culture at an OD600 of 0.1 were added to the agar plate and spread with a glass spreader. The overlay solution (0.6% agarose mixed with equal volumes of 2× ACCM-2) was layered over the agar plate containing the bacteria. Plates were placed in the modular chamber with the premixed gas specified above and incubated for 10–14 days before colonies were enumerated. Additional bacterial quantification was determined using the genomic equivalence (GE) of the cultured suspension (Millar et al., 2017). One milliliter of bacterial suspension (OD600 = 0.10) was collected by centrifugation for 20 min at 16,000 xg, and DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Germantown, MD). The genome copies were calculated as previously described (Brennan and Samuel, 2003). DNA was quantified using a NanoDrop (Thermo Fisher Scientific), and the genome equivalence was determined based on the genome size of 1,987,028 base pairs for CBNMIIC4, and the average mass for a DNA base pair (615 Da).
2.2. Bacteria and culture method
2.4. Cell culture
Coxiella burnetii, nine mile phase II clone 4 (CBNMIIC4) was kindly provided by Dr. Robert Heinzen (Rocky Mountain Laboratories, St, Hamilton, MT) (Vodkin and Williams, 1986). To prepare stock suspensions, 50 μl of the original CBNMIIC4 seed was added to a T75 tissue culture flask with a vented cap containing 25 ml ACCM-2. The flasks were placed into a modular hypoxic chamber (Billups-Rothenberg, Del Mar, CA) and sealed according to the manufacturer's instructions (Fig. 1). Seventy-five liters of premixed gas containing 5% CO2, 2.5% O2, and 92.5% N2 (Airgas, Cherry Hill, NJ) were introduced into the
RAW264.7 murine macrophage-like cells (ATCC TIB-71) were grown at 37 °C in 5% CO2 in low glucose DMEM medium containing 10% FBS, 1% L-glutamine, 1% non-essential amino acids, and 1% HEPES buffer (complete DMEM). Cells above passage number 15 were not utilized for assays. 2.5. High-throughput screening assay The OD600 of CBNMIIC4 cultures grown in ACCM-2 was recorded
Fig. 1. Modular incubator chamber for culturing C. burnetii. Modular incubator chamber sealed with premixed gas containing 5% CO2, 2.5% O2, and 92.5% N2. A) Four T75 flasks containing CBNMIIC4 cultures can be grown in one chamber. B) Fifteen Petri dishes can be incubated in one chamber and the chambers are stackable. 2
Journal of Microbiological Methods 169 (2020) 105813
C.N. Miller, et al.
candidates are then separated as “Classified Spots” based on two parameters: 1) relative spot intensity (ratio of the spot peak intensity to the mean intensity of the cell region where the spot is searched) and 2) corrected spot intensity (mean spot intensity minus spot background intensity). Equations for signal-to-noise ratio and percent coefficient of variation are used for data analyses. The robustness of the assay was determined using Z' on a per plate basis (Z' = (1- (3 X STDEV of Signalmax (neutral control) + 3 X STDEV Signalmin (inhibitor control))/ ABS|mean Signalmax – mean of Signalmin|) (Zhang and McElvain, 1999). Only those plates that had a Z' > 0.3 were considered for data analysis. Acapella generated cell data was imported to GeneData (Basel, Switzerland) and used to calculate the Z' values. The percent infection was calculated using Acapella with the formula, % infection = ((cells with > 1 bacteria (spot)/total number of cells)) x 100. Cytotoxicity was scored by quantifying the loss in cell number per well compared to the infected control as detected by specifying nuclear region with Hoechst staining and whole cell region with CellMask Deep Red staining.
and the cells were harvested by centrifugation at 1,000 xg (4 °C) for 45 min. Pellets were resuspended in complete DMEM medium to an OD600 = 0.1, from which dilutions were prepared for cell infection studies. RAW264.7 macrophages were seeded in 384-well plates (4,000 cells/well) one day prior to infection using the automated Multidrop Combi Reagent Dispenser (Thermo Scientific). Compounds were added to the tissue culture cells using the JANUS automated workstation powered by Packard Innovations (Perkin Elmer, Waltham MA). After 2 h pretreatment with the compounds, macrophages were infected with C. burnetii at an MOI of 80 based upon the OD600 0.1 = 6.8 × 108 bacteria/ml using the automated ViaFill (Integra). The plates were then centrifuged at 400 xg for 10 min to initiate contact between the bacteria and the macrophages and then placed into a 37 °C incubator with 5% CO2. After 24 h, cells were fixed in 10% formalin diluted in PBS. 2.6. Immunofluorescence staining Following formalin fixation, cells were washed and stained using the automated EL 406 Washer Dispenser equipped with the Biostack 3 microplate stacker and vacuumbrand 8 (Bio Tek, Winooski VT). The cells were rinsed with PBS and cell membranes were made permeable by a 30 min treatment of PBS containing 0.1% Triton X-100. Cells were washed with PBS and blocked in PBS containing 3% bovine serum albumin for 4–24 h. After blocking, cells were incubated for 1 h with a 1:10,000 dilution of rabbit anti-Coxiella IgG (AB-R-COX, BEI Resources, Manassas, VA). The excess unbound antibody was removed, cells washed 3 times with PBS, and then incubated for 1 h with a 1:5,000 dilution of anti-rabbit IgG conjugated with DyLight 488 (Thermo Fisher Scientific). Excess antibody was removed and cells were stained with Hoechst nuclear dye (Thermo Fisher Scientific) and the host cell cytoplasmic stain CellMask Deep Red (Thermo Fisher Scientific), both at a 1:10,000 dilution.
2.8. Dose response curves Compounds were tested in quadruplicate in an 8-point 3-fold serial dilution that started at 30 μM. C. burnetii cultures were prepared as previously described for the high-throughput screening assay. The RAW264.7 macrophages were seeded in 384-well plates (4,000 cells/ well) one day before infection, treated with compounds at 2 h pre-infection, and then infected with CBNMIIC4 at an MOI of 80. The EC50 (potency) and CC50 (cytotoxicity) values for the compounds were generated in GeneData software. The EC50 and CC50 values were calculated based on normalized values using the One P Hill fit model. Normalization was done on a per-plate basis for each well the percentof-control (normalized activity) value based on the raw values of the Neutral control (Infected wells only) and a Blank control (cells only/ uninfected wells only). The experiments were repeated at least two independent times.
2.7. Image acquisition and analysis
3. Results
The automated image acquisition was completed with an Opera QEHS confocal system (PerkinElmer) and images analyzed using PerkinElmer's Acapella software as previously described (Kota et al., 2013; Pegoraro et al., 2014). Nine imaging fields per well were acquired using a 10× objective. Acapella's Spot Detection algorithm (PerkinElmer, Waltham MA) was used to detect and quantify internalized and cell-associated bacteria. Bacteria were detected as spots in a specified search region (WholeCell) having a higher intensity than its surroundings. To separate the spatial noise peaks and other artifacts, all spots detected initially were regarded as spot candidates. Spot
3.1. Culture method for C. burnetii To develop a simple culture system for C. burnetii, a modular hypoxic chamber was used for large scale production of the bacteria that resulted in reliable and consistent bacterial growth. C. burnetii undergoes biphasic development, differentiating from the sporogenic form called the small-cell variant (SCV) to the vegetative form called the large-cell variant (LCV) (Sandoz et al., 2014). When grown in ACCM-2,
Fig. 2. C. burnetii growth curve and enumeration A) CBNMIIC4 growth curve in ACCM-2 for 8 days. Left axis) Enumeration of CBNMIIC4 by colony forming units (CFU). Right axis) Optical density measurements at 600 nm (OD600) of the same CBNMIIC4 cultures. Data for OD600 and CFU/ml are generated from at least two independent experiments and error bars designate standard error of the mean. Dotted line indicates sample collection for bacterial quantification by CFU and genomic equivalence (GE). B) CBNMIIC4 enumeration of four day cultures normalized to OD600 of 0.1. To quantify C. burnetii, the OD600 of cultures grown in ACCM-2 was determined after 4 days in hypoxic conditions. The number of bacteria was enumerated by CFU and were compared to GE enumerations. The mean, standard deviation, and the standard error of the mean for each quantification method are shown for at least 15 biological replicates. 3
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environment in which the O2 concentration is maintained without constant airflow through the chamber. The configuration enabled the gas to be distributed evenly throughout the chamber and the tight seal maintained the gas concentration for 72 h (personal communication, Billups-Rothenberg, Inc. (Aminian et al., 2015; Focus, 2015)). In addition to the even distribution of the gases, the interior gas mixture is maintained without the need for a constant air flow, a feature that greatly reduces evaporation of the medium. The reduced evaporation proved particularly advantageous for CFU assays because the agar layers did not dry out during the incubation. The purpose for developing the CBNMIIC4 culture method was to provide reliable and consistent numbers of viable bacteria that were suitable for our screening assays. We demonstrated that cultures harvested at 4 days provided a consistent inoculum for cell infection for the high-throughput assays. After 4 days, however, the correlation between the OD600 and CFU was lost, a factor that may result from nutrient limitations and possible toxic byproducts produced from an initial high inoculum. C. burnetii cultured in ACCM-2 medium showed no drop in bacterial counts in ACCM-2 medium even after 22 days of culture starting with a lower initial inoculum (Sandoz et al., 2014). Sandoz et al. quantified C. burnetii number using focal forming units (FFU) and genomic equivalence (Sandoz et al., 2014). Similarly, Omsland et al. showed no drop in bacterial counts after seven days of growth in ACCM2 medium by quantifying GE (Omsland et al., 2011). It will be important to determine if CFU enumeration straight from ACCM-2 broth to ACCM-2 agar plates could delay the transition from SCV to LCV forms compared to that seen with FFU assay. The FFU assay results in a transition in pH and nutrients during infection, which could trigger the conversion of SCV to LCV that does not occur between ACCM-2 liquid culture and ACCM-2 agar. The drop in CFU could be a consequence of a lack in SCV conversion to LCV. Once infection is established in a host, C. burnetii quickly converts to the LCV (Coleman et al., 2004). The LCV form may be optimal to screen therapeutics against C. burnetii because the organism is more sensitive to potential stresses compared to the SCV. The spore-like SCV is highly stable and resistant to many harsh environmental conditions. While several groups have developed screening methods to identify novel therapeutics for C. burnetii (Czyz et al., 2014;Franklin et al., 2015), these methods do not evaluate potency against both bacteria (EC50) and macrophage cells (CC50) in the same assay. We adapted our high-throughput assay that has been successful in identifying antimicrobial compounds against other intracellular bacteria (Kota et al., 2013; Pegoraro et al., 2014; Madrid et al., 2013), and optimized the assay for C. burnetii. The Opera QEHS confocal system (PerkinElmer), which is a state-of-the-art high-content imaging system with full automation, allows for the quantification of intracellular bacterial number as well as the mammalian cell number from which the potency (EC50) and cellular toxicity (CC50) of the compounds in dose response studies can be determined. The assay was validated using statistical parameters, including Z', that provided a suitable dynamic range with minimal variation (Zhang and McElvain, 1999). Using this system we were able to determine that amiodarone was the most potent (EC50 = 7.1 μM) compound with no observed cytotoxicity (CC50 > 30 μM) against C. burnetii during the early stages (24 h) of macrophage infection when assessed in RAW264.7 cells. On the other hand, pentamidine was not effective in reducing C. burnetii numbers under similar conditions and concentrations above 1 μM were cytotoxic to the macrophages. The differences in the efficacy of the compounds to reduce C. burnetii number in our assay compared to those published previously (Czyz et al., 2014; Minnick et al., 2010), may be due to the different assays used, the different cell lines, and the different timing of infection. Czyz et al. infected THP-1 cells at an MOI of 20 for 5 days with fluorescent bacteria constitutively expressing mCherry and measured macrophage viability with a separate MTT assay (Czyz et al., 2014). While, Minnick et al. infected Vero cells at an MOI of 664 for 4 days, detected bacteria
C. burnetii is predominantly in the LCV during exponential growth until day four (Fig. 2). The initial optical density (OD600) of the culture was < 0.005 but reached approximately 0.15 ± 0.006 by day 4 at which point the bacteria reached stationary phase (Fig. 2A, right axis). The culture method presented here reaches stationary phase with different kinetics compared to previous methods and these differences could be due to the higher initial inoculation density used here (3 × 106 bacteria/ml) (Omsland et al., 2011; Sandoz et al., 2014). Comparison of the absorbance measurements with CFU enumeration (Fig. 2A, left axis) showed that beyond 4 day cultures, the number of CFUs significantly dropped while the optical density remained relatively constant, indicating that OD measurements did not correlate with the number of viable bacteria in stationary phase. Samples of the cultures were collected on day four and diluted to an OD600 of 0.1 for CFU and GE bacterial enumeration (Fig. 2B). Average viable bacterial counts were estimated to be 6.8 × 108 CFU/ml, while the GE were estimated to be 8.73 × 108 bacteria/ml. Together these results suggest that the 4 day bacterial cultures are ideal for cell-based infection studies as they contain high numbers of viable bacteria. 3.2. Optimization of multiplicity of infection for CBNMIIC4 strain in RAW264.7 macrophages After confirming that the bacterial culture system was consistent, the infection in RAW264.7 macrophages with CBNMIIC4 was optimized. To determine the ideal infection conditions, RAW264.7 macrophages were infected for 24 h with various MOIs of CBNMIIC4, as calculated by the CFU/ml determined above. There was an MOI-dependent increase in the number of bacteria per well, although only the MOIs of 40 and 80 were significantly different from the uninfected control (Fig. 3A). Similarly, an MOI-dependent increase in percent infection was also observed, and only MOIs of 16, 40 and 80 were significantly higher than the untreated control cells (Fig. 3B). The number of murine macrophages was unchanged between the uninfected control cells and those infected with any MOI, an indication that the bacteria were not killing the macrophages at the concentrations tested (Fig. 3C). The robustness of the assay was determined by calculating the Z' for bacterial number (Fig. 3D). Z' score > 0.3 are optimal for highthroughput assays, and the MOIs below 40 did not reach that score (Kota et al., 2013). The MOI of 80 gave the highest Z' score (0.57 ± 0.103), and therefore, was selected for subsequent studies. 3.3. Validation of the high-throughput screening assay for C. burnetii To validate our cell-based assay, four compounds were tested in dose-response studies, including several host-targeting compounds (bepridil, loperamide, and amiodarone) and one antimicrobial compound, (pentamidine) that were previously identified to reduce C. burnetii intracellular numbers (Czyz et al., 2014; Minnick et al., 2010). The dose response curves generated in these studies determined compound activity against both bacteria (EC50) and macrophage cells (CC50) (Fig. 4). Loperamide hydrochloride, amiodarone hydrochloride, and bepridil hydrochloride had average EC50 values of 11.35 μM, 7.1 μM, and 11.8 μM, respectively, and CC50 values of > 30 μM. Pentamidine did not exhibit a concentration-dependent effect on bacterial number and had a CC50 of 6.2 μM. From these studies, amiodarone was the most potent compound against C. burnetii and, therefore, will serve as a reference control in future screenings of small molecule chemical libraries. 4. Discussion Critical for the axenic culture of C. burnetii is the ability to maintain a 2.5% O2 environment. We have demonstrated that use of a modular hypoxic chamber instead of specialized incubators is sufficient for the growth of C. burnetii in vitro. The hypoxic chamber provides a controlled 4
Journal of Microbiological Methods 169 (2020) 105813
C.N. Miller, et al.
Fig. 3. Multiplicity of infection optimization for C. burnetii in RAW264.7 macrophages. A) The number of bacteria per well, B) the percent of cells infected with C. burnetii at 24 h post-infection, and C) the number of RAW264.7 macrophages. D) The robustness of the assay was determined by calculating the Z'. The mean from seven independent repeats were plotted and error bars represent standard error of the mean. Statistical significance was determined using ordinary one-way ANOVA using Dunnett's multiple comparisons test (GraphPad Prism, La Jolla, CA).
Funding
using genome equivalents, and measured macrophage viability by quantitative PCR. Both screens focused on late stages of infection where C. burnetii have replicated. Our primary goal was to identify antimicrobial agents that inhibit early stages of C. burnetii infection before bacterial replication and before the formation of the SCV. With this goal in mind, we are including C. burnetii in our high-throughput screening platform to identify compounds that disrupt host-pathogen interactions and thereby reduce bacteria number early during infection in macrophages.
This work was supported by the National Academy of Science, National Research Council fellowship funded through Defense Threat Reduction Agency (project 132141744 to CNM). Author statement The bacterial cell culture design and method was conceived by MLH. SAA and MLH developed modifications to the ACCM-2. The experiments were conceived and designed by CNM and MLH. CNM, MK, and MLH performed the experiments. CNM, RGP, and MLH analyzed and interpreted the data. CNM, MK, and MLH drafted the article. RGP and KK critically revised the article for important intellectual content.
5. Conclusions A simple method for culturing the bacterium was developed and a robust screening platform has been designed to identify host-directed compounds that reduce C. burnetii numbers in macrophage cells during early stages of infection. In our assay system, amiodarone was found to be a suitable control compound for high-throughput screening against C. burnetii. Currently, compound libraries are being screened to identify novel therapeutics to treat Q fever. This high-throughput assay can be easily adapted for the BSL-3 virulent strain of C. burnetii, to identify compounds directed at host pathways, which reduce bacteria number early during infection in macrophages. This assay can also be easily adapted for later times post-infection to identify compounds that inhibit C. burnetii intracellular replication. New compound that reduces C. burnetii number early during infection can be an effective treatment for doxycycline-resistant bacteria.
Declaration of Competing Interest We declare that no financial interests exist. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army. Acknowledgments We would like to thank Tara Kenny, Katlin Recabo, Glenn Gomba, Rouzbeh Zamani, and Sena Gul for their technical assistance. We would also like to thank Jennifer Scarff and Rachel Washart for their critical editing of the manuscript. We declare that no financial interests exist. Opinions, interpretations, conclusions, and recommendations are those 5
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Fig. 4. Dose response curves for host-directed compound activity against C. burnetii in RAW264.7 macrophages. The potency EC50 (green line) and the cytotoxicity CC50 (yellow line) were generated using an 8-point 3-fold dilution of compound. A) Amiodarone hydrochloride, B) Bepridil hydrochloride, C) Loperamide hydrochloride, and D) Pentamidine isethionate. Representative graphs from experiments that were repeated at least two independent times. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
of the authors and are not necessarily endorsed by the U.S. Army.
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Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Development of a Coxiella burnetii culture method for high-throughput assay to identify host-directed therapeutics
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Cheryl N. Millera, , Maisha Khana,b, S. Ashraf Ahmedc, Krishna Kotaa, Rekha G. Panchala, Martha L. Halea a
Countermeasures Division, United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, United States Department of Chemistry and Physics, Hood College, 401 Rosemont Ave, Frederick, MD 21701, United States c Systems and Structural Biology, United States Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, United States b
A R T I C LE I N FO
A B S T R A C T
Keywords: Coxiella burnetii High-content imaging Host-directed therapeutics Axenic culture method
The intracellular Gram-negative bacterium, Coxiella burnetii, is a worldwide zoonotic pathogen and the causative agent of Q fever. The standard of care for C. burnetii infections involves extended periods of antibiotic treatment and the development of doxycycline-resistant strains stress the need for new treatment strategies. A previously developed axenic medium has facilitated in vitro growth of the organism. In this study, we have developed a simple culture method that is inexpensive, reliable and utilizes a modular hypoxic chamber system for either small or large scale production of bacteria without the need of a tri-gas incubator. This method provides consistent growth and yields sufficient viable bacteria within four days of culture and can be used for highthroughput screening. The viable bacteria were quantified by counting colony forming units and total bacteria were enumerated using a genomic equivalent method. The characterized bacterial inoculum was then used to optimize cell-based high-throughput immunofluorescence assays with a goal to quantify intracellular bacteria and then screen and identify compounds that inhibit early stages of C. burnetii infection in macrophages.
1. Introduction Coxiella burnetii is a wide-spread zoonotic pathogen and the causative agent of Q fever. The bacterium has a diverse animal reservoir that includes cattle, sheep, and goats as well as rodents, birds, and fish (Gurtler et al., 2014). Inhaled soil and dust that is contaminated with feces, urine, milk, placenta, and amniotic fluids from infected animals provide the primary mechanism for human infection (Gurtler et al., 2014). At least 50% of human C. burnetii infections are asymptomatic or result in nonspecific flu-like symptoms that are often undiagnosed (Dupuis et al., 1985). The acute infection may result in a self-limited fever and flu-like symptoms, and, if diagnosed in time, may be treated with antibiotics such as doxycycline (Kersh, 2013). Complications with the Q fever can be life-threatening and include endocarditis, vasculitis, osteomyelitis, hepatitis, interstitial lung fibrosis, and recurring fever (Gurtler et al., 2014; Dupuis et al., 1985; Melenotte et al., 2018; Harris et al., 2000). Treatment of Q fever endocarditis is among the longest for bacterial diseases. Initial combination therapies with antibiotics such as doxycycline and chloroquine have had some success (Harris et al., 2000; Calza et al., 2002). More recent recommendations for treatment of Q fever endocarditis of native ⁎
valves include antibiotic combinations of doxycycline and hydroxychloroquine for 18 months (Million et al., 2010). With the extended treatment regimes, the risk of development of antibiotic resistance increases. Also with the advancement in genetic manipulation of C. burnetii, there presents a potential risk for engineered antibiotic resistant strains to be developed. Additionally, C. burnetii infections may initiate development of B cell lymphomas, which further emphasize the need for novel medical interventions (Melenotte et al., 2016). As an obligate intracellular pathogen, C. burnetii has been difficult to culture, making the bacterium difficult to study. In mammalian cells, the bacterium replicates in highly acidic phagolysosomal compartments where oxygen concentration is sub-atmospheric (Omsland et al., 2008; Omsland et al., 2009). Development of an axenic culture medium that resembles the acidic environment of the bacterium's intracellular niche has drastically improved the ability to grow and study the microorganism. The low oxygen concentration critical for axenic culture can be difficult to maintain in a conventional CO2-based incubator that requires purging with N2 gas in order to maintain the low oxygen level (Omsland et al., 2008; Omsland et al., 2009; Omsland et al., 2011). In this study, we report the development of a simple culture system that utilizes a modular hypoxic chamber for small or large scale
Corresponding author at: 1425 Porter Street, Fort Detrick, Frederick, MD 21702, United States. E-mail address: [email protected] (C.N. Miller).
https://doi.org/10.1016/j.mimet.2019.105813 Received 6 November 2019; Received in revised form 16 December 2019; Accepted 16 December 2019 Available online 17 December 2019 0167-7012/ © 2019 Published by Elsevier B.V.
Journal of Microbiological Methods 169 (2020) 105813
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production of the bacteria and results in reliable and consistent bacterial growth. This method is inexpensive as it does not require the purchase of a special incubator and can be set up in space constrained laboratories as it does not require two separate gas cylinders or a nitrogen generator. The bacteria generated using this method were characterized using the genomic equivalent and colony forming unit (CFU) methods. Infection conditions of macrophage cells were optimized with a goal to develop a quantitative, high-throughput, imagebased immunofluorescence screening assay that can identify compounds which effectively inhibit early stages of C. burnetii infection in macrophage cells.
chamber at a flowrate of 60 l per minute for four minutes. The chamber was then placed on a rotating platform (30 rpm) in a non-humidified incubator (37 °C). The chamber was gassed every one to two days. After 4 days, the culture was harvested by centrifugation at 1000 xg (4 °C) for 45 min. Culture fluid was removed and the bacterial pellet re-suspended to an OD600 = 0.350 in Dulbecco's modified minimal essential medium (DMEM) containing 20% fetal bovine serum and 20% DMSO and stored at −80 °C. For experimental studies, CBMNIIC4 was cultured as described at the beginning of this section except that CBNMIIC4 stock suspensions were used instead of the original seed suspension by adding 3 × 106 bacteria/ml to ACCM-2 medium.
2. Material and methods
2.3. Bacterial enumeration
2.1. Axenic medium and reagents Acidified cysteine citric medium (ACCM-2) was prepared using the formula developed by Omsland and coworkers (Omsland et al., 2008; Omsland et al., 2009) with minor modification: concentrated stock solutions of most chemicals were prepared and stored at 4 °C until medium preparation. Unless otherwise stated, all chemicals were purchased from Sigma Aldrich (St. Louis MO). When medium was needed, stock solutions were mixed together to yield the final concentrations: 13.4 mM citric acid, 16.1 mM sodium citrate, 3.67 mM potassium phosphate, 1 mM magnesium chloride, 0.02 mM calcium chloride, 0.01 mM iron sulfate (Fisher Scientific, Hampton, NH), 125.4 mM sodium chloride, L-cysteine (1.5 mM), 0.1 g/l Bacto Neopeptone (BD, Franklin Lakes, NJ), 2.5 g/l casamino acids (Fisher Scientific), methyl beta cyclodextrin (1 g/l), and 125 ml/l RPMI containing glutamax (Thermo Fisher Scientific, Waltham, MA). A 2× solution was prepared for the CFU assays. The pH of both preparations was adjusted to 4.75 with 6 N sodium hydroxide. Media was filter-sterilized, and stored at 4 °C for 1–2 weeks.
Viable bacteria were enumerated with colony forming units (CFU) (Omsland et al., 2011). Equal volumes of the 2× ACCM-2 were mixed with a 2% agarose solution and poured into sterile plastic petri dishes. Dilutions of the culture at an OD600 of 0.1 were added to the agar plate and spread with a glass spreader. The overlay solution (0.6% agarose mixed with equal volumes of 2× ACCM-2) was layered over the agar plate containing the bacteria. Plates were placed in the modular chamber with the premixed gas specified above and incubated for 10–14 days before colonies were enumerated. Additional bacterial quantification was determined using the genomic equivalence (GE) of the cultured suspension (Millar et al., 2017). One milliliter of bacterial suspension (OD600 = 0.10) was collected by centrifugation for 20 min at 16,000 xg, and DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Germantown, MD). The genome copies were calculated as previously described (Brennan and Samuel, 2003). DNA was quantified using a NanoDrop (Thermo Fisher Scientific), and the genome equivalence was determined based on the genome size of 1,987,028 base pairs for CBNMIIC4, and the average mass for a DNA base pair (615 Da).
2.2. Bacteria and culture method
2.4. Cell culture
Coxiella burnetii, nine mile phase II clone 4 (CBNMIIC4) was kindly provided by Dr. Robert Heinzen (Rocky Mountain Laboratories, St, Hamilton, MT) (Vodkin and Williams, 1986). To prepare stock suspensions, 50 μl of the original CBNMIIC4 seed was added to a T75 tissue culture flask with a vented cap containing 25 ml ACCM-2. The flasks were placed into a modular hypoxic chamber (Billups-Rothenberg, Del Mar, CA) and sealed according to the manufacturer's instructions (Fig. 1). Seventy-five liters of premixed gas containing 5% CO2, 2.5% O2, and 92.5% N2 (Airgas, Cherry Hill, NJ) were introduced into the
RAW264.7 murine macrophage-like cells (ATCC TIB-71) were grown at 37 °C in 5% CO2 in low glucose DMEM medium containing 10% FBS, 1% L-glutamine, 1% non-essential amino acids, and 1% HEPES buffer (complete DMEM). Cells above passage number 15 were not utilized for assays. 2.5. High-throughput screening assay The OD600 of CBNMIIC4 cultures grown in ACCM-2 was recorded
Fig. 1. Modular incubator chamber for culturing C. burnetii. Modular incubator chamber sealed with premixed gas containing 5% CO2, 2.5% O2, and 92.5% N2. A) Four T75 flasks containing CBNMIIC4 cultures can be grown in one chamber. B) Fifteen Petri dishes can be incubated in one chamber and the chambers are stackable. 2
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candidates are then separated as “Classified Spots” based on two parameters: 1) relative spot intensity (ratio of the spot peak intensity to the mean intensity of the cell region where the spot is searched) and 2) corrected spot intensity (mean spot intensity minus spot background intensity). Equations for signal-to-noise ratio and percent coefficient of variation are used for data analyses. The robustness of the assay was determined using Z' on a per plate basis (Z' = (1- (3 X STDEV of Signalmax (neutral control) + 3 X STDEV Signalmin (inhibitor control))/ ABS|mean Signalmax – mean of Signalmin|) (Zhang and McElvain, 1999). Only those plates that had a Z' > 0.3 were considered for data analysis. Acapella generated cell data was imported to GeneData (Basel, Switzerland) and used to calculate the Z' values. The percent infection was calculated using Acapella with the formula, % infection = ((cells with > 1 bacteria (spot)/total number of cells)) x 100. Cytotoxicity was scored by quantifying the loss in cell number per well compared to the infected control as detected by specifying nuclear region with Hoechst staining and whole cell region with CellMask Deep Red staining.
and the cells were harvested by centrifugation at 1,000 xg (4 °C) for 45 min. Pellets were resuspended in complete DMEM medium to an OD600 = 0.1, from which dilutions were prepared for cell infection studies. RAW264.7 macrophages were seeded in 384-well plates (4,000 cells/well) one day prior to infection using the automated Multidrop Combi Reagent Dispenser (Thermo Scientific). Compounds were added to the tissue culture cells using the JANUS automated workstation powered by Packard Innovations (Perkin Elmer, Waltham MA). After 2 h pretreatment with the compounds, macrophages were infected with C. burnetii at an MOI of 80 based upon the OD600 0.1 = 6.8 × 108 bacteria/ml using the automated ViaFill (Integra). The plates were then centrifuged at 400 xg for 10 min to initiate contact between the bacteria and the macrophages and then placed into a 37 °C incubator with 5% CO2. After 24 h, cells were fixed in 10% formalin diluted in PBS. 2.6. Immunofluorescence staining Following formalin fixation, cells were washed and stained using the automated EL 406 Washer Dispenser equipped with the Biostack 3 microplate stacker and vacuumbrand 8 (Bio Tek, Winooski VT). The cells were rinsed with PBS and cell membranes were made permeable by a 30 min treatment of PBS containing 0.1% Triton X-100. Cells were washed with PBS and blocked in PBS containing 3% bovine serum albumin for 4–24 h. After blocking, cells were incubated for 1 h with a 1:10,000 dilution of rabbit anti-Coxiella IgG (AB-R-COX, BEI Resources, Manassas, VA). The excess unbound antibody was removed, cells washed 3 times with PBS, and then incubated for 1 h with a 1:5,000 dilution of anti-rabbit IgG conjugated with DyLight 488 (Thermo Fisher Scientific). Excess antibody was removed and cells were stained with Hoechst nuclear dye (Thermo Fisher Scientific) and the host cell cytoplasmic stain CellMask Deep Red (Thermo Fisher Scientific), both at a 1:10,000 dilution.
2.8. Dose response curves Compounds were tested in quadruplicate in an 8-point 3-fold serial dilution that started at 30 μM. C. burnetii cultures were prepared as previously described for the high-throughput screening assay. The RAW264.7 macrophages were seeded in 384-well plates (4,000 cells/ well) one day before infection, treated with compounds at 2 h pre-infection, and then infected with CBNMIIC4 at an MOI of 80. The EC50 (potency) and CC50 (cytotoxicity) values for the compounds were generated in GeneData software. The EC50 and CC50 values were calculated based on normalized values using the One P Hill fit model. Normalization was done on a per-plate basis for each well the percentof-control (normalized activity) value based on the raw values of the Neutral control (Infected wells only) and a Blank control (cells only/ uninfected wells only). The experiments were repeated at least two independent times.
2.7. Image acquisition and analysis
3. Results
The automated image acquisition was completed with an Opera QEHS confocal system (PerkinElmer) and images analyzed using PerkinElmer's Acapella software as previously described (Kota et al., 2013; Pegoraro et al., 2014). Nine imaging fields per well were acquired using a 10× objective. Acapella's Spot Detection algorithm (PerkinElmer, Waltham MA) was used to detect and quantify internalized and cell-associated bacteria. Bacteria were detected as spots in a specified search region (WholeCell) having a higher intensity than its surroundings. To separate the spatial noise peaks and other artifacts, all spots detected initially were regarded as spot candidates. Spot
3.1. Culture method for C. burnetii To develop a simple culture system for C. burnetii, a modular hypoxic chamber was used for large scale production of the bacteria that resulted in reliable and consistent bacterial growth. C. burnetii undergoes biphasic development, differentiating from the sporogenic form called the small-cell variant (SCV) to the vegetative form called the large-cell variant (LCV) (Sandoz et al., 2014). When grown in ACCM-2,
Fig. 2. C. burnetii growth curve and enumeration A) CBNMIIC4 growth curve in ACCM-2 for 8 days. Left axis) Enumeration of CBNMIIC4 by colony forming units (CFU). Right axis) Optical density measurements at 600 nm (OD600) of the same CBNMIIC4 cultures. Data for OD600 and CFU/ml are generated from at least two independent experiments and error bars designate standard error of the mean. Dotted line indicates sample collection for bacterial quantification by CFU and genomic equivalence (GE). B) CBNMIIC4 enumeration of four day cultures normalized to OD600 of 0.1. To quantify C. burnetii, the OD600 of cultures grown in ACCM-2 was determined after 4 days in hypoxic conditions. The number of bacteria was enumerated by CFU and were compared to GE enumerations. The mean, standard deviation, and the standard error of the mean for each quantification method are shown for at least 15 biological replicates. 3
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environment in which the O2 concentration is maintained without constant airflow through the chamber. The configuration enabled the gas to be distributed evenly throughout the chamber and the tight seal maintained the gas concentration for 72 h (personal communication, Billups-Rothenberg, Inc. (Aminian et al., 2015; Focus, 2015)). In addition to the even distribution of the gases, the interior gas mixture is maintained without the need for a constant air flow, a feature that greatly reduces evaporation of the medium. The reduced evaporation proved particularly advantageous for CFU assays because the agar layers did not dry out during the incubation. The purpose for developing the CBNMIIC4 culture method was to provide reliable and consistent numbers of viable bacteria that were suitable for our screening assays. We demonstrated that cultures harvested at 4 days provided a consistent inoculum for cell infection for the high-throughput assays. After 4 days, however, the correlation between the OD600 and CFU was lost, a factor that may result from nutrient limitations and possible toxic byproducts produced from an initial high inoculum. C. burnetii cultured in ACCM-2 medium showed no drop in bacterial counts in ACCM-2 medium even after 22 days of culture starting with a lower initial inoculum (Sandoz et al., 2014). Sandoz et al. quantified C. burnetii number using focal forming units (FFU) and genomic equivalence (Sandoz et al., 2014). Similarly, Omsland et al. showed no drop in bacterial counts after seven days of growth in ACCM2 medium by quantifying GE (Omsland et al., 2011). It will be important to determine if CFU enumeration straight from ACCM-2 broth to ACCM-2 agar plates could delay the transition from SCV to LCV forms compared to that seen with FFU assay. The FFU assay results in a transition in pH and nutrients during infection, which could trigger the conversion of SCV to LCV that does not occur between ACCM-2 liquid culture and ACCM-2 agar. The drop in CFU could be a consequence of a lack in SCV conversion to LCV. Once infection is established in a host, C. burnetii quickly converts to the LCV (Coleman et al., 2004). The LCV form may be optimal to screen therapeutics against C. burnetii because the organism is more sensitive to potential stresses compared to the SCV. The spore-like SCV is highly stable and resistant to many harsh environmental conditions. While several groups have developed screening methods to identify novel therapeutics for C. burnetii (Czyz et al., 2014;Franklin et al., 2015), these methods do not evaluate potency against both bacteria (EC50) and macrophage cells (CC50) in the same assay. We adapted our high-throughput assay that has been successful in identifying antimicrobial compounds against other intracellular bacteria (Kota et al., 2013; Pegoraro et al., 2014; Madrid et al., 2013), and optimized the assay for C. burnetii. The Opera QEHS confocal system (PerkinElmer), which is a state-of-the-art high-content imaging system with full automation, allows for the quantification of intracellular bacterial number as well as the mammalian cell number from which the potency (EC50) and cellular toxicity (CC50) of the compounds in dose response studies can be determined. The assay was validated using statistical parameters, including Z', that provided a suitable dynamic range with minimal variation (Zhang and McElvain, 1999). Using this system we were able to determine that amiodarone was the most potent (EC50 = 7.1 μM) compound with no observed cytotoxicity (CC50 > 30 μM) against C. burnetii during the early stages (24 h) of macrophage infection when assessed in RAW264.7 cells. On the other hand, pentamidine was not effective in reducing C. burnetii numbers under similar conditions and concentrations above 1 μM were cytotoxic to the macrophages. The differences in the efficacy of the compounds to reduce C. burnetii number in our assay compared to those published previously (Czyz et al., 2014; Minnick et al., 2010), may be due to the different assays used, the different cell lines, and the different timing of infection. Czyz et al. infected THP-1 cells at an MOI of 20 for 5 days with fluorescent bacteria constitutively expressing mCherry and measured macrophage viability with a separate MTT assay (Czyz et al., 2014). While, Minnick et al. infected Vero cells at an MOI of 664 for 4 days, detected bacteria
C. burnetii is predominantly in the LCV during exponential growth until day four (Fig. 2). The initial optical density (OD600) of the culture was < 0.005 but reached approximately 0.15 ± 0.006 by day 4 at which point the bacteria reached stationary phase (Fig. 2A, right axis). The culture method presented here reaches stationary phase with different kinetics compared to previous methods and these differences could be due to the higher initial inoculation density used here (3 × 106 bacteria/ml) (Omsland et al., 2011; Sandoz et al., 2014). Comparison of the absorbance measurements with CFU enumeration (Fig. 2A, left axis) showed that beyond 4 day cultures, the number of CFUs significantly dropped while the optical density remained relatively constant, indicating that OD measurements did not correlate with the number of viable bacteria in stationary phase. Samples of the cultures were collected on day four and diluted to an OD600 of 0.1 for CFU and GE bacterial enumeration (Fig. 2B). Average viable bacterial counts were estimated to be 6.8 × 108 CFU/ml, while the GE were estimated to be 8.73 × 108 bacteria/ml. Together these results suggest that the 4 day bacterial cultures are ideal for cell-based infection studies as they contain high numbers of viable bacteria. 3.2. Optimization of multiplicity of infection for CBNMIIC4 strain in RAW264.7 macrophages After confirming that the bacterial culture system was consistent, the infection in RAW264.7 macrophages with CBNMIIC4 was optimized. To determine the ideal infection conditions, RAW264.7 macrophages were infected for 24 h with various MOIs of CBNMIIC4, as calculated by the CFU/ml determined above. There was an MOI-dependent increase in the number of bacteria per well, although only the MOIs of 40 and 80 were significantly different from the uninfected control (Fig. 3A). Similarly, an MOI-dependent increase in percent infection was also observed, and only MOIs of 16, 40 and 80 were significantly higher than the untreated control cells (Fig. 3B). The number of murine macrophages was unchanged between the uninfected control cells and those infected with any MOI, an indication that the bacteria were not killing the macrophages at the concentrations tested (Fig. 3C). The robustness of the assay was determined by calculating the Z' for bacterial number (Fig. 3D). Z' score > 0.3 are optimal for highthroughput assays, and the MOIs below 40 did not reach that score (Kota et al., 2013). The MOI of 80 gave the highest Z' score (0.57 ± 0.103), and therefore, was selected for subsequent studies. 3.3. Validation of the high-throughput screening assay for C. burnetii To validate our cell-based assay, four compounds were tested in dose-response studies, including several host-targeting compounds (bepridil, loperamide, and amiodarone) and one antimicrobial compound, (pentamidine) that were previously identified to reduce C. burnetii intracellular numbers (Czyz et al., 2014; Minnick et al., 2010). The dose response curves generated in these studies determined compound activity against both bacteria (EC50) and macrophage cells (CC50) (Fig. 4). Loperamide hydrochloride, amiodarone hydrochloride, and bepridil hydrochloride had average EC50 values of 11.35 μM, 7.1 μM, and 11.8 μM, respectively, and CC50 values of > 30 μM. Pentamidine did not exhibit a concentration-dependent effect on bacterial number and had a CC50 of 6.2 μM. From these studies, amiodarone was the most potent compound against C. burnetii and, therefore, will serve as a reference control in future screenings of small molecule chemical libraries. 4. Discussion Critical for the axenic culture of C. burnetii is the ability to maintain a 2.5% O2 environment. We have demonstrated that use of a modular hypoxic chamber instead of specialized incubators is sufficient for the growth of C. burnetii in vitro. The hypoxic chamber provides a controlled 4
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Fig. 3. Multiplicity of infection optimization for C. burnetii in RAW264.7 macrophages. A) The number of bacteria per well, B) the percent of cells infected with C. burnetii at 24 h post-infection, and C) the number of RAW264.7 macrophages. D) The robustness of the assay was determined by calculating the Z'. The mean from seven independent repeats were plotted and error bars represent standard error of the mean. Statistical significance was determined using ordinary one-way ANOVA using Dunnett's multiple comparisons test (GraphPad Prism, La Jolla, CA).
Funding
using genome equivalents, and measured macrophage viability by quantitative PCR. Both screens focused on late stages of infection where C. burnetii have replicated. Our primary goal was to identify antimicrobial agents that inhibit early stages of C. burnetii infection before bacterial replication and before the formation of the SCV. With this goal in mind, we are including C. burnetii in our high-throughput screening platform to identify compounds that disrupt host-pathogen interactions and thereby reduce bacteria number early during infection in macrophages.
This work was supported by the National Academy of Science, National Research Council fellowship funded through Defense Threat Reduction Agency (project 132141744 to CNM). Author statement The bacterial cell culture design and method was conceived by MLH. SAA and MLH developed modifications to the ACCM-2. The experiments were conceived and designed by CNM and MLH. CNM, MK, and MLH performed the experiments. CNM, RGP, and MLH analyzed and interpreted the data. CNM, MK, and MLH drafted the article. RGP and KK critically revised the article for important intellectual content.
5. Conclusions A simple method for culturing the bacterium was developed and a robust screening platform has been designed to identify host-directed compounds that reduce C. burnetii numbers in macrophage cells during early stages of infection. In our assay system, amiodarone was found to be a suitable control compound for high-throughput screening against C. burnetii. Currently, compound libraries are being screened to identify novel therapeutics to treat Q fever. This high-throughput assay can be easily adapted for the BSL-3 virulent strain of C. burnetii, to identify compounds directed at host pathways, which reduce bacteria number early during infection in macrophages. This assay can also be easily adapted for later times post-infection to identify compounds that inhibit C. burnetii intracellular replication. New compound that reduces C. burnetii number early during infection can be an effective treatment for doxycycline-resistant bacteria.
Declaration of Competing Interest We declare that no financial interests exist. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army. Acknowledgments We would like to thank Tara Kenny, Katlin Recabo, Glenn Gomba, Rouzbeh Zamani, and Sena Gul for their technical assistance. We would also like to thank Jennifer Scarff and Rachel Washart for their critical editing of the manuscript. We declare that no financial interests exist. Opinions, interpretations, conclusions, and recommendations are those 5
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Fig. 4. Dose response curves for host-directed compound activity against C. burnetii in RAW264.7 macrophages. The potency EC50 (green line) and the cytotoxicity CC50 (yellow line) were generated using an 8-point 3-fold dilution of compound. A) Amiodarone hydrochloride, B) Bepridil hydrochloride, C) Loperamide hydrochloride, and D) Pentamidine isethionate. Representative graphs from experiments that were repeated at least two independent times. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
of the authors and are not necessarily endorsed by the U.S. Army.
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