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AnintegratedsurrogatemodelforscreeningofdrugsagainstMycobacteriumtuberculosis

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AntimaGupta

Birkbeck,UniversityofLondon

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SanjibBhakta

Birkbeck,UniversityofLondon

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An integrated surrogate model for screening of drugs againstMycobacterium tuberculosis

Antima Gupta and Sanjib Bhakta*

Mycobacteria Research Laboratory, Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck,University of London, Malet Street, London WC1E 7HX, UK

*Corresponding author. Tel: +44-(0)20-7631-6355 (Office) and +44-(0)20-7079-0799 (Lab); Fax: +44-(0)20-7631-6246; E-mail: s.bhakta@bbk.ac.uk

Received 9 November 2011; returned 26 December 2011; revised 20 January 2012; accepted 31 January 2012

Objectives: The intracellularly surviving and slow-growing pathogen, Mycobacterium tuberculosis, adapts thehost cell environment for its active and dormant life cycle. It is evident that the lack of appropriate high-throughput screening of inhibitors within host cells is an impediment for the early stages of anti-tuberculardrug discovery. We aimed to develop an integrated surrogate model that enhances the screening of largeinhibitor libraries.

Methods: Different mycobacterial species were compared for their growth, drug susceptibility and intracellularuptake. A 6-well plate solid agar-based spot culture growth inhibition (SPOTi) assay was developed into a higherthroughput format. The uptake and intracellular survival of Mycobacterium aurum within mouse macrophagecells (RAW 264.7) were optimized using 24/96-well plate formats.

Results: Fast-growing, non-pathogenic M. aurum was found to have an antibiotic-susceptibility profile similar tothat of M. tuberculosis. The sensitivity to an acidic pH environment and the ability to multiply inside RAW 264.7macrophages provided additional advantages for employing M. aurum in intracellular drug screening methods.A selection of anti-tubercular drugs inhibited the growth and viability of M. aurum inside the macrophages atdifferent levels.

Conclusions: We present a rapid, convenient, high-throughput surrogate model, which provides a comprehen-sive evaluation platform for new chemical scaffolds against different physiological stages of mycobacteriawithin the primary cell environment of the host. The results using anti-tubercular drugs validate this modelfor screening libraries of existing and novel chemical entities.

Keywords: drug susceptibility testing, Mycobacterium aurum, RAW 264.7 macrophages, SPOTi assay, surrogate

IntroductionTuberculosis (TB) is an ancient infectious disease that is stillresponsible for the highest human mortality worldwide from itssingle causal pathogen, Mycobacterium tuberculosis.1 In spiteof intensive efforts towards eradicating TB, it remains a challen-ging problem, not only for developing countries but also for thedeveloped world. According to the latest data, 1.6 milliondeaths from TB have been reported.1 Furthermore, the situationis becoming even more complicated due to issues such as(i) long, complex and ineffective chemotherapy against newlyemerging, extensively drug-resistant (XDR)/totally drug-resistant(TDR) TB strains, and (ii) incompatibilities between anti-TB andanti-HIV drugs. Therefore, development of a new and moreeffective drug treatment against TB is urgently required.

A concerted effort has been recently made worldwidetowards developing new anti-TB drugs by different consortia

and organizations, such as the TB Alliance (http://www.tballiance.org/), TBDUK (http://www.tbd-uk.org.uk/), Open SourceDrug Discovery (OSDD) (http://www.osdd.net/), New MedicinesFor Tuberculosis (NM4TB) (http://www.nm4tb.org/), the WorkingGroup on New TB Drugs (WGND) (http://www.newtbdrugs.org/)and More Medicines for Tuberculosis (MM4TB) (http://www.mm4tb.org/), resulting in a large number of novel chemicallibraries in the pipeline waiting for comprehensive evaluationat the preclinical stage of TB drug development.2 – 6

During infection, M. tuberculosis is able to survive inside thehost’s primary immune cells even under different stress condi-tions for an undefined period.7 Whilst the standard practice forscreening inhibitors is to test them against actively dividingbacilli in vitro, most growth inhibitor hits fail at a later stage ofthe extremely expensive in vivo clinical trials.8 Furthermore, theslow growth, highly infectious nature of M. tuberculosis and thecomplex infrastructure pose major obstacles towards developing

# The Author 2012. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.For Permissions, please e-mail: journals.permissions@oup.com

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a comprehensive high-throughput, intracellular, drug screeningprocedure.

To avoid the use of the highly pathogenic, slow-growingM. tuberculosis in the early drug screening process, different myco-bacterial species, such as Mycobacterium smegmatis,9 Mycobac-terium fortuitum,10,11 Mycobacterium phlei,11 Mycobacteriumaurum,10 Mycobacterium abcessus,11 Mycobacterium marinum12

and Mycobacterium bovis BCG13 have been utilized as surrogates.Of these, the non-pathogenic fast-growing M. aurum possessesa similar cell wall profile to M. tuberculosis,14 as well as sharingintracellular therapeutic targets,15 gene organization16 anddrug resistance patterns.17 Here, we compared M. aurum withM. smegmatis mc2155, Mycobacterium neoaurum (anotherspecies of the M. aurum clade) and M. bovis BCG for suitability indrug screening systems. M. smegmatis mc2155, which does notprovide a consistent and reliable model for drug screening, waschosen as a negative control because it does not have the abilityto survive inside macrophages.18

Mouse macrophage cell line RAW 264.7 has been consideredsuitable for drug screening because of its high phagocytic activ-ity, adherence capability and ease of culturing.19 The choice ofmouse macrophages is also supported by the wide use of miceas an animal model for in vivo anti-TB drug testing. We have pre-viously used the spot culture growth inhibition (SPOTi) assay, asolid agar-based method that has advantages in drug screeningover liquid-based colorimetric assays, and methods that employcfu counting (the ‘gold standard’ quantification method).20,21

In this study, we have integrated M. aurum (a surrogate forM. tuberculosis), RAW 264.7 macrophages (surrogates forprimary immune cells) and SPOTi (a method for testing inhibi-tors) to develop a rapid and convenient intracellular-screeningplatform. This is the first report of such a comprehensivemodel for testing inhibitor libraries.

In developing the surrogate method for screening novelantimycobacterials against M. tuberculosis within a host cell en-vironment, our objectives were: (i) to validate M. aurum as a goodintracellular model for testing bacteriological inhibitors; (ii) tocharacterize the uptake and survival of M. aurum inside RAW264.7 macrophage cells; and (iii) to examine the reliability ofthe screening system using known anti-TB drugs on M. auruminside the RAW 264.7 macrophages.

Materials and methods

Selected anti-TB drugsThe known anti-TB front-line drugs [isonicotinic acid hydrazide (isoniazid),rifampicin, pyrazinamide and ethambutol] and second-line drugs(ethionamide, p-aminosalicylic acid, D-cycloserine, streptomycin, ofloxa-cin, phosphomycin, cephalosporin and metronidazole) were boughtfrom Sigma. Isoniazid, streptomycin and cephalosporin were dissolvedin water, and the others were dissolved in DMSO, to a concentration of50 g/L. All antibiotics were sterilized using 0.2 mm filters (Sartorius). Allchemicals were purchased from Sigma, unless otherwise stated.

Growth and maintenance of mycobacterial speciesBiosafety level 2 mycobacterial species22 M. neoaurum (NC 10440) andM. aurum (NC 10437) were purchased from the UK National Collectionof Type Cultures (NCTC). These mycobacterial cells, M. smegmatismc2155 (ATCC 700084) and M. bovis BCG Pasteur (ATCC 35734) were

grown in Middlebrook (MB) 7H9 medium (BD Biosciences) enriched with10% (v/v) albumin/dextrose/catalase (ADC; BD Biosciences) for liquidgrowth, and in MB7H10 (BD Biosciences) with 10% (v/v) oleic acid/albumin/dextrose/catalase (OADC; BD Biosciences) for solid agar growthat 378C. Stock cultures of log-phase cells were maintained in glycerol(25% final concentration of glycerol) at 2808C.

Development of a higher throughput extracellularSPOTi assayThe solid agar-based MIC-determining SPOTi screening method20,21

was extended from a 6-well to a 24-well format. Log-phase cultures ofM. smegmatis mc2155, M. neoaurum, M. aurum and M. bovis BCG[Optical Density (OD)600≈1] were first checked for quality control usingcold Ziehl–Neelsen (ZN) staining (also called ‘acid fast staining’; TB-colourstaining kit, BDH/Merck) according to the manufacturer’s protocol. Then�103 cells (2 mL) were placed with 2 mL of MB7H10/OADC (containingantibiotics at 0, 0.39, 1.5, 6.25, 25 or 100 mg/L) in each well of a24-well Costar plate (Appleton Woods). In order to obtain accurate MICvalues, the MICs determined from the first set of experiments wereused to select a narrower concentration range in a subsequent experi-ment. The cell growth at 378C was recorded after 2 days for M. smegma-tis mc2155, 3 days for M. neoaurum, 5 days for M. aurum and 14 days forM. bovis BCG. Wells with DMSO or H2O were used as negative controls.

Growth inhibition of mycobacteria at different pHM. neoaurum and M. aurum cultures (OD600≈1.0) were harvested, and2 mL of cells (undiluted, ×10 and ×100 diluted) were spotted on Petridishes containing Sauton’s agar medium. The Sauton’s agar mediumwas prepared by adding 4.0 g L-asparagine, 2.0 g citric acid, 0.5 gKH2PO4, 0.5 g MgSO4.7H2O, 0.05 g ferric ammonium citrate, 15.0 g agarand 60 mL glycerol in deionized water. It was adjusted to different pHvalues (5.5, 5.8, 6.0, 7.0 and 7.2) using 5 M NaOH. The volume was adjustedto 1 L and then sterilized. To observe the antibiotic susceptibility underacidic stress, M. neoaurum cells (OD600≈1.0) were washed with PBS (pH7.0) twice before being stressed by placing in acidic Sauton’s liquidmedium for 1 h at 378C.23 Stressed cells (2 mL, 106 cells/mL) werespotted onto 24-well plates containing Sauton’s agar medium at pH 5.5,5.8, 6.0 and 7.0) with antibiotics at 0, 0.39, 1.5, 6.25, 25 and 100 mg/L.

Growth and maintenance of mouse macrophage cell line(RAW 264.7)RAW 264.7 cells (NCTC #91062702) were maintained in 25 or 75 cm2

tissue culture flasks (BD Biosciences) containing RPMI-1640 completemedium [RPMI-1640 medium supplemented with 2 mM L-glutamineand 10% heat-inactivated fetal bovine serum (378C, humidified 5%CO2)], and passaged twice before the assay. Before passage, cells werewashed twice with 1× PBS to remove unattached cells. Adhered cellswere detached using a lidocaine/EDTA mixture (10 mM lidocaine HCl,10 mM EDTA in 1× PBS) at room temperature for 10 min followed byhitting the side of the flask against the palm of the hand, and then dilut-ing with an equal volume of fresh medium.20 Cells were then centrifugedat 1000 g for 5 min and resuspended in fresh medium. The number ofviable cells was counted using a Trypan Blue assay. Stock cultures ofthe cells were maintained at 2808C by adding actively growing cells toan equal volume of BambankerTM Cell Freezing Media (Anachem).

Cell toxicity assay using RAW 264.7 macrophagesThe assay was performed in 96-well cell culture flat-bottom plates(Costar; Appleton Woods) in triplicate. First, 20 mL samples of the 50 g/L

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stock solutions of antimicrobial compounds were placed in wells contain-ing 200 mL of RPMI-1640 complete medium in the first row, and then2-fold serial dilution was done six times (the last well did not containany antibiotic and was used as the control). To each well, 100 mL ofdiluted macrophage cells (5×105 cells/mL) was added. After 48 h of incu-bation, the monocytes were washed twice with PBS, and fresh RPMI-1640complete medium was added. Plates were then treated with 30 mL of afreshly prepared 0.01% resazurin solution and incubated overnight at378C. The following day the change in colour was observed and the fluor-escence intensity was measured (lex560 nm, lem590 nm, FLUOstarOPTIMA microplate reader; BMG LABTECH GmbH).4 The 50% growth in-hibitory concentration (GIC50) was determined based on a resazurinfluorescence assay, and the selectivity index (SI) was determined, asthe ratio between the GIC50 against RAW 264.7 macrophages and theMIC against M. aurum, for all tested drugs.

Optimizing the uptake of mycobacteria in RAW 264.7macrophagesM. smegmatis mc2155, M. neoaurum, M. aurum and M. bovis BCGwere labelled with fluorescein 5(6)-isothiocyanate (FITC) by adding108–109 cells to 1 mL of 0.01% FITC in 0.5 M carbonate/bicarbonatebuffer pH 9.5 (0.5 M Na2CO3, 1 volume; 0.5 M NaHCO3 3 volumes) andincubating for 30 min at 258C.20 The bacteria were then washed twicein RPMI-1640 complete medium to remove surplus dye and suspendedin RPMI-1640 complete medium. Macrophages (100 mL, 1×106 cells/mL) in a 96-well clear-bottom black plate (Corning, Appleton Woods)were infected with an equal number of unlabelled and labelled bacilliat 10:1 multiplicity of infection (moi) ratio. At 2 h post-infection, theinfected macrophages were washed with PBS containing 0.05% Tween80, and this was followed by a wash with PBS alone. Cells were then sus-pended in 100 mL of PBS and the fluorescence was measured(lex485 nm, lem520 nm). For optimizing the uptake of M. aurum, macro-phage cells were infected with unlabelled and labelled bacilli at differentmois (5:1, 10:1 and 20:1) at 2 h post-infection, and at different time-points (0, 30, 60 and 120 min) with a fixed moi (10:1). Only-macrophages and only-bacteria wells were used as controls, andonly-PBS/RPMI-1640 was used as a blank. Fluorescence per mg ofprotein was calculated by equating the fluorescence intensity withprotein concentration of the respective well. The protein concentrationwas measured using the Bradford colorimetric assay.

Optimizing the infection of M. aurum in RAW 264.7macrophagesA macrophage cell suspension was adjusted to 1×106 cells/mL, and 2 mLwas added onto circular coverslips (Appleton Wood) of 6-well cell cultureflat-bottom plates. After 15 min of incubation, 10, 20 or 40 mL ofmid-log phase M. aurum cells (1×109 cells/mL) harvested in RPMI-1640complete medium were added to each well at different mois (5:1, 10:1and 20:1) 2 h post-infection, and at different times (30, 60 and120 min) with a fixed moi (10:1). Treated coverslips were washed thricewith RPMI-1640 medium containing 0.05% Tween 80 and assessed byZN staining using a TB-colour staining kit. Co-infection was optimized bycounting bacteria per macrophage cell under a bright field microscope(Zeiss) with coverslips mounted onto slides. Acid fast-stained phagocy-tosed mycobacteria images were captured at ×1000 magnification.

Optimizing the survival of M. aurum in RAW 264.7macrophagesFor the survival assay, macrophages cells infected with M. aurum werelysed at different times (0, 24, 48 and 72 h) in distilled water at room

temperature for 10 min followed by scrubbing using a syringe plunger.The lysed cells were spread onto plates containing MB7H10 OADC agar,and then incubated at 378C to determine the cfu.

Development of 24-well plate SPOTi assay with RAW264.7 macrophagesFor the killing assay, macrophages (5×105 cells per well) were infectedwith M. aurum at 10:1 moi for 1 h at 378C in a 24-well plate. Theculture was washed with RPMI-1640 thrice and incubated with differentconcentration of inhibitors (0, 0.39, 1.5, 6.25, 25 and 100 mg/L) inRPMI-1640 complete medium. Inhibitors were incubated for differenttimes (2 h, 24 h, 48 h and 72 h). Cells were then washed twice withRPMI-1640 and lysed in 500 mL of distilled water at room temperaturefor 10 min. The lysed cells were centrifuged and suspended in 50 mL ofdistilled water. Then, 5 mL was spotted onto wells of a 24-well plate con-taining MB7H10/OADC/agar and incubated at 378C for 5 days to deter-mine intracellular survival.

Results

M. aurum as a bacteriological model for testingantimycobacterials

Comparative growth curves of mycobacterial species

Based on cell morphology, generation time, antibiotic susceptibilityand intracellular survival ability, we compared M. smegmatismc2155, M. neoaurum, M. aurum and M. bovis BCG in order toestablish a bacteriological model for this study. The mycobacterialspecies were first observed for their morphology using cold ZNstaining. M. neoaurum and M. aurum showed a similar pattern toM. bovis BCG, while M. smegmatis mc2155 cells were more elongated(acid fast-stained cells at mid-log phase; Figure 1). The myco-bacterial species were also studied for their physiological propertiesby plotting their growth curves (OD600 against time). As bothM. neoaurum and M. aurum are scotochromogenic species, a spec-trum analysis was performed to exclude possible interferenceby chromogenic substrate at 600 nm. The growths of mycobacterialspecies were observed at three different growth rates: active,moderate, and slow. Although M. smegmatis mc2155 andM. neoaurum showed the fastest growth, comparative growthcurves showed that M. aurum also take less time to enter the logphase compared with M. bovis BCG (Figure 1). For M. aurum, log-phase cells were collected after 36 h, SPOTi assays were performedafter 5 days and colonies were obtained on plates after 8 days; forM. bovis BCG the corresponding values were 3–4 days, 14 daysand 28 days (Table 1).

Growth of mycobacterial species in the presenceof anti-TB drugs (MIC)

A solid agar-based SPOTi assay was performed in a 24-well plateformat (Figure 2). The MICs of known antibiotics were determinedfor complete inhibition for M. smegmatis mc2155, M. neoaurum,M. aurum and M. bovis BCG. When we tested MICs of knownanti-TB drugs at or below 100 mg/L values (extracellular), 5 outof 12 antibiotics were not active against M. smegmatis mc2155,while 4 were not active against M. neoaurum and only 3 werenot active against M. aurum, M. bovis BCG and M. tuberculosisH37Rv (Table 2). In comparison with M. smegmatis mc2155

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and M. neoaurum, the extracellular MICs of each of 9 of the12 tested anti-TB drugs were within narrow ranges forM. aurum, M. bovis BCG and M. tuberculosis H37Rv: isoniazid(0.02–0.4 mg/L), rifampicin (0.1–0.5 mg/L), ethambutol (0.47–2.0 mg/L), ethionamide (0.6–5.0 mg/L), p-aminosalicylic acid(1.0 mg/L), D-cycloserine (5.0–25 mg/L), streptomycin (0.1–1.0 mg/L), ofloxacin (0.2–2.0 mg/L) and cephalosporin (25 mg/L),

while the other three, phosphomycin, metronidazole and pyrazi-namide, were not active (Table 2).

Tolerance of mycobacterial species to pH

In order to select an intracellularly survivable bacteriologicalmodel, M. neoaurum and M. aurum were analysed at acidic pH

M. smegmatis mc2155 M. neoaurum

M. aurum M. bovis BCG

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Figure 1. Comparative growth curves of (a) M. smegmatis mc2155, (b) M. neoaurum, (c) M. aurum and (d) M. bovis BCG. Each point represents themean value of optical density from three independent experiments. The standard deviation of the mean for each is shown as an error bar.Microscopic observation of log phase (OD600 �1.0) acid fast stained (e) M. smegmatis mc2155 (cfu: 1×109/mL), (f) M. neoaurum (cfu: 1×108/mL),(g) M. aurum (cfu: 1.2×108/mL) and (h) M. bovis BCG (cfu: 1×109/mL). This figure appears in colour in the online version of JAC and in black andwhite in the print version of JAC.

Table 1. Comparative growth pattern of M. smegmatis mc2155, M. neoaurum, M. aurum, M. bovis BCG and M. tuberculosis H37Rv

ParametersM. smegmatis

mc2155 M. neoaurum M. aurumM. bovis BCG/

M. tuberculosis H37Rv

Generation time (h) 2.7 4.56 9.6 20Time to observe single colony (days) 3 4 8 28Time to observe spot (days) 2 3 5 14Level of bio-safety containment22 1 2 2 2/3Growth rate fast slow

Generation time was calculated according to (t22t1)×ln(2)/ln(q2/q1) where t2 is the final time, t1 is the beginning of growth (end of lag phase), q2 isthe cell number at t2, and q1 is the cell number at t1.37

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for their survival ability, and the results were compared withearlier reports for M. smegmatis mc2155, M. bovis BCG andM. tuberculosis H37Rv.24 We found that, similarly to M. bovisBCG and M. tuberculosis H37Rv, M. aurum was sensitive toacidic pH (Figure 3b): growth was undetected at ≤pH 6.0, andfull growth was seen only at pH 7.2 (Figure 3b). In contrast, simi-larly to M. smegmatis mc2155, M. neoaurum was not sensitive topH 5.8, 6.0, 7.0 or 7.2, but we observed retarded growth at pH 5.5(Figure 3a). We also performed the anti-TB drug susceptibilitytest against M. neoaurum in acidic stress conditions (pH 5.5–7.0)and found that the rifampicin, D-cycloserine and cephalosporinhad increased effectivity in acidic conditions (Table 3), which indi-cates higher antibiotic susceptibility at low pH. We also observedmodulation in MICs between an enriched medium (MB7H10) andminimal medium (Sauton’s) (Tables 2 and 3). Rifampicin,p-aminosalicylic acid, cephalosporin and ofloxacin were moreactive against M. neoaurum in enriched medium while isoniazidwas more active against M. neoaurum in minimal medium.

RAW 264.7 for an ex vivo infection model

We observed that monolayers of RAW 264.7 spread uniformly,adhered well and started differentiating into active macrophageswithin 15 min. The 80% viability of macrophage cells was deter-mined by using a Trypan Blue assay.

Toxicity of anti-TB drugs against RAW 264.7

Macrophages in logarithmic growth phase were used fortesting the cytotoxicity of various antibiotics against RAW 264.7macrophage cells before testing compounds for intracellular in-hibition. All anti-TB drugs were found to be non-toxic to RAW264.7 macrophages, as determined by GIC50 values (cephalo-sporin 500 mg/L, rifampicin 700 mg/L, ethionamide 1000 mg/L,ofloxacin 1500 mg/L and others ≥3000 mg/L), resulting in SIvalues .20 for all drugs tested (Table 4).

Uptake of mycobacterial species into RAW 264.7macrophages

When phagocytosis of M. smegmatis mc2155, M. neoaurum,M. aurum and M. bovis BCG were compared by an FITC fluorescenceuptake assay (2 h infection time), we found that M. aurum hadsimilar uptake to M. bovis BCG, while M. smegmatis mc2155 andM. neoaurum had 3–5 times lower uptake (Figure 4c). Thisfurther supported the selection of M. aurum as a fast-growingmycobacterial model organism for drug screening.

Infection of M. aurum within RAW 264.7 macrophages

A small aliquot of M. aurum culture was used for screening againstextracellular bacilli by performing a SPOTi assay, and the remainderwas used further to optimize the uptake and killing assay. Uptake ofM. aurum was assessed using ZN staining of infected and non-infected macrophages, and by a quantitative fluorescence assay.Staining results showed the presence of engulfed pink (carbol-fuchsin-stained) M. aurum inside blue (methylene-blue-stained)macrophage cells (Figure 5). Phagocytosis was observed between30 min and 2 h (moi 10:1), while increasingly clumped bacilliwere seen with increasing moi at fixed timepoints (Figure 5).

Quantitative analysis of unlabelled and FITC-labelledM. aurum-infected macrophages showed that maximum uptakewas observed at 10:1 moi and 1 h infection time (Figure 4a and b).

Killing of M. aurum within RAW 264.7 macrophages

Intracellular survival was evaluated by cfu counting in theabsence or presence of a subinhibitory concentration of isoniazid(Figure 4d). M. aurum was able to survive intracellularly andentered into logarithmic growth phase in 48 h, with a 10-foldincrease in bacilli in the following 24 h (Figure 4d). Intracellulargrowth inhibition of M. aurum was observed at different timeintervals in the presence and absence of front-line anti-TB

INHmg/L

0

0.39

1.56

6.25

25

100

RIF PYZ ETH DCS PHO ETM STR PAS CEP MET OFX

Figure 2. Extracellular susceptibility of M. aurum to anti-TB drugs using the 24-well plate format SPOTi assay. The assay was performed with threebiological replicates. INH, isoniazid; RIF, rifampicin; PYZ, pyrazinamide; ETH, ethionamide; DCS, D-cycloserine; PHO, phosphomycin; ETM, ethambutol;STR, streptomycin; PAS, p-aminosalicylic acid; CEP, cephalosporin; MET, metronidazole; OFX, ofloxacin.

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Table 2. Comparison of extracellular MICs of anti-tubercular drugs for M. smegmatis mc2155, M. neoaurum, M. aurum and M. bovis BCG using the SPOTi assay in MB7H10 mediumand M. tuberculosis H37Rv (acquired from the literature)

Anti-TB drugs Structure Mol. weight (g/M)

MICs, mg/L (mM)

M. smegmatis mc2155 M. neoaurum M. aurum M. bovis BCG M. tuberculosis H37Rv

INH O

NH2

HN

N

137.14 5 (36.46) 2 (14.58) 0.40 (3.65) 0.10 (0.73) 0.02–0.2 (0.15–1.46)38

RIF OH

O

OH

OH

OH

NH

N

N

OH

O

O

O

O

O

O

H

822.94 8 (9.72) 1 (1.22) 0.10 (0.12) 0.50 (0.61) 0.10 (0.12)39

PYZN

O

N

NH2

123.11 .100 (.812.26) .100 (.812.26) .100 (.812.26) .100 (.812.26) .100 (.812.26)40

ETM OH

NH

NH

HO

204.31 2 (9.79) 10 (48.95) 0.5 (2.45) 2 (9.79) 0.47 (2.30)41

ETH NH2

CH3

N

S 166.24 50 (300.76) 100 (601.53) 5 (30.08) 5 (30.08) 0.6–2.5 (3.60–15.04)42

PAS

H2N OH

OH

O 153.14 .100 (.653.02) .100 (.653.02) 1 (6.53) 1 (6.53) 1 (6.53)38

DCS H2N

NH

O

O 102.09 75 (734.63) 25 (244.88) 25 (244.88) 5 (48.98) 5–25 (48.97–244.88)43,44

STR

CH3

H2N

NH2

CH3

HNHO

HO

HOHO

OH

OHN

H

HOO

O

O

O

O

581.57 1 (1.72) 0.4 (0.69) 0.2 (0.34) 1 (1.72) 0.1–0.5 (0.17–0.86)43,45

OFX

N

N

F

O

O

N

361.37 1 (2.77) 0.2 (0.55) 0.2 (0.55) 1 (2.77) 1–2 (2.77–5.53)43,46

PHO O

O

HO

OH

CH3P

138.06 .200 (.1448.66) .200 (.1448.66) .200 (.1448.66) .200 (.1448.66) .200 (.1448.66)46

CEP H H

S

R2

O

N

N

O

O O

478.79 .100 (208.86) 50 (104.43) 25 (52.21) 25 (52.21) 25 (52.21)47

MET N

N

HOO

N

O171.15 .200 (.1168.56) .200 (.1168.56) .200 (.1168.56) .200 (.1168.56) .200 (.1168.56)47

INH, isoniazid; RIF, rifampicin; PYZ, pyrazinamide; ETM, ethambutol; ETH, ethionamide; PAS, p-aminosalicylic acid; DCS, D-cycloserine; STR, streptomycin; OFX, ofloxacin; PHO,phosphomycin; CEP, cephalosporin; MET, metronidazole.MIC was determined for complete inhibition.

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Neat

10–1

10–2

5.8 6.0 7.0 7.2 5.5 5.8 6.0 7.0 7.2

Figure 3. Growth of (a) M. neoaurum and (b) M. aurum in plates containing Sauton’s medium of different pH (5.5–7.2). The assay was performed withthree biological replicates.

Table 3. MICs of anti-TB drugs for M. neoaurum in acidic environments using Sauton’s medium

Anti-TB drug Drug targets

MICs, mg/L (mM)

pH 5.5 pH 5.8 pH 6.0 pH 7.0

INH mycolic acid biosynthesis38 0.39 (2.84) 0.39 (2.84) 0.39 (2.84) 0.39 (2.84)RIF RNA synthesis38 0.39 (0.47) 0.39 (0.47) 1.56 (1.86) 1.56 (1.86)PYZ fatty acid synthesis pathway-i38 .100 (.812.26) .100 (.812.26) .100 (.812.26) .100 (.812.26)DCS peptidoglycan biosynthesis43 6.25 (61.22) 6.25 (61.22) 25 (244.87) 25 (244.88)PAS tetrahydrofolate pathway and

mycobactin biosynthesis430.39 (2.55) 0.39 (2.55) 0.39 (2.55) 0.39 (2.55)

CEP penicillin binding proteins43 100 (208.86) .100 (.208.86) .100 (.208.86) .100 (.208.86)ETH mycolic acid biosynthesis43 100 (601.53) 100 (601.53) 100 (601.53) 100 (601.53)OFX DNA gyrase/topoisomerase43 0.39 (1.08) 0.39 (1.08) 0.39 (1.08) 0.39 (1.08)

INH, isoniazid; RIF, rifampicin; PYZ, pyrazinamide; DCS, D-cycloserine; PAS, p-aminosalicylic acid; CEP, cephalosporin; ETH, ethionamide; OFX, ofloxacin.

Table 4. Comparison of extracellular and intracellular MICs for M. aurum, 50% eukaryotic cell growth inhibitory concentrations (GIC50s) andintracellular SIs (GIC50/ex vivo MIC) for different anti-TB drugs

Anti-TB drug

M. aurum

GIC50s, mg/L (mM) Intracellular SIsextracellular MICs, mg/L (mM) intracellular MICs, mg/L (mM)

INH 0.39 (2.84) 0.39 (2.84) 3000 (21875.62) 7692.31RIF 0.10 (0.12) 0.39 (0.47) 700 (850.61) 1794.87PYZ .100 .100 3000 (24367.86) ,30DCS 25 (244.87) 25 (244.87) 3500 (34282.80) 140PAS 1 (6.53) 100 (65) 3000 (19590.56) 30CEP 25 (52.21) 25 (52.21) 500 (1044.30) 20ETH 5 (30.08) 6.25 (37.59) 1000 (6015.25) 160ETM 0.5 (2.45) 0.39 (1.91) 3000 (14683.57) 7692.31STR 0.2 (0.34) 1.56 (2.68) 3500 (6018.15) 2243.59OFX 0.2 (0.55) 1.56 (4.32) 1500 (4150.89) 3846.15

INH, isoniazid; RIF, rifampicin; PYZ, pyrazinamide; DCS, D-cycloserine; PAS, p-aminosalicylic acid; CEP, cephalosporin; ETH, ethionamide; ETM,ethambutol; STR, streptomycin; OFX, ofloxacin.

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Figure 4. Uptake fluorescence assay using FITC-labelled mycobacteria. (a) Uptake of M. aurum at different timepoints. (b) Uptake of M. aurum at different moi. (c) Different speciesuptake in 2 h and 10:1 moi. (d) Intracellular survival of M. aurum in the presence and absence of isoniazid (INH). Each bar represents the mean value of fluorescence from threeindependent experiments. The standard deviation of the mean for each is shown as an error bar.

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drugs (isoniazid, rifampicin, pyrazinamide and ethambutol) usingthe SPOTi assay (Figure 6). Significant inhibition by isoniazid andrifampicin was observed after 24 h, and complete inhibition wasobserved after 48 h. Therefore, a 48 h drug-incubation timepointwas chosen for our model (Figure 7). In conclusion, the M. aurumwith murine macrophage high-throughput drug-screening modelwas optimized with the following parameters: 10:1 moi uptake,1 h infection, and 48 h drug incubation.

Intracellular MICs of all anti-TB drugs (isoniazid, rifampicin,pyrazinamide, D-cycloserine, p-aminosalicylic acid, cephalosporin,ethionamide, ethambutol, streptomycin and ofloxacin) for pha-gocytosed M. aurum were calculated, except phosphomycin andmetronidazole, which were not active against extracellular myco-bacteria. We found that rifampicin, p-aminosalicylic acid, ethiona-mide, streptomycin and ofloxacin had slightly higher MIC valueswhen inside the macrophages compared with extracellular values,while ethambutol was more active inside macrophages. It is note-worthy that pyrazinamide was not active and p-aminosalicylicacid was considerably less active against intracellular M. aurum,although the extracellular MIC of the latter was 1 mg/L (Table 4).

DiscussionDuring pathogenesis, M. tuberculosis cells enter the alveolarmacrophage cells of the host, and are often able to survive in

a nutrient-deficient, oxygen-deficient, redox-active and highlyacidic environment, in various physiological states (replicating,not replicating or slowly replicating) for an undefined period oftime.7 Therefore, it is important to have an inhibitor screeningassay for these different physiological bacilli states within thehost cell environment. In addition, the solubility, permeability,stability and eukaryotic cell toxicity of novel chemical entitiesare also relevant for antibacterial selectivity and subsequent pro-gress through the drug development pipeline. Considering these,our study aimed to establish an integrated high-throughputmethod for testing libraries of compounds for in-cell growthinhibition of mycobacteria.

The use of the fast-growing, non-pathogenic, biosafety level 2organism M. aurum as surrogate model organism provides arapid, less hazardous and more economic platform for theprimary screening of inhibitors. Based on the similarity of itsgene organization and enzymatic activity to those ofM. tuberculosis, recombinant M. aurum has already been estab-lished as a model for screening inhibitors targeting known path-ways.16,17 RAW 264.7 cells were found to have better growthkinetics and ability to phagocytose mycobacteria than othercell lines.19 Our results further support the identification ofM. aurum as an appropriate screening model for M. tuberculosisover other tested fast-growing species, such as M. smegmatismc2155 and M. neoaurum.

8 µm

(d) (e) (f)

(a) (b) (c)

1000 ×8 µm

1000 ×8 µm

1000 ×

8 µm1000 ×

8 µm1000 ×

8 µm1000 ×

Figure 5. Phagocytosis of M. aurum at different moi and times of infection. (a) Control (without infection). (b) 5:1 moi, 2 h of infection. (c) 10:1 moi,2 h of infection. (d) 20:1 moi, 2 h of infection. (e) 10:1 moi, 1 h of infection. (f) 10:1 moi, 30 min of infection. The assay was performed with threebiological replicates.

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In addition, the adaptation of the SPOTi method to a 24-wellplate format provided further saving of resources, including con-sumption of inhibitors. The direct observation of growth inhibitionin the SPOTi method affords reliability and reproducibility ofresults, superior to other available methods where growth canbe compromised by contaminants (radioactivity, fluorescenceor colorimetric counts in automated systems). Furthermore, theSPOTi method saves time compared with the authenticatedbut laborious gold standard method, cfu counting. The

compatability of the SPOTi assay with differing media compo-nents and pH in minimal media was shown, and it can be utilizedfor assaying actively dividing bacilli as well as mycobacteria invarious physiological growth conditions, such as the Hu et al.25

models of dormant and drug-tolerant mycobacteria, theWayne and Hayes26 dormancy model, the oxygen-depletion-induced model27 and the chemostatic model.28 Furthermore,this SPOTi-based model can be used to screen againstdrug-resistant as well as genetically modified mycobacteria.17,29

2 h

mg/L mg/L100 25 6.25 1.56 0.39 0 100 25 6.25 1.56 0.39 0

mg/L mg/L

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PYZ

ETM

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100 25

48 h 72 h

24 h

1.56 0.39 0 100 25 6.256.25 1.56 0.39 0

Figure 6. Growth inhibition of M. aurum inside RAW 264.7 cells at different time intervals for different drug concentrations of front-line anti-TB drugsisoniazid (INH), rifampicin (RIF), pyrazinamide (PYZ) and ethambutol (ETM). The assay was done in three biological replicates.

Growth in liquid Infection and

Intracellular growth

Growth on solid

1. Cell toxicity

Macrophages

RAW 264.7 3. Uptake assay

*

*

*

*2 days

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presence of drug

Spotting of intracellular

bacilli for viability testing

48 h 5 days

Washing

lnhibitor treatment

SPOTi assay

Preparation time

1 h

Uptake

4. Ex vivo SPOTi assay

Figure 7. Schematic representation of the surrogate model.

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Moreover, the natural pigmentation of M. aurum helps to distin-guish its spots from mycobacterial contaminants (if present).

Extracellular antibiotic-susceptibility profiles of first- andsecond-line anti-tubercular drugs against M. aurum showedclose similarity to the MICs for M. tuberculosis H37Rv (Table 2).We found that the sensitivity of M. aurum, unlike M. neoaurumand M. smegmatis, to acidic pH is similar to that of M. tubercu-losis H37Rv (Figure 3). We further justified the use of M. aurumfor testing of inhibitors within infected macrophages (which areknown to be acidic). Moreover, similar uptake of M. aurum andM. bovis BCG into macrophages (Figure 4c) provided additionalvalidation of M. aurum as an appropriate fast-growing, intracellu-larly surviving surrogate.

The developed model involves screening inhibitors againstextracellular bacilli and host macrophages, and then screeningagainst intracellular populations. Using an optimized SPOTi-based M. aurum intracellular model, MIC patterns showed differ-ences in drug susceptibility between actively dividing extracellu-lar bacilli and bacilli at different physiological stages inside thehost macrophage cells (Table 4). Interestingly, mycobacteriashowed no such difference in susceptibility to pyrazinamide,and susceptibility to p-aminosalicylic acid was significantlylower inside macrophages.

Pyrazinamide, a front-line anti-TB drug was found to be inef-fective against actively dividing mycobacteria in vitro, yet activein a mouse model.30 In this study, pyrazinamide was found tobe inactive both against M. neoaurum grown in culturemedium at acidic pH and against M. aurum grown inside macro-phages. Therefore, the effect of the intracellular environmentand the requirement for the presence of other anti-TB drugsfor the activation of the pro-drug are still debatable.31,32

p-Aminosalicylic acid is a second-line anti-TB drug reported tohave more than one intracellular target. As a metabolic analogue,p-aminosalicylic acid inhibits the tetrahydrofolate pathway, andas an antisalicylate compound, it interferes with the conversionof salicylic acid to carboxymycobactin and mycobactin (iono-phores for iron transport). We observed that only p-aminosalicylicacid showed a significant difference in activity againstM. neoaurum when tested in two different culture media (Sauton’sand MB7H10) (Tables 2 and 3). We infer, as MB7H10 mediumhas 8 times higher iron concentration than Sauton’s medium,that more p-aminosalicylic acid is needed to inhibit ironuptake.33,34 p-Aminosalicylic acid inhibits the uptake of iron bytargeting mycobactin. We also found that p-aminosalicylic acidwas not active against M. smegmatis mc2155 or M. neoaurum,while it was active against M. aurum and M. bovis BCG (Table 2).In an earlier study it was found that M. smegmatis mc2155,unlike M. tuberculosis H37Rv or M. bovis BCG, has a second,salicylic acid-independent route of iron uptake; therefore, it isless susceptible to p-aminosalicylic acid.35 Results in this study in-dicate that M. aurum has an iron uptake mechanism similar tothat of M. bovis BCG and M. tuberculosis H37Rv, while the mechan-ism in M. neoaurum is similar to that in M. smegmatis mc2155. Wehave found that p-aminosalicylic acid loses its activity against M.aurum inside the macrophages; this supports a previous findingwhere no intracellular activity was detected in a mouse model.36

The integration of M. aurum-infected RAW 264.7 mousemacrophage cells with the SPOTi assay to yield a cost-effectiveand low-hazard microbiology containment system now providesan effective inhibitor screening method to address the large

number of compound libraries, thus facilitating early stageanti-TB drug discovery.

AcknowledgementsWe thank Professor Siamon Gordon, Glaxo Wellcome Professor of CellularPathology at the University of Oxford, for providing the RAW 264.7 cellline. We would like to thank Dr Katherine Thompson for criticallyreading the manuscript prior to submission. We would also like toacknowledge Dr Ranjana Srivastava, Dr Brahm Shankar Srivastava andDr Dimitrios Evangelopoulos for helpful discussions.

FundingThis work was supported by UNESCO-L’OREAL (co-sponsored internation-al fellowship to A. G.) and the Medical Research Council (New InvestigatorResearch Grant, Code: BSR00BSA03 to S. B.).

Transparency declarationsNone to declare.

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