Green tea polyphenol inhibitsMycobacterium tuberculosissurvival within human macrophages

Paras K. Ananda, Deepak Kaula,∗, Meera Sharmab

Abstract

Lack of maturation of phagosomes containing pathogenicMycobacterium tuberculosis within macrophages has been widelyrecognized as a crucial factor for the persistence of mycobacterial pathogen. Host molecule tryptophan-aspartate containing coatprotein (TACO) has been shown to play a crucial role in the arrest of such a maturation process. The present study was addressedto understand whether or not polyphenols derived from green tea could down-regulate TACO gene transcription. And if yes, whatimpact TACO gene down-regulation has on the uptake/survival ofM. tuberculosis within macrophages. The reverse-transcriptasepolymerase chain reaction and reporter assay technology, employed in this study, revealed that the major component of greentea polyphenols, epigallocatechin-3-gallate had the inherent capacity to down-regulate TACO gene transcription within humanmacrophages through its ability to inhibit Sp1 transcription factor. We also found out that TACO gene promoter does contain Sp1binding sequence using bioinformatics tools. The down-regulation of TACO gene expression by epigallocatechin-3-gallate was

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accompanied by inhibition of mycobacterium survival within macrophages as assessed through flow cytometry and coloBased on these results, we propose that epigallocatechin-3-gallate may be of importance in the prevention of tuberculosi

Keywords: Green tea polyphenols; Epigallocatechin-3-gallate; TACO gene; Transcription; Phagosome; Tuberculosis

1. Introduction

The resurgence of concern about tuberculosis hasresulted in the molecular dissection of the membranetrafficking pathway to study the travel route ofMycobac-terium tuberculosis within mycobacterial phagosome.Intracellular pathogens likeM. tuberculosis have evolvednovel mechanisms to inhibit their fusion with the lyso-somes (Meresse et al., 1999). Specifically, mycobacte-ria containing phagosomes, also known as ‘mycobacte-

rial phagosome’, inhibits its lysosomal delivery becaof a block in the endosomal trafficking that prevephagosome maturation (Vergne, Chua, Singh, & Dereti2004). Phagosomal maturation involves a seriessequential fusion events with various vesicles fromendocytic pathway, by which nascent phagosomes amicrobicidal properties (Desjardins, Huber, Parton,Griffiths, 1994; Koul, Hergert, Klebl, & Ullrich, 2004).During this process, the phagosomes lose early endmal markers such as Rab5 and acquire markers olate endosomal/lysosomal pathway such as Rab7 (ciated with vesicular fusion) and cathepsin D (involin acidification of the phagosome) and thus beca ‘phagolysosome’ (Koul et al., 2004; Kusner, 200Vergne et al., 2004). However, the shuttling betwe

these Rab markers is altered in phagosomes carryingpathogenic mycobacteria (Via et al., 1997). Similarly,reduced or altered recruitment was observed for anotherRab5 effector, early endosome antigen 1 (EEA1), thatserves as an organelle tethering molecule between mem-branes destined for fusion (Christoforidis et al., 1999;Fratti, Backer, Gruenberg, Corvera, & Deretic, 2001;Simonsen et al., 1998). EEA1 interacts with Rab5 as wellas FYVE domain of phosphatidylinositol-3-phosphate(PI3P), generated on endosomal membranes by hVPS34,a type III phosphatidylinositol-3-kinase (Christoforidiset al., 1999; Simonsen et al., 1998). Consequently, phos-phatidylinositides have also become a focus of recentstudies aimed at identifying the molecular events caus-ing M. tuberculosis phagosome maturation arrest (Chua& Deretic, 2004; Vergne et al., 2005).

Another molecule that has been shown to inhibitendosomal/lysosomal fusion and therefore acidifica-tion, is host protein TACO (tryptophan-aspartate con-taining coat protein), also known as coronin-1, whichimparts non-fusogenic property to specifically mycobac-teria containing phagosomes (Ferrari, Langen, Naito,& Pieters, 1999). TACO/coronin-1 protein is acquiredfrom the plasma membrane at the time of phagocyto-sis and persists around phagosomes carrying pathogenicmycobacteriaMycobacterium bovis BCG (Ferrari et al.,1999; Gatfield & Pieters, 2000). The active retentionof TACO correlates well with the mycobacterial eva-sion of the endocytic pathway and thus the intracel-lular survival of the pathogen (Pieters, 2001, 2002).I beenot ingsT ur-v n-t COi oft iningl iningm CO( 1T inp cte-rhiA ate braned ACOc ande hat

TACO helps both in entry as well as intracellular survivalof M. tuberculosis.

Considerable interest has been expressed in the bio-logical activity of green tea (Camellia sinensis) andits impact to human health (Cos et al., 2004). Greentea (GT) is well known because of its antioxidantand free radical scavenging abilities associated withits active components polyphenols (Higdon & Frei,2003). The polyphenol fraction of GT includes epicat-echin (EC), epicatechin gallate (ECG), epigallocatechin(EGC), and epigallocatechin-3-gallate (EGCG) of whichEGCG generally accounts for greater than 40% of thetotal and is considered the most potent component ofthe green tea polyphenols (Orzechowski, Ostaszewski,Jank, & Berwid, 2002). EGCG in particular has beenshown to modulate different signaling pathways thatprevent/control various pathological processes (Kaul,Sikand, & Shukla, 2004b; Park & Dong, 2003). EGCGhas been shown to modulate MAP-kinase pathway andcytokine signaling through various transcription factorslike Sp1, NF-�B and AP-1 (Manson et al., 2000). Sp1, aDNA-binding protein, is known to enhance the transcrip-tion by binding to Sp1 response elements in the promoterregion of target genes (Boisclair, Browns, Casola, &Rechler, 1993; Kadonaga, Jones, & Tjian, 1986). Specif-ically, EGCG has been shown to block this activity thusacting as a negative regulator of Sp1-dependent genes(Ren, Zhang, Mitchell, Butler, & Young, 2000; Yeh,Chen, Chiang, Lin-Shiau, & Lin, 2003).

Keeping in view the above-mentioned findings, anct of, onthatCOrther,nism

andsti-COl.

ommanors

ingion.llateBS)

ncreased TACO recruitment and retention has alsobserved in phagosomes carrying pathogenicHelicobac-

er pylori strain in comparison to phagosomes carrytrains that lack virulent factors (Zheng & Jones, 2003).his indicates that TACO may be of importance in sival of other intracellular microbes as well. In corast to this, one study indicated that although TAs involved in M. bovis BCG uptake, persistencehis protein was not observed in phagosomes contaess than five bacteria although phagosomes conta

ore than five bacteria conspicuously retained TASchuller, Neefjes, Ottenhoff, Thole, & Young, 200).ACO protein in association with cholesterol withlasma membrane also facilitates entry of mycobaia within macrophages (Gatfield & Pieters, 2000). Weave proposed that a cholesterol specific receptor-Ck can

n part contribute to the entry of mycobacteria (Kaul,nand, & Verma, 2004a). Therefore, mycobacteria thnter macrophages at cholesterol rich plasma memomains are subsequently sequestered within Toated phagosomes, preventing lysosomal deliverynsuring their intracellular survival. This indicates t

attempt has been made to understand the effeGT-derived polyphenols (GTPs), especially EGCGTACO gene transcription. Such a study revealedEGCG has the inherent ability to down-regulate TAgene transcription in a dose-dependent manner. Futhe study has been extended to find out the mechaof this down-regulation. Since TACO helps in entryintracellular survival of mycobacteria, we also invegated the effect of this EGCG down-regulation of TAgene transcription onM. tuberculosis entry and surviva

2. Materials and methods

2.1. Cells and materials

THP-1 and Jurkat cell lines were obtained frNational Centre for Cell Science (NCCS), Pune. Humonocytes were isolated from blood of healthy donwho were not on any kind of medication, includherbal medicines for 2 weeks prior to blood donatPurified epicatechin (EC), epigallocatechin-3-ga(EGCG), culture media and fetal bovine serum (F

were obtained from SIGMA. cDNA synthesis kit waspurchased from MBI Fermentas and PCR core kit wasobtained from QIAGEN. TOPO-reporter kit, Lipofec-tamine 2000 and�-galactosidase assay kit were pur-chased from Invitrogen. All other reagents were of high-est quality available. Anti-LAM monoclonal antibodyCS-35 was kindly provided by Drs J.T. Belisle and L.Johnson (Colorado State University, Fort Collins, CO)through NIH, NIAID Contract NO1 AI-75320.

2.2. Green tea polyphenol isolation andHPLC-fractionation

Green tea polyphenols (GTPs) were isolated fromdried green tea leaves by using standard method (Ahmad,Feyes, Nienminen, Agarwal, & Mukhtar, 1997). Theextract containing GTPs was further fractionated using0–3 M KCl gradient on C18-column attached to watersHPLC system. The peak fractions were collected anddialysed overnight against normal saline. The fractionswere then vacuum concentrated. Ethanol stocks of thesefractions were used in culture experiments.

2.3. Cell culture, RNA isolation and RT-PCR

Human mononuclear cells isolated by ficoll-hypaquedensity gradient method were propagated in six-well cul-ture dishes at the desired dentsity in Dulbecco’s ModifiedEagle’s Medium (DMEM) supplemented with 10% fetalbovine serum (FBS), 100 U/ml pencillin and 100�g/ml

dyeper-h to

chd fornoli,intoNAcific

rs

2.3.2. PCR for β2McDNA was amplified during 20 cycles of 94◦C

for 30 s, 60◦C for 30 s and 72◦C for 60 s, using theprimers (forward: 5′-GAATTGCTATGTGTCTGGGT-3′; reverse: 5′-CATCTTCAAACCTCCATGATG-3′) asdescribed previously (Xu, Gruber, Peterson, & Pisa,1998).

The number of cycles used for RT-PCR was standard-ized in preliminary experiments. The amplicons resolvedon ethidium bromide stained agarose gels were pho-tographed and analysed by densitometry scanning usingthe public domain NIH ImageJ gel analysis program(developed at the U.S. National Institutes of Health).The mRNA expression of TACO gene was normalizedto invariant control�2M mRNA. Each experiment wasrepeated thrice and similar results were obtained.

2.4. In silico deduction of Sp1 transcription factorbinding site

A 2 kb promoter sequence upstream of transcriptionalstart site of TACO gene was extracted from GenBank DB(Benson et al., 2000; GenBank, 2004). The TRANSFACdatabase search for transcription factor (TF) bindingsites in this sequence was performed using the programMatInspector professional (MatInspector, 2004). MatIn-spector (Quandt, Frech, Karas, Wingender, & Werner,1995) utilizes a library of precompiled weight matri-ces for TF-binding sites in the TRANSFAC database(Wingender et al., 2001) to scan potential binding

idualtches

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pre-

n ofctor,

Sp1ted in

streptomycin at 37◦C in humidified 5% CO2 atmo-sphere. Cell viability was assayed by trypan-blueexclusion method and found to be >95% in all the eximents. The cells were subsequently exposed for 24either (i) GTP extract 0–120�g/ml; or (ii) HPLC puri-fied peaks PI and PII ; or (iii) Epicatechin 60�g/ml; or(iv) EGCG 0–60�g/ml.

At the end of this incubation, the cells from eawell were again assayed for viability and processeRNA isolation by acid guanidium-thiocyanate phechloroform extraction method (Chomczynski & Sacch1987). The isolated RNA was reverse transcribedcDNA using M-MuL V reverse transcriptase. This cDwas then subjected to PCR amplification using speprimers for genes coding TACO and�2M.

2.3.1. PCR for TACOcDNA was amplified during 22 cycles of 94◦C for

45 s, 60◦C for 45 s and 72◦C for 90 s, using the prime(forward: 5′-CCAGTGCTATGAGGATGTGCGCG-3′;reverse: 5′-GACACGACTCGCTTGTCACGGC-3′) asdescribed previously (Anand & Kaul, 2003).

sequences of TFs. The program determines an indivscore for each sequence fragment and reports mathat score above a user-defined threshold. Since dsimilarity values (core similarity: 0.75) of MatInspetor may produce high number of false positives,used vertebrate matrix with strict threshold values (similarity: 1.0; matrix similarity optimized) for efficienanalysis of promoter sequences.

2.5. Transfection and reporter plasmid assay

Transfection and reporter assay was done asviously described (Kaul & Anand, 2003). In order toascertain whether or not EGCG-dependent regulatioTACO gene is mediated through Sp1 transcription fawe employed the following experimental design:

1. Jurkat cell line being a pure CD4+ T-cell line withhigh expression of c-myc gene.

2. The reporter cassette comprising of c-myc andresponse elements was designed and incorpora�-gal reporter plasmid (Invitrogen).

Jurkat cells transfected with this reporter plasmidshowed higher basal levels of�-galactosidase due tointrinsic over expression of c-myc in these cells. Conse-quently, EGCG-dependent regulation of�-galactosidaseactivity in these cells could specifically and selec-tively reflect its interaction with Sp1 dependent acti-vation of reporter plasmid. Sp1 response element‘TGGCGGGCGGGCCTG’ present in the TACO genepromoter and c-myc response sequence ‘GTCACGT-GCCTT’ was incorporated in the TOPO vector withthe help of TOPO-cloning reaction as per manufac-turer’s instruction. TOPO-vector (without insert) wasused as a control. Jurkat cells were seeded at thedesired density in DMEM media and the above-mentioned vector was transfected using Lipofectamine2000. The cells were then treated with 60�g/ml ofpurified EGCG. A 72 h post-transfection, the cellswere lysed and supernatant was assayed for�-galreporter activity. The experiment was repeated threetimes and the results are represented as relative�-galactivity.

2.6. Monolayer culture and treatment

THP-1 cells were seeded at the desired density in atotal volume of 3.5 ml DMEM media. The THP-1 mono-cytes were then induced to become macrophages byincubation with 16 nM of PMA (phorbol 12-myristate13-acetate) for 16–18 h. Macrophages in culture wellswere either (i) pre-treated, i.e., EGCG treatment beforeM Gt -d n of6

2

lay-i .5 mlo tibi-o st ll for2 )o withP ntedw arb nter-nw rew ola-b ibedb

2.8. Immunoflourescent labeling, flow cytometryand colony counts

At each measurement time point, the control andEGCG treated cells were removed from tissue cul-ture wells and washed three times with PBS. Cellswere then fixed and permeabilized for 20 min with4% paraformaldehyde/0.1% Triton-X 100 in PBS. Pri-mary monoclonal antibody against lipoarabinomannan(LAM), a major component of the outer surface ofmycobacteria, was then added at appropriate dilution(1:1500) in PBS for 1 h at 37◦C. THP-1 macrophageswere then washed three times in PBS and then over-laid with FITC-labeled anti-mouse IgG, diluted 1:50in PBS, for 1 h at 37◦C (Barker, George, Falkow, &Small, 1997; Heesemann & Laufs, 1985). The cellswere again rinsed with PBS and analysed by FACSCalibur for mean fluorescence intensity associated witheach sample. The results are shown as FACS histogramswith percentage LAM fluorescence (indicating numberof bacteria present). Each experiment was performedat least three times. A duplicate set of this experimentwas performed in THP-1 macrophages and viableM.tuberculosis was enumerated by colony counting (CFU)as described previously (Kisich, Higgins, Diamond,

COr cellsl-am.te.omide

. tuberculosis infection or (ii) post-treated, i.e., EGCreatment afterM. tuberculosis infection. In both conitions, EGCG was used at a fixed concentratio0�g/ml.

.7. M. tuberculosis invasion assay

Prior to invasion assays, the DMEM media overng the macrophage cultures was replaced with 3f prewarmed tissue culture media containing no antics or FBS.M. tuberculosis virulent strain H37Rv wa

hen added to macrophage monolayers in each weh at 37◦C, to obtain a multiplicity of infection (MOIf 10:1. Assays were terminated by washing onceBS and replacing the overlying medium supplemeith 100�g/ml of gentamicin that kills extracellulacteria to remove any non-specific adhesion or ialization (Lee & Falkow, 1990). After 2 h incubationith gentamicin at 37◦C, the cell monolayers weashed once with PBS and processed for immuneling and flow cytometry or colony counts as descrelow.

Fig. 1. Effect of green tea derived polyphenols (GTPs) on TAgene expression in human mononuclear cells. The mononucleawere exposed to 0–120�g/ml of polyphenolic extract. (A) Normaized mRNA levels for TACO obtained using NIH ImageJ progrEach point represents mean± CI of the experiments done in triplica(B) Representative agarose gel photograph showing ethidium brstained RT-PCR products.

& Heifets, 2002). The experiment was repeated atleast three times and results represented as percentsurvival± confidence interval (CI). EGCG was alsochecked for its direct ability to inhibit mycobacterialgrowth by adding it at appropriate concentration in 7H9medium (supplemented with 10% ADC and 2 ml/l glyc-erol) inoculated with H37Rv and observing the growthover next 48 h.

3. Results

3.1. EGCG inhibits TACO gene transcription

Exposure of human blood mononuclear cells topolyphenolic extract derived from green tea at concen-trations 0–120�g/ml lead to dose-dependent oscillatorypattern of TACO mRNA expression (Fig. 1). In orderto resolve this oscillatory effect, the crude polypheno-lic extract was subjected to HPLC and this purificationresolved these polyphenols into two peaks PI and PII

(Fig. 2). Exposure of mononuclear cells to either PI orPII showed a 48% and 72% reduction in TACO mRNAlevel, respectively (Fig. 2). In order to find out the natureof polyphenols that caused this effect, the mononuclearcells in culture were exposed to highly pure epicatechin(EC) and epigallocatechin-3-gallate (EGCG). AlthoughEC did not show any effect on TACO gene transcrip-tion (Fig. 3), EGCG had the inherent capacity to down-regulate TACO gene transcription in a dose-dependentfashion (Fig. 3).

3.2. Sp1 binding sequence is present within TACOgene promoter

Keeping in view that EGCG is a known inhibitorof transcription factor Sp1 activity, we examined thepromoter sequence of TACO gene using bioinformat-ics approach to predict putative transcription factorbinding sites. This investigation revealed Sp1 bindingsequence within the promoter region of TACO gene

ks PI and P tea

Fig. 2. HPLC fractionation and the effect of HPLC-derived pea (GT)-derived polyphenols on C18 column attached to HPLC system usinpolyphenols on the transcriptional expression of gene coding for TACOcontrol, PI : peak I and PII : peak II). (C) Representative agarose gel photo

II on TACO gene expression. (A) Fractionation profile of green

g KCl gradient (0–3 M). (B) Effect of peak fractions of green tea (GT)-. Each bar represents mean± CI of the experiments done in triplicate (C:graphs showing ethidium bromide stained RT-PCR products.* p < 0.05.

Fig. 3. Effect of epicatechin (EC) and epigallocatechin-3-gallate (EGCG) on TACO gene expression in human mononuclear cells. (A) NormalizedmRNA levels of TACO in cells treated with EC. (B) Dose-dependent effect of EGCG on TACO gene expression in human mononuclear cells usingNIH ImageJ program. Each point represents mean± CI of the experiments done in triplicate.* p < 0.05.

(Fig. 4), thereby making Sp1 a potential candidate forthe observed EGCG activity in relation to TACO genetranscription (Fig. 3). To conform that the observedEGCG effect on TACO gene transcription is throughthe predicted Sp1 binding sequence, we performed�-galactosidase reporter assay. The activity of such areporter construct, carrying Sp1 response sequence cou-pled with c-myc sequence, decreased by greater thanthree-fold as compared to control cells that were notstimulated with EGCG (Fig. 4).

Fig. 4. (A) Predicted Sp1 binding site in the promoter region of TACOgene on the basis of in silico analysis using MatInspector ver 7.2.2 in theTRANSFAC database. (B) TACO promoter dependent�-galactosidasereporter activity in Jurkat T-cell exposed to EGCG. Each bar representsmean± CI of the experiments done in triplicate.* p < 0.05.

3.3. EGCG inhibits M. tuberculosis survival withinhuman macrophages

The transcriptional down-regulation of TACO geneby EGCG and the importance of TACO inM. tubercu-losis survival can be appreciated only if such suppres-sion leads to inhibition of mycobacterial entry/survival.To investigate the same, we performed mycobacterialinvasion of THP-1 macrophage cultures for differenttime periods. To investigate the entry and intracellularsurvival of M. tuberculosis in TACO down-regulatedmacrophages, the cells were pre-treated with EGCGand then infected with liveM. tuberculosis H37Rv.When such a culture was analysed by flow cytome-try (FACS) to detect FITC-labeled mycobacterial LAM(indicating number of bacteria present), we observedthat there was 18% inhibition of mycobacterial entry insuch cells. However, by 12 h only 26% of the bacteriasurvived as compared to control cells (Fig. 5). Further,to investigate the bacterial survival when TACO geneis down-regulated, liveM. tuberculosis H37Rv infectedmacrophages were post-treated with EGCG for 24 h. Insuch an experiment, 82% of the survival was observedtill 12 h, which reduced to 77% till 24 h. At 48 h ofinfection, survival decreased significantly to 55% ascompared to that in control cells (Fig. 5). However,colony counts (CFU) of the same pre-treated experi-ment in THP-1 macrophages revealed that although theentry of bacteria decreased by only 25%, at 12 h of

Fig. 5. Percent LAM fluorescence (indicating number of bacteria present) within macrophages at indicated time periods. (A) Percent LAM floures-cence of mycobacteria in macrophages exposed to EGCG for 24 h prior to infection. (B) Percent LAM flourescence of mycobacteria in macrophagesthat were exposed to EGCG for 24 h afterM. tuberculosis infection.

infection no live bacteria were observed as determinedby CFU (Fig. 6). In the case of post-treated culture,there was reduction in mycobacterial colony counts from25% at 12 h to 50% at 48 h (Fig. 6). To confirm thatthe observed effect is not because of antimycobacte-rial effect of EGCG, we incubated EGCG with freegrowing mycobacteria. Although a slight reduction wasobserved in mycobacterial growth but it was insignificant(Fig. 7) and thus confirmed that the observed effect onTACO expression is because of EGCG inhibition of Sp1activity.

4. Discussion

M. tuberculosis is among the most successfulpathogens able to survive and replicate within thedefense machinery of its host, i.e. macrophage. The mostcrucial event for the persistent infection ofM. tuberculo-sis is the inhibition of the phagosome–lysosome fusion.

Fusion of phagosome to lysosome results in killing ofmycobacteria. But, before the two organelles can fuse,budding and fusion machinery needs to be recruitedto the mycobacterial phagosome. However, the activeretention of TACO around the mycobacterial phagosomeprevents the delivery of this machinery and thus thepathogen continues to survive and replicate within theTACO armored phagosome (Ferrari et al., 1999). Thismakes TACO a strategic molecule to be modulated. Aninteresting level of modulation is at the transcriptionallevel. The present study was based on the hypothesisthat the transcriptional down-regulation of this gene willlead mycobacterial phagosome vulnerable to the deliveryof H+-ATPase complex that initiates acidification. Thiswill also leave the phagosomes (carrying mycobacteria)exposed for the recruitment of fusion machinery as inthe case of any other phagosome. Such a state will resultin phagosome–lysosome fusion and subsequent killingof the pathogen.

Fig. 6. Percent colony forming units (CFU) ofM. tuberculosis in THP-1 macrophages at indicated time periods. (A) Percent CFU of mycobacteriain THP-1 macrophages exposed to EGCG for 24 h prior to infection. (B) Percent CFU of mycobacteria in THP-1 macrophages that were exposedto EGCG for 24 h afterM. tuberculosis infection. Each bar represents mean± CI of the experiments done in triplicate.* p < 0.05.

Recent reports have suggested that green tea polyphe-nols (GTPs), besides their antioxidant properties, havefar greater impact on the regulation of gene expressionthan previously appreciated (Kaul et al., 2004b). This canbe judged by the fact that the most potent component ofGTPs, EGCG has been shown to modulate different tran-scription factors as final targets in various signal trans-

F 8 hp

duction cascades (Manson et al., 2000; Park & Dong,2003). It is in this context that the results reported hereassume importance. We observed that GTPs showed anoscillatory effect on TACO mRNA expression (Fig. 1).This effect may be due to the fact that the polyphenolfraction obtained from green tea contains different com-ponents and it is likely that the different componentsinteract with each other and thus may not be equallyeffective to bring a net down-regulation of TACO geneexpression. In order to assess the individual components,we HPLC purified the extract and again tested the twomain peaks (Fig. 2). The observed 72% reduction withPII confirmed that the fraction might contain componentsthat could suppress TACO expression to significant lev-els (Fig. 2). Although, EC did not have any effect, EGCGwas observed to have a dose-dependent effect on TACOgene expression (Fig. 3).

In order to find out the possible mechanism of TACOgene down-regulation by EGCG, we did in silico analysisof the TACO gene promoter sequence using MatInspec-tor program and found a putative Sp1 transcription factorbinding sequence (Fig. 4). To prove that Sp1 inhibitionby EGCG leads to TACO gene transcriptional down-regulation, we performed�-galactosidase reporter assayusing reporter plasmid (carrying c-myc coupled withSp1 response site) in a homogenous Jurkat T-cell linethat shows intrinsic c-myc expression. EGCG treatmentof reporter plasmid carrying cells resulted in greaterthan three-fold decrease in�-galactosidase activity ascompared to control cells (Fig. 4). Cellular transcrip-

ding

ig. 7. Effect of EGCG on free growing H37Rv bacilli over a 4eriod. tion factor Sp1 has been recognized as a DNA-bin

protein known to enhance transcription by binding toGC-rich sequences in the promoter region of target genes(Boisclair et al., 1993; Kadonaga et al., 1986). In con-trast to this, EGCG has been shown to block Sp1 bindingin the promoter region of fatty acid synthase gene (FAS)that significantly suppressed its transcription and trans-lation products (Yeh et al., 2003). Similarly, anotherstudy indicated that EGCG treatment decreased the DNAbinding activity as well as protein expression of Sp1(Ren et al., 2000). This decreased the androgen receptormRNA levels thus indicating a potential transcriptionalregulation of various genes by Sp1 (Ren et al., 2000).The �-galactosidase reporter assay coupled with theabove-mentioned studies prove that loss of Sp1 func-tion following EGCG treatment makes it unavailable asa positive regulator of TACO gene expression. This resultis in accordance with Sp1 site observed in the promoterregion of TACO gene using bioinformatics approach.We have also recently predicted VDR/RXR binding sitein the regulatory region of TACO gene in accordancewith the down-regulation of this gene by synergisticaction of Vitamin D and retinoic acid (Anand & Kaul,2003).

To evaluate the effect of TACO down-regulation onM.tuberculosis entry and survival, we performed mycobac-terial invasion experiments in THP-1 macrophagestreated with EGCG (Figs. 5 and 6). Such experi-ments revealed that EGCG pre-treatment inhibited themycobacterial entry by 18% as analysed by FACS todetect percent LAM fluorescence (indicating number

ainstedsur-

-1tionACSrs theflu-eriar theithindeedal

reat-se-m forSp1ow

GCG

may prove useful as an effective preventive therapyagainst tuberculosis.

Acknowledgements

Anti-LAM monoclonal antibody CS-35 was procuredthrough NIH, NIAID Contract NO1 AI-75320. PKAthanks Indian Council of Medical Research (ICMR),New Delhi for a senior research fellowship.

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