Evaluation of Epigallocatechin Gallate and Related Plant Polyphenols as Inhibitors of the FabG and FabI Reductases of Bacterial Type II Fatty-acid Synthase
2004; Elsevier BV; Volume: 279; Issue: 30 Linguagem: Inglês
10.1074/jbc.m403697200
ISSN1083-351X
AutoresYongmei Zhang, Charles O. Rock,
Tópico(s)Tannin, Tannase and Anticancer Activities
ResumoEpigallocatechin gallate (EGCG) is the major component of green tea extracts and possesses antibacterial, antiviral, and antitumor activity. Our study focused on validating the inhibition of the bacterial type II fatty acid synthesis system as a mechanism for the antibacterial effects of EGCG and related plant polyphenols. EGCG and the related tea catechins potently inhibited both the FabG and FabI reductase steps in the fatty acid elongation cycle with IC50 values between 5 and 15 μm. The presence of the galloyl moiety was essential for activity, and EGCG was a competitive inhibitor of FabI and a mixed type inhibitor of FabG demonstrating that EGCG interfered with cofactor binding in both enzymes. EGCG inhibited acetate incorporation into fatty acids in vivo, although it was much less potent than thiolactomycin, a validated fatty acid synthesis inhibitor, and overexpression of FabG, FabI, or both did not confer resistance. A panel of other plant polyphenols was screened for FabG/FabI inhibition and antibacterial activity. Most of these inhibited both reductase steps, possessed antibacterial activity, and inhibited cellular fatty acid synthesis. The ability of the plant secondary metabolites to interfere with the activity of multiple NAD(P)-dependent cellular processes must be taken into account when assessing the specificity of their effects. Epigallocatechin gallate (EGCG) is the major component of green tea extracts and possesses antibacterial, antiviral, and antitumor activity. Our study focused on validating the inhibition of the bacterial type II fatty acid synthesis system as a mechanism for the antibacterial effects of EGCG and related plant polyphenols. EGCG and the related tea catechins potently inhibited both the FabG and FabI reductase steps in the fatty acid elongation cycle with IC50 values between 5 and 15 μm. The presence of the galloyl moiety was essential for activity, and EGCG was a competitive inhibitor of FabI and a mixed type inhibitor of FabG demonstrating that EGCG interfered with cofactor binding in both enzymes. EGCG inhibited acetate incorporation into fatty acids in vivo, although it was much less potent than thiolactomycin, a validated fatty acid synthesis inhibitor, and overexpression of FabG, FabI, or both did not confer resistance. A panel of other plant polyphenols was screened for FabG/FabI inhibition and antibacterial activity. Most of these inhibited both reductase steps, possessed antibacterial activity, and inhibited cellular fatty acid synthesis. The ability of the plant secondary metabolites to interfere with the activity of multiple NAD(P)-dependent cellular processes must be taken into account when assessing the specificity of their effects. Plants are renowned for containing compounds of medicinal interest, and there is a continuing debate over the clinical value and safety of herbal remedies (1De Smet P.A. N. Engl. J. Med. 2002; 347: 2046-2056Google Scholar). Botanical extracts include a large variety of low molecular weight secondary metabolites derived from isoprenoid, phenylpropanoid, or fatty acid/polyketide pathways. The rich diversity of these compounds is thought to arise from an evolutionary process driven by the acquisition of resistance to microbiological attack (2Dixon R.A. Nature. 2001; 411: 843-847Google Scholar). Plants and their natural enemies co-evolve (3Rausher M.D. Nature. 2001; 411: 857-864Google Scholar), so it is also expected that bacterial defenses have arisen to combat these agents. The majority of plant pathogens are Gram-negative bacteria, and consistent with the co-evolutionary hypothesis, Gram-positive bacteria are generally more susceptible to the plant secondary metabolites than Gram-negative bacteria (4Lewis K. J. Mol. Microbiol. Biotechnol. 2001; 3: 247-254Google Scholar, 5Yam T.S. Shah S. Hamilton-Miller J.M. FEMS Microbiol. Lett. 1997; 152: 169-174Google Scholar). For example, most Gram-negative bacteria are refractory to plant secondary metabolites when tested in standard susceptibility tests producing MIC 1The abbreviations used are: MIC, minimal inhibitory concentration; EGCG, (–)-epigallocatechin gallate; TLM, thiolactomycin; ACP, acyl carrier protein; FabB, β-ketoacyl-ACP synthase I; FabH, β-ketoacyl-ACP synthase III; FabG, β-ketoacyl-ACP reductase; FabI, trans-2-enoyl-ACP reductase; 3HC, 2,2′,4′-trihydroxychalcone. values in the range of 0.1–1 mg/ml. A primary underlying cause for the resistance of Gram-negative bacteria to a wide range of plant toxins is the existence of efflux pumps that prevent the intracellular accumulation of polyphenols (4Lewis K. J. Mol. Microbiol. Biotechnol. 2001; 3: 247-254Google Scholar, 6Tegos G. Stermitz F.R. Lomovskaya O. Lewis K. Antimicrob. Agents Chemother. 2002; 46: 3133-3141Google Scholar). Genetic elimination of these pumps can increase the efficacy of the plant metabolites by 1–2 orders of magnitude (6Tegos G. Stermitz F.R. Lomovskaya O. Lewis K. Antimicrob. Agents Chemother. 2002; 46: 3133-3141Google Scholar). Green tea and the individual compounds purified from tea extracts are among the best known plant polyphenols and possess numerous biological activities (including antimicrobial activity) against a variety of organisms (7Hamilton-Miller J.M. Antimicrob. Agents Chemother. 1995; 39: 2375-2377Google Scholar). The main components of a cup of green tea (200 ml) are the well characterized catechins consisting of (–)-epigallocatechin gallate (EGCG) (140 mg), (–)-epigallocatechin (65 mg), (–)-epicatechin gallate (28 mg), and (–)-epicatechin (17 mg) (8Yang C.S. Wang Z.Y. J. Natl. Cancer Inst. 1993; 85: 1038-1049Google Scholar). Avid tea drinkers consume several cups a day, and although the potency of the catechins is low, the large quantities of the polyphenols that are ingested make it reasonable to think that they have potential for antibacterial activity. An example is the correlation between the in vivo and in vitro susceptibility of Helicobacter pylori to green tea (9Yee Y.K. Koo M.W. Aliment. Pharmacol. Ther. 2000; 14: 635-638Google Scholar, 10Mabe K. Yamada M. Oguni I. Takahashi T. Antimicrobiol. Agents Chemother. 1999; 43: 1788-1791Google Scholar). There is a huge amount of literature on the biological and biochemical activities of green tea extracts and isolated compounds, but a unifying hypothesis that accounts for their biological activities has not emerged, and the underlying biochemical targets remain unidentified (8Yang C.S. Wang Z.Y. J. Natl. Cancer Inst. 1993; 85: 1038-1049Google Scholar, 11Adhami V.M. Ahmad N. Mukhtar H. J. Nutr. 2003; 133: 2417S-2424SGoogle Scholar, 12Higdon J.V. Frei B. Crit. Rev. Food Sci. Nutr. 2003; 43: 89-143Google Scholar). For example, EGCG and related polyphenols inhibit the fungal and mammalian type I fatty-acid synthase system, most likely by interacting with their reductase and perhaps condensation subdomains (13Wang X. Tian W. Biochem. Biophys. Res. Commun. 2001; 288: 1200-1206Google Scholar, 14Wang X. Song K.S. Guo Q.X. Tian W.X. Biochem. Pharmacol. 2003; 66: 2039-2047Google Scholar, 15Li B.H. Tian W.X. J. Biochem. (Tokyo). 2004; 135: 85-91Google Scholar, 16Li X.C. Joshi A.S. el-Sohly H.N. 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Commun. 2002; 292: 1155-1166Google Scholar), in contrast to the mammalian fatty-acid synthase, which is a homodimer of single multifunctional polypeptide derived from a single gene (19Smith S. Witkowski A. Joshi A.K. Prog. Lipid Res. 2003; 42: 289-317Google Scholar). The difference between the bacterial and mammalian synthases has been exploited to establish fatty acid synthesis as a target for antibacterial drug discovery (20Heath R.J. White S.W. Rock C.O. Prog. Lipid Res. 2001; 40: 467-497Google Scholar, 21Campbell J.W. Cronan Jr., J.E. Annu. Rev. Microbiol. 2001; 55: 305-332Google Scholar). There are several naturally produced antibiotics, such as cerulenin (22Vance D.E. Goldberg I. Mitsuhashi O. Bloch K. Omura S. Nomura S. Biochem. Biophys. Res. Commun. 1972; 48: 649-656Google Scholar), thiolactomycin (TLM) (23Hayashi T. Yamamoto O. Sasaki H. Okazaki H. J. Antibiot. (Tokyo). 1984; 37: 1456-1461Google Scholar), and CT2108A (24Laakso J.A. Raulli R. McElhaney-Feser G.E. Actor P. Underiner T.L. Hotovec B.J. Mocek U. Cihlar R.L. Broedel Jr., S.E. J. Nat. Prod. 2003; 66: 1041-1046Google Scholar), and synthetic molecules (such as isoniazid (25Banerjee A. Dubnau E. Quémard A. Balasubramanian V. Um K.S. Wilson T. Collins D. de Lisle G. Jacobs Jr., W.R. Science. 1994; 263: 227-230Google Scholar) and triclosan (26McMurray L.M. Oethinger M. Levy S. Nature. 1998; 394: 531-532Google Scholar, 27Heath R.J. Yu Y.-T. Shapiro M.A. Olson E. Rock C.O. J. Biol. Chem. 1998; 273: 30316-30321Google Scholar)), all of which specifically target this pathway. Some of these, such as cerulenin (28Omura S. Microbiol. Rev. 1976; 40: 681-697Google Scholar), inhibit both the type I and type II fatty-acid synthases, whereas others, such as thiolactomycin (23Hayashi T. Yamamoto O. Sasaki H. Okazaki H. J. Antibiot. (Tokyo). 1984; 37: 1456-1461Google Scholar), are selective for the bacterial type II system and are potentially useful therapeutics (29Miyakawa S. Suzuki K. Noto T. Harada Y. Okazaki H. J. Antibiot. (Tokyo). 1982; 35: 411-419Google Scholar). The goals of this study were to determine whether the tea catechins and related plant polyphenols were inhibitors of bacterial type II fatty-acid synthase and to determine whether this inhibition was related to their antibacterial properties. We find that EGCG is a potent inhibitor of both the β-ketoacyl-ACP reductase (FabG) and the trans-2-enoyl-ACP reductase (FabI) components in the bacterial type II fatty-acid synthase system, a property that is common to a broad range of plant polyphenols. However, the inhibition of fatty acid synthesis was not the sole reason for the antibacterial activity of the tested compounds in the Escherichia coli model system. Materials—Amersham Biosciences supplied [1-14C]acetyl-CoA (60 μCi/μmol), [1-14C]acetate (54 μCi/μmol), and [2-14C]malonyl-CoA (52 μCi/μmol); Sigma supplied acetoacetyl-CoA, malonyl-CoA, ACP, NADPH, NADH, and chloramphenicol. The trans-2-octanoyl-N-acetylcysteamine was the generous gift of Rocco Gogliotti and John Domagala (Parke-Davis). His-tagged FabB, FabH, FabG, and FabI from E. coli were purified as described previously (30Heath R.J. Rock C.O. J. Biol. Chem. 1995; 270: 26538-26542Google Scholar, 31Heath R.J. Rubin J.R. Holland D.R. Zhang E. Snow M.E. Rock C.O. J. Biol. Chem. 1999; 274: 11110-11114Google Scholar). All other reagents were of the highest grade available. Green tea extract compounds, including EGCG, (–)-epigallocatechin, (–)-epicatechin gallate, (–)-epicatechin, (–)-gallocatechin gallate, (–)-gallocatechin, (–)-catechin gallate, (+)-catechin, propyl gallate, gallic acid, butein, isoliquirtigenin, resveratrol, piceatannol, fisetin, quercetin, rhein, and plumbagin were purchased from Sigma. Fustin, taxifolin, 2,2′,4′-trihydroxychalcone, and 7,3′,4′-trihydroxyisoflavone were purchased from Indofine Chemicals Co. The compounds were dissolved in Me2SO at 10 mm. Bacterial Strains—Strain ANS1 (metB1 relA1 spoT1 gyrA216 tolC::Tn10 λ– λR F–) cultured in Tryptone broth (1% Tryptone) was used to determine the minimal inhibitory concentrations (32Jackowski S. Zhang Y.-M. Price A.C. White S.W. Rock C.O. Antimicrob. Agents Chemother. 2002; 46: 1246-1252Google Scholar). Chemically defined M9 medium (33Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1972Google Scholar) was used in the [14C]-acetate labeling experiments described below. ANS1 was constructed by P1-mediated transduction of the tolC::Tn10 element in strain EP1581 into strain UB1005 followed by selection for tetracycline resistance. The TolC-dependent type I secretion and multidrug efflux systems are defective in ASN1 (32Jackowski S. Zhang Y.-M. Price A.C. White S.W. Rock C.O. Antimicrob. Agents Chemother. 2002; 46: 1246-1252Google Scholar). Plasmid expressing fabG or fabI was constructed by moving the XbaI-BamHI fragment of the His-tagged version of either the fabG or fabI gene (34Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 10996-11000Google Scholar), respectively, in pET15-b into pBluescript II KS(+). To select for the presence of both reductases, the pBluescript plasmid carrying the enoyl-ACP reductase gene from Clostridium acetobutylicum was modified to replace the AmpR gene with and KanR cassette (35Marrakchi H. DeWolf Jr., W.E. Quinn C. West J. Polizzi B.J. So C.Y. Holmes D.J. Reed S.L. Heath R.J. Payne D.J. Rock C.O. Wallis N.G. Biochem. J. 2003; 370: 1055-1062Google Scholar). The His-tagged versions were used to confirm expression using an anti-tag antibody. β-Ketoacyl-ACP Reductase (FabG) Assay—The disappearance of NADPH, the cofactor for FabG reaction, was measured spectrophotometrically at 340 nm as described previously (36Zhang Y.-M. Wu B. Zheng J. Rock C.O. J. Biol. Chem. 2003; 278: 52935-52943Google Scholar). The reaction mixture contained 0.5 mm acetoacetyl-CoA, 0.2 mm NADPH, 4 μg of FabG protein, 0.1 m sodium phosphate buffer, pH 7.4, in a final volume of 300 μl. Test compounds were added to the reaction mixture at the concentrations indicated in the text and figure legends. The reaction was initiated by the addition of acetoacetyl-CoA. A decrease in the absorbance at 340 nm was recorded for 2 min. The initial rate was used to calculate the enzymatic activity. Reactions with Me2SO solvent alone were used as controls. Enoyl-ACP Reductase (FabI) Assay—Reduction of the trans-2-octanoyl-N-acetylcysteamine substrate analog was measured spectrophotometrically by following the utilization of NADH at 340 nm at 25 °C as described previously (30Heath R.J. Rock C.O. J. Biol. Chem. 1995; 270: 26538-26542Google Scholar). Briefly, a standard reaction contained 0.1 m sodium phosphate, pH 7.5, 100 μm trans-2-octanoyl-N-acetylcysteamine, 200 μm NADH, and 12 μg of FabI in a final volume of 300 μl. A decrease in absorbance at 340 nm was measured at 25 °C for the linear period of the assay (usually the first 1–2 min). Test compounds were added in the assay to the indicated concentrations as described in the text and figure legends. An equal volume of the solvent was used for the untreated control. β-Ketoacyl-ACP Synthase I (FabB) and III (FabH) Assay—The coupled assay of FabH as described previously (34Heath R.J. Rock C.O. J. Biol. Chem. 1996; 271: 10996-11000Google Scholar) contained 25 μm ACP, 1 mm β-mercaptoethanol, 65 μm malonyl-CoA, 45 μm [1-14C]acetyl-CoA (specific activity, 60 μCi/μmol), 1 μg of purified FabD, 0.1 m sodium phosphate buffer, pH 7.0, and 20 ng of FabH protein in a final volume of 40 μl. The FabD protein was present to generate the malonyl-ACP substrate for the reaction. The ACP, β-mercaptoethanol, and buffer were preincubated at 37 °C for 30 min to ensure the complete reduction of ACP. The reaction was initiated by the addition of FabH. After incubation at 37 °C for 15 min, 35 μl of the reaction mixture was removed and dispensed onto a paper filter disc (Whatman No. 3MM filter paper). The disc was washed successively with ice-cold 10, 5, and 1% trichloroacetic acid with 20 min for each wash and 20 ml of wash solution per disc. The filter discs were dried and counted for 14C isotope in 3 ml of scintillation fluid. The condensation assay for FabB was the same as described previously (37Garwin J.L. Klages A.L. Cronan Jr., J.E. J. Biol. Chem. 1980; 255: 11949-11956Google Scholar). Briefly, FabB assays contained 45 μm myristoyl-ACP, 50 μm [2-14C]malonyl-CoA (specific activity, 52 μCi/μmol), 100 μm ACP, 1 μg of FabD, and 25 ng of FabB in a final volume of 20 μl. ACP was reduced by 0.3 mm dithiothreitol before the other reaction components were added. The reaction was initiated by the addition of the enzyme. After incubation at 37 °C for 15 min, the reaction was stopped by adding 0.4 ml of the reducing reagent containing 0.1 m K2HPO4, 0.4 m KCl, 30% tetrahydrofuran, and 5 mg/ml NaBH4. The reaction contents were vigorously mixed after the addition of the reducing reagent and incubated at 37 °C for 40 min before being extracted into 0.4 ml of toluene. The 14C isotope in the upper phase was quantitated by scintillation counting. IC50 values were determined at a series of concentrations. A line was drawn between the points, and the IC50 was the interpolated concentration that gave 50% inhibition (see Fig. 1). Determination of the MIC—The MICs of the test compounds against E. coli strain ANS1 were determined by a broth microdilution method. ANS1 was grown to midlog phase in 1% Tryptone broth and then diluted 30,000-fold in the same medium. A 10-μl aliquot of the diluted cell suspension (3,000–5,000 colony-forming units) was used to inoculate each well of a 96-well plate (U-bottom with a low evaporation lid) containing 100 μl of Tryptone broth with the indicated concentration of inhibitors. The plate was incubated at 37 °C for 20 h before being read with a Fusion™ universal microplate analyzer (Packard) at 600 nm. The absorbance was normalized to the solvent-treated control, which was considered to be 100%. [1-14C]Acetate Labeling—Strain ANS1 was grown to midlog phase in M9 minimal medium supplemented with 0.1% casamino acids, 0.4% glycerol, and 0.0005% thiamin. A 1-ml aliquot of cells was treated with the antimicrobial compounds for 10 min at the indicated concentrations as described in the figure legends. An equal volume of the solvent Me2SO was added to the untreated control. Cells were then labeled with 10 μCi of [1-14C]acetate for 1 h before being harvested by centrifugation. The cell pellets were washed with phosphate-buffered saline and resuspended in 100 μl of M9 medium. The total cellular lipids were extracted, and the incorporated 14C isotope in lipids in the chloroform phase was quantitated by scintillation counting. Results reflect duplicate experiments. Inhibition of FabG and FabI by Tea Catechins—The enzymes of the type II fatty-acid synthase were assayed for inhibition by EGCG, the major catechin of green tea (Fig. 1A). The elongation condensing enzyme (FabB) was refractory to EGCG inhibition, whereas FabH, the initiating condensing enzyme, was inhibited by EGCG with an IC50 of 40 μm. The most potently inhibited enzyme was FabG, the NADPH-dependent ketoreductase in the pathway, which exhibited an IC50 of 5 μm. FabI, the NADH-dependent enoyl reductase, was inhibited by EGCG with an IC50 of 15 μm. FabG was used as a model enzyme to determine the inhibitory potency of the other significant green tea catechins (Fig. 1B). EGCG was the most potent, but the other catechins, (–)-epicatechin gallate, (–)-gallocatechin gallate, and (–)-catechin gallate, all exhibited activity against FabG with IC50 values ranging from 5 to 15 μm. Similarly, the four compounds exhibited activity against FabI with IC50 values between 5 and 15 μm (Table I). Also, we found that the gallate substitution was critical for the inhibitory activity of the compounds (Table I). Removal of the galloyl moiety from any of the tea catechins resulted in complete loss of inhibitory activity in vitro. The galloyl group itself has no significant inhibitory activity as shown by the lack of FabG and FabI inhibition by propyl gallate and gallic acid (Table I). These data establish that green tea catechins have significant inhibitory activity against the reductase enzymes of the bacterial type II fatty-acid synthase. Furthermore, the structure-activity relationship shows that the portion of the EGCG molecule required for inhibition is defined by the boxed area in Scheme 1.Table IInhibitory effects of green extract compounds on the activity of fatty acid synthetic enzymes (FabG and FabI) and bacterial growth Open table in a new tab Mechanism for Tea Catechin Inhibition of FabG and FabI— The kinetic mechanism for the inhibition of FabG and FabI was determined using EGCG as the model compound (Fig. 2). Both FabG (38Price A.C. Zhang Y.-M. Rock C.O. White S.W. Structure. 2004; 12: 417-428Google Scholar) and FabI (39Sivaraman S. Zwahlen J. Bell A.F. Hedstrom L. Tonge P.J. Biochemistry. 2003; 42: 4406-4413Google Scholar) have compulsory ordered mechanisms with the nucleotide cofactors as the leading substrates. This knowledge allowed us to design a kinetic analysis that would distinguish between the three possible outcomes; EGCG could bind to the free enzyme, the enzyme-substrate complex, or both to prevent catalysis. In the first case, the inhibition pattern with respect to the cofactor would be competitive; in the second, the inhibition pattern would be non-competitive; and in the third case, mixed-type inhibition would be observed (40Cornish-Bowden A. Fundamentals of Enzyme Kinetics. Portland Press Ltd., London1995: 95-101Google Scholar). The inhibition of FabG by EGCG was mixed with respect to NADPH (Fig. 2A). Thus, EGCG binds to both the free enzyme to prevent the binding of the nucleotide cofactor and also to the FabG-NADPH complex to prevent the binding of the substrate. In contrast, EGCG was a competitive inhibitor of FabI with respect to NADH (Fig. 2B), meaning that EGCG interferes with activity by binding to the free enzyme and preventing the binding of NADH. These data illustrate that EGCG inhibits both enzymes by association with the nucleotide cofactor binding site, and with FabG, EGCG has the additional property of binding to the enzyme-cofactor complex. Antimicrobial Effect of EGCG—As reported previously (5Yam T.S. Shah S. Hamilton-Miller J.M. FEMS Microbiol. Lett. 1997; 152: 169-174Google Scholar, 7Hamilton-Miller J.M. Antimicrob. Agents Chemother. 1995; 39: 2375-2377Google Scholar), EGCG has moderate antibacterial activity. Most Gram-negative bacteria are resistant to plant polyphenols; therefore we used our E. coli strain ANS1 (tolC) to eliminate the activity of a major class of multidrug efflux pumps (6Tegos G. Stermitz F.R. Lomovskaya O. Lewis K. Antimicrob. Agents Chemother. 2002; 46: 3133-3141Google Scholar, 32Jackowski S. Zhang Y.-M. Price A.C. White S.W. Rock C.O. Antimicrob. Agents Chemother. 2002; 46: 1246-1252Google Scholar). Such tolC mutants are used routinely as a platform to investigate the mechanism of drug action (41Sulavik M.C. Houseweart C. Cramer C. Jiwani N. Murgolo N. Greene J. DiDomenico B. Shaw K.J. Miller G.H. Hare R. Shimer G. Antimicrob. Agents Chemother. 2001; 45: 1126-1136Google Scholar). In strain ANS1, EGCG had an MIC of 500 μm (Fig. 3A), although the parent wild-type strain UB1005 had the same MIC in this case. To evaluate whether the antibacterial properties of the catechins could be attributed to their effects on fatty acid synthesis, first we determined whether the compounds blocked the incorporation of acetate into membrane fatty acids (Fig. 3B). EGCG indeed attenuated fatty acid synthesis in vivo compared with the untreated control cells and cells treated with chloramphenicol, a protein synthesis inhibitor. However, the effect of EGCG was not nearly as great as the effect of TLM, an established inhibitor of fatty acid synthesis at the elongation condensing enzyme step (42Tsay J.-T. Rock C.O. Jackowski S. J. Bacteriol. 1992; 174: 508-513Google Scholar, 43Price A.C. Choi K.H. Heath R.J. Li Z. Rock C.O. White S.W. J. Biol. Chem. 2001; 276: 6551-6559Google Scholar). These data demonstrate that EGCG inhibits fatty acid synthesis in vivo, although it is not as potent when present at double its MIC compared with a bona fide inhibitor added to the cells at the same relative concentration, suggesting that fatty acid synthesis may not be the sole pathway inhibited by EGCG. Overexpression of individual genes and the isolation of resistant mutants are powerful genetic tools for target validation in vivo. For example, FabB was unequivocally established as the critical in vivo target for TLM by demonstrating that overexpression of FabB increased the resistance to TLM and that a point mutation conferring resistance to the drug was localized to the fabB gene (32Jackowski S. Zhang Y.-M. Price A.C. White S.W. Rock C.O. Antimicrob. Agents Chemother. 2002; 46: 1246-1252Google Scholar, 42Tsay J.-T. Rock C.O. Jackowski S. J. Bacteriol. 1992; 174: 508-513Google Scholar). Similarly, FabI was validated as the triclosan target through analyzing the effects of FabI overexpression and the isolation of resistant mutants in the fabI gene (26McMurray L.M. Oethinger M. Levy S. Nature. 1998; 394: 531-532Google Scholar, 27Heath R.J. Yu Y.-T. Shapiro M.A. Olson E. Rock C.O. J. Biol. Chem. 1998; 273: 30316-30321Google Scholar). Unfortunately, there are no known specific inhibitors against FabG to corroborate the function of our FabG expression plasmid, but the construct increases FabG expression, and like the FabB and FabI constructs with identical promoter elements is anticipated to shift the dose-response curve for an antibacterial compound that selectively targets FabG. Therefore, we examined the effect of the overexpression of FabG, FabI, or both on the MIC for EGCG in strain ANS1 (Fig. 3A). Unlike other drugs that primarily target lipid synthesis, none of these plasmids increased the resistance of strain ANS1 to EGCG. We also attempted to raise EGCG-resistant mutants using the techniques described previously to isolate mutants specifically resistant to TLM (32Jackowski S. Zhang Y.-M. Price A.C. White S.W. Rock C.O. Antimicrob. Agents Chemother. 2002; 46: 1246-1252Google Scholar) or triclosan (27Heath R.J. Yu Y.-T. Shapiro M.A. Olson E. Rock C.O. J. Biol. Chem. 1998; 273: 30316-30321Google Scholar). However, we were unable to obtain colonies resistant to 1 mm EGCG. These data do not support fatty acid synthesis as a primary target for the action of EGCG, and the inability to isolate specific mutants is consistent with the existence of multiple targets in vivo. FabG/FabI Inhibition by Other Plant Natural Products—To determine whether the inhibition of FabG and FabI was a property of other natural products with the biphenyl core structure diagramed in Scheme 1, we tested a panel of plant polyphenols (Table II). The MIC values were obtained using strain ANS1 to eliminate the effects of efflux pumps, and the MICs for a wild-type and tolC isogenic pair were reported previously (6Tegos G. Stermitz F.R. Lomovskaya O. Lewis K. Antimicrob. Agents Chemother. 2002; 46: 3133-3141Google Scholar). Some of the natural products (such as coumestrol, rhein, and plumbagin) had potent antibacterial activity but were not significant inhibitors of either the FabG or FabI reductases. However, in general all of the tested natural products with the biphenyl chalcone nucleus inhibited both enzymes. Resveratrol, piceatannol, fustin, taxifolin, and 7,3′,4′-trihydroxyisoflavone were good inhibitors for FabG and FabI, but their MIC values were high, indicating only weak activity against any target in vivo.Table IIInhibitory effects of polyphenol compounds on the activity of fatty acid synthetic enzymes (FabG and FabI) and bacterial growth Open table in a new tab The remaining polyphenols with comparable reasonable MIC values (Table II) were further evaluated for their effects on fatty acid synthesis at 4 times their MICs. The inhibition of [14C]acetate incorporation by butein, 3HC, fisetin, and quercetin was between 20 and 50% in comparison to cells treated with Me2SO (Table II). The highest inhibition was observed with isoliquirtigenin, which reduced the [14C]acetate incorporation by about 75%. The FabG/FabI inhibitor with the lowest MIC against strain ANS1, 3HC, was selected for more detailed investigation to determine whether its antibacterial action could be linked to blocking fatty acid synthesis (Fig. 4). FabH was not inhibited by 3HC. FabB, FabG, and FabI were all inhibited by 3HC with IC50 values of 100, 25, and 40 μm, respectively (Fig. 4A). Thus, 3HC was less potent than EGCG in vitro. The MIC for 3HC was 6.25 μm, and the MIC was not shifted by the overexpression of either FabG or FabI (Fig. 4B). Finally, acetate incorporation studies showed that fatty acid synthesis was inhibited only at concentrations significantly higher than the MIC for the compound (Fig. 4C). Although like most of the other plant polyphenols, 3HC inhibited the reductase steps in fatty acid synthesis in vitro, these data establish that the most effective antibacterial examined in the screen did not primarily inhibit cell growth by blocking the type II fatty acid synthesis. Likewise, we examined the inhibition of fatty acid synthesis in vivo using four other analogs with MICs below 75 μm (Table II). Although all of the compounds inhibited acetate incorporation into cellular fatty acids, none were as potent as the TLM control. Thus, these polyphenols reduced fatty acid synthesis, but the extent of inhibition was not consistent with fatty acid synthesis as the primary target for their antibacterial activity. The emergence of multidrug resistance in pathogenic bacteria i
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