DprE1 Is a Vulnerable Tuberculosis Drug Target Due to Its Cell Wall

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DprE1 Is a Vulnerable Tuberculosis Drug Target Due to Its Cell Wall Localization Miroslav Brecik,†,§ Ivana Centárová,†,§ Raju Mukherjee,‡,§,∥ Gael̈ le S. Kolly,‡ Stanislav Huszár,† Adela Bobovská,† Emöke Kilacsková,† Veronika Mokošová,† Zuzana Svetlíková,† Michal Šarkan,† Joaõ Neres,‡ Jana Korduláková,† Stewart T. Cole,*,‡ and Katarína Mikušová*,† †

Department of Biochemistry, Faculty of Natural Sciences, Comenius University in Bratislava, 842 15 Bratislava, Slovakia Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland

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S Supporting Information *

ABSTRACT: The flavo-enzyme DprE1 catalyzes a key epimerization step in the decaprenyl-phosphoryl D-arabinose (DPA) pathway, which is essential for mycobacterial cell wall biogenesis and targeted by several new tuberculosis drug candidates. Here, using differential radiolabeling with DPA precursors and high-resolution fluorescence microscopy, we disclose the unexpected extracytoplasmic localization of DprE1 and periplasmic synthesis of DPA. Collectively, this explains the vulnerability of DprE1 and the remarkable potency of the best inhibitors.

DprE2; DprE1 uses FAD to oxidize DPR to a ketointermediate, which, in turn, is reduced to DPA by DprE2 using NADH as a cofactor10 (Figure 1a). Since its initial discovery as a target of BTZs,4 DprE1 has been rediscovered in numerous whole-cell screens of chemical libraries and is inhibited by compounds as diverse as dinitrobenzamides, benzoquinoxalines, nitro-substituted triazoles, and the benzothiazole derivative TCA1, among others.11 Given its inhibition by multiple pharmacophores representing diverse chemical space, DprE1 was designated as a “promiscuous” target.12 The vulnerability of DprE1 was also demonstrated by genetic studies with conditional knock-down mutants, in which down-regulation of dprE1 resulted in faster depletion of the enzyme and a stronger bactericidal effect in vitro than was seen following knock-downs of the other DPA pathway genes.9 Here, we show that DprE1 is localized in the periplasmic space of the mycobacterial cell wall, which makes it more accessible to drugs and could be a critical factor contributing to its vulnerability.

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), a contagious air-borne disease of humans. Despite the fact that most of the cases are curable, TB claimed 1.5 million lives in 2013 and accounted for an estimated 9 million incident cases.1 The current 6-month combination therapy was introduced 40 years ago, but due to the lengthy treatment duration, drug−drug interactions, undesirable side-effects, and the emergence of drug resistance, more effective drugs and a better regimen are needed.2 Benzothiazinones (BTZs), such as PBTZ169, are among the most potent and best-characterized novel candidates currently in the TB drug development pipeline.3,4 PBTZ169 covalently binds to an active site cysteine residue (Cys387 in M. tuberculosis, Cys394 in Mycobacterium smegmatis) in the essential enzyme DprE1, thereby causing quantitative and irreversible inactivation.3,5,6 DprE1 is the flavoprotein subunit of the enzyme, decaprenylphosphoryl D-ribose epimerase, that produces decaprenylphosphoryl arabinose (DPA), a unique sugar donor for biogenesis of the vital mycobacterial cell wall polysaccharides arabinogalactan and lipoarabinomannan (Figure 1a). DPA biosynthesis starts when the UbiA enzyme transfers ribose-5-phosphate from 5-phosphoribose pyrophosphate (PRPP) to the lipid carrier decaprenyl phosphate.7 The resultant product, decaprenylphosphoryl ribose-5-phosphate (DPRP), is then dephosphorylated to decaprenylphosphoryl ribose (DPR). The rv3807c gene product was proposed to serve as the phosphatase catalyzing this reaction, but its role in the DPA pathway remains unclear.8,9 Epimerization of DPR to DPA is accomplished by the concerted action of DprE1 and © XXXX American Chemical Society

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RESULTS AND DISCUSSION The first indication of possible cell wall localization of DPA synthesis within mycobacteria emerged from cell-free assays monitoring incorporation of the radioactive, water-soluble substrate P[14C]RPP into the lipid-linked intermediates Received: February 16, 2015 Accepted: April 23, 2015

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DOI: 10.1021/acschembio.5b00237 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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composition but are still cross-contaminated. However, the greater activity of the cell envelope fraction in converting P[14C]RPP to DP[14C]A suggests that the enzymes responsible for DPA biosynthesis are associated with the cell wall. We envision that the cell envelope fraction is composed of plasma membrane remnants associated with cell wall components comprising, among other things, the cell wall synthesizing machinery, including the enzymes of the DPA pathway. Indeed, colocalization, or interaction of several enzymes involved in the biosynthesis of the cell wall components, peptidoglycan, arabinogalactan, and mycolates, was recently reported by several authors.5,18,19 Initially, we focused on DprE1 and attempted to reveal its localization by producing His-tagged versions in M. smegmatis mc2155. As shown in Supporting Information Figure 2, recombinant N-terminally or C-terminally His-tagged DprE1 was not detected in the cytoplasm. However, DprE1 bearing a His-tag at the C-terminus was found mainly in the cell envelope fraction, and this finding is consistent with the strong enzymatic activity of the cell envelope in producing DP[14C]A from P[14C]RPP (Figure 1b; Supporting Information Table 1). Nterminally His-tagged DprE1 also localized to the membrane fraction. However, since the occurrence of DprE1 in the membrane fraction does not reveal whether it is associated with the cytoplasmic or periplasmic side, and the position of the Histag may have affected the subcellular distribution of DprE1, we concluded that a combination of enzymology and microscopy with the native protein could be more physiologically relevant to disclose its cellular localization. To establish whether the biosynthetic reactions that produce DPA took place on the outer surface of the mycobacterial plasma membrane, we generated spheroplasts, which are known to be metabolically active and a suitable tool for investigating cell wall biosynthesis.20,21 Spheroplasting resulted in major destruction of the outer membrane as evidenced by a reduction of up to 70% in the arabinogalactan and peptidoglycan content (Supporting Information Table 2), as well as by a morphological change (Supporting Information Figure 3). When cell envelope fractions prepared from spheroplasts were used, P[14C]RPP was incorporated less efficiently, as compared to the same fractions prepared from untreated bacteria. Furthermore, DP[14C]R was the last intermediate in the DPA pathway produced as seen on the TLC profile (Figure 1b). Apparently, the lack of DPR epimerization observed in spheroplasts could be related to cell wall damage, implying that both the DprE1 and DprE2 enzymes might be associated with this compartment. Next, we explored the capacity of intact spheroplasts to metabolize P[14C]RPP. We observed that spheroplasts incorporated P[14C]RPP into DP[14C]A precursors, as did intact untreated cells of M. smegmatis mc2155, which were used as a control in this experiment (Figure 1b). As was seen with the cell envelope fraction, epimerization of DP[14C]R did not take place in spheroplasts, contrary to untreated cells that produced DP[14C]A. These findings were unexpected, since UbiA, the enzyme responsible for the PRPP to DPRP conversion, was proposed to act inside the plasma membrane,22 and DprE1 and DprE2 were always considered to be peripherally associated with the cytoplasmic side of the plasma membrane.2,23 In this model, DPA would then flip across the membrane for incorporation into arabinogalactan and lipoarabinomannan.

Figure 1. P[14C]RPP incorporation into DP[14C]A and its precursors by whole cells and cell lysates. (a) The DPA biosynthetic pathway, intermediates, and key enzymes. (b) Autoradiogram of TLC-separated products after incorporation of P[14C]RPP (15 000 dpm) into DP[14C]A and its precursors by membranes (Me; 300 μg) and cell envelopes (CE; 300 μg) prepared from untreated cells (UC) and spheroplasts (S), respectively, or by whole untreated cells and spheroplasts. ∗, unidentified lipid. Reaction products were separated on a silica gel TLC plate developed in CHCl3/CH3OH/concentrated NH4OH/1 M ammonium acetate/H2O (180:140:9:9:23, v/v).

DP[14C]RP, DP[14C]R, DP[14C]X, and the final product DP[14C]A.13,14 Enzymatically active membranes and/or cell envelopes were obtained from sonicated M. smegmatis mc2155 by differential centrifugation.15 When these two fractions were examined for their ability to synthesize DP[14C]A from P[14C]RPP, the cell envelope fraction was considerably more efficient (Figure 1b; Supporting Information Table 1). To assess the level of cross-contamination that inevitably results from subcellular fractionation,16,17 we used NADH oxidase activity and mycolates as markers for the plasma membrane and cell wall, respectively.17 As expected, the membrane fraction showed much higher NADH oxidase specific activity (25 ± 5 nmol min−1 mg−1) compared to the cell envelope fraction (7 ± 0.5 nmol min−1 mg−1; Supporting Information Figure 1a) whereas mycolates were found predominantly in the cell envelope (Supporting Information Figure 1b). These data demonstrate that the two fractions clearly differ in their B


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Figure 2. P[14C]RPP incorporation into water-soluble and lipid products using whole cells and cell lysates. (a) Reactions were performed with 20 mg of M. smegmatis mc2155 or lysates prepared from 20 mg of cells and 15 000 dpm of P[14C]RPP. C, control; U, supplemented with 200 μM uracil; 5FU, supplemented with 200 μM 5-fluorouracil. The spots labeled [14C]UMP and 5F-[14C]UMP comigrated with nonradioactive standards of UMP and 5-FUMP visualized by UV. PEI cellulose TLC plate was developed in 0.9 M guanidine hydrochloride. Spot 1 corresponds to PRPP based on migration of cold standard; spots 2 and 3 were not identified. (b) Autoradiogram of the TLC analysis of the organic extracts from the experiment shown in a. TLC was carried out on Silica gel TLC plate in CHCl3/CH3OH/concentrated NH4OH/1 M ammonium acetate/H2O (180:140:9:9:23,v/v). (c) Schematic representation of the sites of DPA synthesis and pyrimidine salvage pathways. Upp - uracil phosphoribosyltransferase.

or with 5-fluorouracil (5F-U), respectively. No changes in the TLC profiles were observed when whole cells were used (Figure 2a). In contrast, the addition of uracil to the reaction mixture containing cell lysate led to an increase in the intensity of the spot comigrating with UMP, while supplementing with 5F-U resulted in the synthesis of a new species whose Rf value corresponded to that of 5F-UMP (Figure 2a). These data confirmed that P[14C]RPP was only accessible to Upp in the cytoplasm when the cells were broken open. Quantification of the radioactivity and TLC analysis of the organic extracts clearly showed that P[14C]RPP was converted to DP[14C]A, and its precursors, when whole cells were used (Figure 2b). This conversion also took place in the cell lysates because they contain plasma membrane and cell envelopes, but it is much less efficient due to competition for the P[14C]RPP substrate with cytoplasmic enzymes. We can thus conclude that the cells remained intact during P[14C]RPP incorporation to DP[14C]A and, consequently, that all the reactions in the DPA pathway likely occurred on the periplasmic side (Figure 2c). Based on our data, exogenous PRPP can gain access to the active site of UbiA from outside the plasma membrane. Furthermore, the active site must also be accessible to endogenously produced PRPP, and this would either require a transport system for PRPP or, alternatively, the transmembrane UbiA protein could effect this function itself. UbiA also appears to be able to use both the periplasmically exposed source of decaprenyl phosphate that arises in the last stages of peptidoglycan and arabinogalactan biosynthesis in a possible recycling step as well as the nascent form synthesized inside the cell. To confirm the periplasmic localization of the DPA biosynthetic system, we took advantage of a novel, highly specific fluorescent probe for DprE1, a derivative of PBTZ169 labeled with TAMRA (Supporting Information Figure 5a). PBTZ-TAMRA arose from structure−activity relationship studies of a series of fluorescently labeled benzothiazinone

Metabolic activity of spheroplasts was also examined with UDP-[14C]GlcNAc, a substrate unrelated to the DPA pathway (Supporting Information Figure 4). When intact or lysed spheroplasts were incubated with UDP-[14C]GlcNAc and TDP-Rha, we observed biosynthesis of glycolipid 1 (GL1, decaprenyl−P-P-[14C]GlcNAc) and glycolipid 2 (GL2, decaprenyl-P-P-[14C]GlcNAc-Rha), which serves as a primer for galactan polymerization in mycobacteria.15 In lysed or intact spheroplasts, production of GL1/2 was comparable, indicating both the metabolic activity and also the permeability of the spheroplasts in this experiment. On the contrary, when untreated whole cells or lysates were used, higher levels of production of GL1/2 were observed in lysed cells as compared to intact cells. These data support the presumed cytoplasmic localization of the active sites of the enzymes producing GL1/ GL2, but they also reflect the considerable level of integrity of the untreated whole cells and underline the validity of performing enzymology with intact and lysed untreated cells for investigation of cytoplasmic vs periplasmic localization of the enzyme reactions in the DPA pathway. In the experiment with the whole untreated cells and P[14C]RPP, we addressed the possibility that this substrate could enter the cytoplasm by measuring the activity of uracil phosphoribosyltransferase (Upp) since this cytoplasmic enzyme, from the pyrimidine salvage pathway, synthesizes UMP from uracil and PRPP.24 Reactions were performed with untreated whole cells or the equivalent amount of crude cell lysate. TLC analysis of the products produced by cell lysates following incubation with P[14C]RPP revealed the presence of a radiolabeled spot comigrating with a UMP standard (Figure 2a), confirming that the reaction occurred in disrupted mycobacteria. This spot was not present when whole cells were used. In order to obtain further confirmation of Upp activity in the cell lysates, we supplemented the reaction mixtures with uracil C


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Figure 3. Extracytoplasmic localization of DprE1. (a) Representative images of M. smegmatis cells where the plasma membrane is labeled with GFP (green) fused to Rv3789, an integral membrane protein, and stained with PBTZ-TAMRA (red). (b) Close-up of two cells that have just divided. (c) M. smegmatis MN84 is a C394S point mutant of DprE1 that does not bind PBTZ-TAMRA. further use at −20 °C, or directly used in the reactions performed with whole cells and crude lysates. Preparation and Analysis of Spheroplasts. Spheroplasts were prepared by a modified procedure.21 M. smegmatis cells were grown in NB medium supplemented with 0.05% (v/v) Tween 80 and 1% (w/v) glucose at 37 °C and shaken at 120 rpm until the OD600 reached 0.8− 1. Glycine was added at this point to a concentration of 1.2% (w/v) and incubation continued for a further 24−48 h at 37 °C, with shaking at 120 rpm. The cells were then harvested in sterile conditions, washed with SMM buffer (0.5 M sucrose, 20 mM maleate buffer, pH 6.6, 20 mM MgCl2), and suspended in the original volume of NB-SMM medium supplemented with 1.2% (w/v) glycine and 50 μg/mL lysozyme. After 24 h of shaking at 120 rpm at 37 °C, the cells were harvested and separated by centrifugation at 27000g for 1.5 h at RT using 60% (v/v) Percoll (GE Healthcare) in SMM buffer. The upper fraction corresponding to spheroplasts was harvested and gently washed with SMM buffer by 20 min centrifugation at 4500g at RT. The arabinogalactan and peptidoglycan content of the untreated cells and spheroplasts was assessed by quantification of monosacharides and m-diaminopimelic acid by GC/MS as described.26 Preparation and Analysis of Enzyme Fractions. Disintegration of the untreated cells or spheroplasts by probe sonication (15 cycles of 30 s pulses and 90 s cooling) was carried out in 1 g aliquots suspended in 5 mL of buffer A (50 mM MOPS buffer, pH 7.9, 10 mM MgCl2, 5 mM 2-mercaptoethanol) mixed with SMM buffer to obtain final concentrations of 39 mM MOPS, 8.8 mM maleate, 220 mM sucrose, 18.8 mM MgCl2, and 5 mM 2-mercaptoethanol. Preparation of enzymatically active membrane and cell envelope fractions was as described previously.14 NADH oxidase activity was measured in reaction mixtures containing 3 μg of protein, 100 μM NADH, and buffer A in a final volume of 200 μL. The reaction was started by the addition of proteins, and the decrease in absorbance at 340 nm was followed for 15 min at 37 °C. The assay was carried out in 96-well plates. Analysis of mycolic acids was performed as described previously27 on material corresponding to 200 μg of proteins. Reaction Mixtures, Incubation Conditions, Fractionation, and Analysis of Reaction Products. P[14C]RPP was prepared as described previously13 from [14C]-glucose (specific activity 290 mCi/ mmol, American Radiolabeled Chemicals, Inc.). The reaction mixtures for assessing P[14C]RPP incorporation into DP[14C]A and its precursors contained 300 μg of membrane or cell envelope proteins, 250 μM NADH, 10 000 or 15 000 dpm of P[14C]RPP, and buffer A in a final volume of 80 μL. Alternatively, the reactions were performed with 20 mg of whole untreated cells or spheroplasts suspended in 100 μL of buffer A. The reactions were incubated for 1 h and stopped by adding 1.5 or 3 mL of CHCl3/CH3OH (2:1). After 10 min of rocking

analogues (Neres et al, in preparation) and not only binds covalently to purified DprE1 in vitro but also retains significant antimycobacterial activity. Its minimal inhibitory concentration for M. smegmatis is ∼114-fold higher than that of PBTZ169 despite addition of the bulky fluorophore. PBTZ-TAMRA was incubated with an M. smegmatis strain expressing the membrane protein Rv3789 fused to GFP. This fusion protein uniformly labels the plasma membrane, and its C-terminal GFP moiety is cytoplasmic25 (Figure 3a). Using high-resolution fluorescence microscopy, we confirmed that DprE1 is located at the old pole of the cells,5 but thanks to the higher resolution and the contrast with the GFP-labeled plasma membrane, it was now clear that DprE1 labeled with PBTZ-TAMRA was localized beyond the plasma membrane (Figure 3a,b). This labeling was not observed when PBTZ-TAMRA was incubated with the BTZ-resistant strain M. smegmatis MN84 (Figure 3c), where covalent binding cannot occur due to the Cys394Ser mutation in DprE1.4 Likewise, no polar labeling was observed when a simple propyl-TAMRA analogue was used (Supporting Information Figure 5b). Conclusions. The present investigation provides compelling evidence that DPA biosynthesis in mycobacteria occurs in part outside the cytoplasmic membrane, contrary to current thought.2,23 Our finding that DprE1 is located in the periplasm explains the remarkable vulnerability and the recently found “promiscuity” of this target to various classes of inhibitors, since these do not have to enter the cytoplasm to exert their effects on DprE1. Consequently, drugs targeting DprE1 will escape the action of the efflux pumps, trapping, and potential cytoplasmic inactivation mechanisms that might confer intrinsic resistance. However, some intriguing questions now arise. First, how are DprE1 and by extension DprE2, two proteins lacking known export signals, translocated across the cytoplasmic membrane and, second, how are their nucleotide cofactors replenished? Answering these questions could lead to the identification of additional TB drug targets.

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METHODS

Growth of Bacteria. Mycobacterium smegmatis mc2155 was grown in nutrient broth (NB) at 37 °C with constant shaking to mid log phase. The cells were harvested by centrifugation and stored until D


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at RT, H2O was added to achieve the ratio CHCl3/CH3OH/H2O (4:2:1). Centrifugation (3000g) of the mixtures resulted in two phases. The bottom organic phase was dried under a stream of N2 at RT, dissolved in 50 μL of CHCl3/CH3OH/H2O/NH4OH (65:25:3.6:0.5, solvent I), and quantified for radioactivity in a Tri Carb 2900TR liquid scintillation analyzer (PerkinElmer) using scintillation liquid EcoLite (MP Biomedicals). Reaction products were separated by thin layer chromatography (TLC) on Silica Gel TLC plates (Merck) in solvent II [CHCl 3 /CH 3 OH/NH 4 OH/1 M ammonium acetate/H 2 O (180:140:9:9:23,v/v)] and visualized by autoradiography (Biomax MR-1 film, Kodak) for 7 days (Figures 1 and 2). To measure Upp activity, the cells were disintegrated as above in buffer A. The reactions were performed with 20 mg of untreated whole cells suspended in 100 μL of buffer A or with 120 μL crude lysates after sonication. The reaction mixtures contained 250 μM NADH, 15 000 dpm of P[14C]RPP, and 200 μM uracil (Sigma) or 5fluorouracil (Sigma) and buffer A to a final volume of 125 μL. After incubation, reactions were stopped by adding 3 mL of CHCl3/ CH3OH (2:1); H2O was added to achieve the ratio CHCl3/CH3OH/ H2O (4:2:1) and the lower organic phase removed and analyzed as described above. A total of 100 μL of each upper aqueous phase was dried under a stream of N2 and redissolved in 5 μL of deionized H2O. The samples were loaded on PEI Cellulose TLC plate (Merck), along with 20 nmol each of UMP, UDP, UTP, and 5F-UMP standards and a radiolabeled standard containing [14C]-5F-uracil analogs of UMP, UDP, UTP, and UDP-containing sugar nucleotides. These were prepared as described.28 The plate was soaked in CH3OH for 20 min, dried, developed in 0.9 M guanidine hydrochloride,29 and subjected to autoradiography (Biomax MR-1 film, Kodak) for seven days. Nucleotide standards were detected under UV light (λ = 260 nm). Fluorescence Microscopy. Wild type M. smegmatis strain mc2155 and BTZ-resistant strain MN844 were grown at 37 °C in Middlebrook 7H9 broth (Difco) supplemented with 2% Albumin-Dextrose-Catalase (ADC), 0.2% glycerol, and 0.05% Tween 80. M. smegmatis mc2155 expressing Rv3789-GFP was grown as above in the presence of kanamycin (25 μg/mL). Cells were incubated for 4.5 h at 37 °C with 400 nM JN108, a TAMRA-linked PBTZ-derivative (MIC of 89 nM against M. smegmatis Neres et al. in preparation), or 400 nM JN111 (Propyl-TAMRA), which were added to the growth medium. After washing the cells once with PBS containing 0.05% Tween 80, 5 μL of live bacteria were spotted on a microscope slide, and a coverslip was placed over the sample. Slides were imaged on a Zeiss LSM 700 inverted microscope equipped with a Plan-Apochromat 63X/1.4 oil objective and Axiocam MRm (B/W) camera. Images were acquired upon excitation (488 nm for GFP and 555 nm for TAMRA) and emission filters were set at 490−530 nm for GFP and 560−700 nm for TAMRA using ZEN 2010B SP1 software. Images were processed with ZEN 2011 software (Zeiss).

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The authors declare the following competing financial interest(s): S.T.C. is a named inventor on patents pertaining to this paper.

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ACKNOWLEDGMENTS The authors thank Arne Seitz (EPFL bioimaging and optics platform), Jaroslav Blaško (Institute of Chemistry, FNS, CU, Bratislava for GC/MS analyses), Vinayak Singh (University of Cape Town for providing 5F-UMP), Peter Polčic (Department of Biochemistry, FNS, CU, Bratislava for graphics), Neeraj Dhar, Jérémie Piton, and Caroline Foo (EPFL for helpful discussions). J.N. was the recipient of an International Incoming Marie Curie fellowship (252802 − DPRETB) from the European Commission. The research leading to these results received funding from the European Community’s Seventh Framework Programme (Grant 260872) and the Slovak Research and Development Agency (Contract No. DO7RP-0015-11, K.M.).

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(1) Global tuberculosis report 2014, World Health Organization, Geneva. (2) Zumla, A., Nahid, P., and Cole, S. T. (2013) Advances in the development of new tuberculosis drugs and treatment regimens. Nat. Rev. Drug Discovery 12, 388−404. (3) Makarov, V., Lechartier, B., Zhang, M., Neres, J., van der Sar, A. M., Raadsen, S. A., Hartkoorn, R. C., Ryabova, O. B., Vocat, A., Decosterd, L. A., Widmer, N., Buclin, T., Bitter, W., Andries, K., Pojer, F., Dyson, P. J., and Cole, S. T. (2014) Towards a new combination therapy for tuberculosis with next generation benzothiazinones. EMBO Mol. Med. 6, 372−383. (4) Makarov, V., Manina, G., Mikusova, K., Mollmann, U., Ryabova, O., Saint-Joanis, B., Dhar, N., Pasca, M. R., Buroni, S., Lucarelli, A. P., Milano, A., De Rossi, E., Belanova, M., Bobovska, A., Dianiskova, P., Kordulakova, J., Sala, C., Fullam, E., Schneider, P., McKinney, J. D., Brodin, P., Christophe, T., Waddell, S., Butcher, P., Albrethsen, J., Rosenkrands, I., Brosch, R., Nandi, V., Bharath, S., Gaonkar, S., Shandil, R. K., Balasubramanian, V., Balganesh, T., Tyagi, S., Grosset, J., Riccardi, G., and Cole, S. T. (2009) Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis. Science 324, 801−804. (5) Neres, J., Pojer, F., Molteni, E., Chiarelli, L. R., Dhar, N., BoyRottger, S., Buroni, S., Fullam, E., Degiacomi, G., Lucarelli, A. P., Read, R. J., Zanoni, G., Edmondson, D. E., De Rossi, E., Pasca, M. R., McKinney, J. D., Dyson, P. J., Riccardi, G., Mattevi, A., Cole, S. T., and Binda, C. (2012) Structural basis for benzothiazinone-mediated killing of Mycobacterium tuberculosis. Sci. Transl. Med. 4, 150ra121. (6) Trefzer, C., Rengifo-Gonzalez, M., Hinner, M. J., Schneider, P., Makarov, V., Cole, S. T., and Johnsson, K. (2010) Benzothiazinones: prodrugs that covalently modify the decaprenylphosphoryl-beta-Dribose 2′-epimerase DprE1 of Mycobacterium tuberculosis. J. Am. Chem. Soc. 132, 13663−13665. (7) Huang, H., Scherman, M. S., D’Haeze, W., Vereecke, D., Holsters, M., Crick, D. C., and McNeil, M. R. (2005) Identification and active expression of the Mycobacterium tuberculosis gene encoding 5-phosphoα-d-ribose-1-diphosphate: decaprenyl-phosphate 5-phosphoribosyltransferase, the first enzyme committed to decaprenylphosphoryl-darabinose synthesis. J. Biol. Chem. 280, 24539−24543. (8) Jiang, T., He, L., Zhan, Y., Zang, S., Ma, Y., Zhao, X., Zhang, C., and Xin, Y. (2011) The effect of MSMEG_6402 gene disruption on the cell wall structure of Mycobacterium smegmatis. Microb. Pathog. 51, 156−160. (9) Kolly, G. S., Boldrin, F., Sala, C., Dhar, N., Hartkoorn, R. C., Ventura, M., Serafini, A., McKinney, J. D., Manganelli, R., and Cole, S. T. (2014) Assessing the essentiality of the decaprenyl-phospho-d-

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S Supporting Information *

Supplementary methods, tables, figures, and references. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.5b00237.

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Corresponding Authors

*E-mail: stewart.cole@epfl.ch. *E-mail: [email protected]. Present Address ∥

MRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Institute of Infectious Disease and Molecular Medicine and Division of Medical Microbiology, Faculty of Health Sciences, University of Cape Town, South Africa Author Contributions §

These authors contributed equally to this work. E


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DprE1 Is a Vulnerable Tuberculosis Drug Target Due to Its Cell Wall

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