Mechanism of Fluorinated Anthranilate-Induced Growth Inhibition in

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The mechanism of fluorinated anthranilate-induced growth inhibition in Mycobacterium tuberculosis M. Nurul Islam, Reese Hitchings, Santosh Kumar, Fabio Levi Fontes, J Shaun Lott, Nicole A Kruh-Garcia, and Dean C. Crick ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00092 • Publication Date (Web): 08 Nov 2018 Downloaded from http://pubs.acs.org on November 12, 2018

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ACS Infectious Diseases

The mechanism of fluorinated anthranilate-induced growth inhibition in Mycobacterium tuberculosis

M. Nurul Islam, Reese Hitchings, Santosh Kumar, Fabio L. Fontes, J. Shaun Lott1*, Nicole A. Kruh-Garcia and Dean C. Crick*

Mycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, United States

1Current

address: School of Biological Sciences & Maurice Wilkins Centre for Molecular

Biodiscovery, The University of Auckland, Private Bag 92019, Auckland, New Zealand.

* Corresponding authors: [email protected], [email protected]

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The biosynthesis of tryptophan in Mycobacterium tuberculosis is initiated by the transformation of chorismate to anthranilate, catalysed by anthranilate synthase (TrpE/TrpG). Five additional enzymes are required to complete tryptophan biosynthesis.

M. tuberculosis strains

auxotrophic for tryptophan, an essential amino acid in the human diet, are avirulent. Thus, tryptophan synthesis in M. tuberculosis has been suggested as a potential drug target and it has been reported that fluorinated anthranilate is lethal to the bacillus. Two mechanisms that could explain the cellular toxicity were tested: 1) the inhibition of tryptophan biosynthesis by a fluorinated intermediate or 2) formation of fluorotryptophan and its subsequent effects. Here M. tuberculosis mc2 6230 cultures were treated with anthranilates fluorinated at positions 4, 5 and 6. These compounds inhibited bacterial growth on tryptophan-free media, with 4-fluoroanthranilate being more potent than 5-fluoroanthranilate or 6-fluoroanthranilate. LC-MS based analysis of extracts from bacteria treated with these compounds did not reveal accumulation of any of the expected fluorinated intermediates in tryptophan synthesis. However, in all cases significant levels of fluorotryptophan were readily observed, suggesting that the enzymes involved in the conversion of fluoro-anthranilate to fluorotryptophan were not being inhibited. Inclusion of tryptophan in cultures treated with the fluoroanthranilates obviated the cellular toxicity. Bacterial growth was also inhibited in a dose-dependent manner by exposure to tryptophan substituted with fluorine at positions 5 or 6. Thus, the data suggests that fluorotryptophan rather than fluoro-anthranilate or intermediates in the synthesis of fluorotryptophan causes the inhibition of M. tuberculosis growth.

Key Words:

fluorotryptophan, tryptophan, mycobacteria, auxotroph, tuberculosis, feedback

inhibition


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Tuberculosis (TB) remains one of the most significant worldwide causes of death from a communicable disease; the World Health Organization (WHO) estimates that around 1.7 million people died from TB in 2016, with an estimated 10.4 million new cases.1 The disease is curable with currently available therapy and mortality has been slowly declining in recent years - the number of TB deaths reduced by 22% between 2000 and 2015. However, there is a significant concern that the emergence of multidrug-resistant (MDR) and extensively-drug-resistant (XDR) strains of Mycobacterium tuberculosis, the causative agent of the disease, may roll back this progress. It is estimated that almost half a million people developed MDR-TB in 2016, and over 40,000 people developed XDR-TB. The success rate for treatment of MDR-TB is generally little better 50%, and for XDR-TB it is less than 30%, leading to a pressing need for new anti-TB therapies with novel modes of action against the bacterium. Interfering with M. tuberculosis tryptophan biosynthesis represents a novel point of intervention in the pathogenesis of the bacterium. Tryptophan auxotroph mutants of M. tuberculosis are strikingly avirulent: they fail to cause disease, and are cleared from the body, in both immunocompetent (DBA/2) and immunodeficient (SCID) mice,2 with a particular sensitivity to macrophage activation by CD4+ T-cells.3 The first committed steps of tryptophan synthesis in M. tuberculosis are the production of anthranilate from chorismate and the subsequent reaction of anthranilate with 5′‑phosphoribosyl1′-pyrophosphate (PRPP) to produce phosphoribosylanthranilate (PRA). These biosynthetic steps are catalysed by anthranilate synthase (AS; TrpE/TrpG, a heterodimeric enzyme encoded by the open reading frames Rv1609 and Rv0013) and anthranilate phophoribosyltransferase (AnPRT; TrpD, encoded by Rv2192c) respectively (Fig. 1).4 It was recently reported that the fluorinated anthranilate derivatives 5‑fluoro-anthranilate (5‑FA) and 6‑fluoro-anthranilate (6‑FA) are cytotoxic to M. tuberculosis in vitro, with a minimum inhibitory concentration (MIC) of 5 μM in Middlebrook 7H9 broth.3 Additionally, 6‑FA was reported to be an antitubercular compound in vivo. A significant reduction in bacterial load was shown, especially in the spleen, in C57BL/6 mice infected by aerosol with M. tuberculosis and subsequently treated with 200 mg/kg 6‑FA or its ester.



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However, the mechanism by which fluoro-anthranilates might cause cellular toxicity is unclear. The toxic effects of 6‑FA in vitro could be reversed by the addition of 1 mM tryptophan to the medium,3 indicating that tryptophan biosynthesis was the target pathway. Resistance mutants were isolated which contained a variation in the allosteric binding pocket of TrpE (F68I) and the mutant enzyme is 3-fold more active and 50-fold less sensitive to allosteric feedback inhibition by tryptophan than the wild-type enzyme, indicating that an increased flux through the tryptophan biosynthetic pathway is sufficient to overcome the toxicity of 6‑FA. We have previously shown that all of the possible positional isomers of fluoro-anthranilate (3‑FA, 4‑FA, 5‑FA and 6‑FA) are effective substrates in vitro for M. tuberculosis TrpD, producing the cognate fluorinated phosphoribosylanthranilates (PRAs), and that the fluorinated PRAs are themselves substrates for E. coli indole-3-glycerol phosphate synthase (InGPS; TrpFC), resulting in the production of fluoroindole-3-glycerol phosphate, but it is unknown whether this is also the case in vivo in M. tuberculosis.5 It is also unknown whether fluoro-indole-3-glycerol phosphate is an effective substrate for indole-3-glycerol phosphate aldolase (InGPA; TrpA), and whether or not the biosynthesis proceeds to produce fluorinated indole and finally fluoro-tryptophan. Therefore, two possible mechanisms for the toxicity of fluoro-anthranilate can be suggested: the inhibition of tryptophan biosynthesis downstream of TrpD by a fluorinated intermediate, leading to tryptophan starvation; or the production of fluorinated tryptophan, which subsequently demonstrates cytotoxic effects due to its incorporation into proteins, as has previously been observed in E. coli6 and cultured mouse fibroblasts.7 In order to determine the mechanism of action of fluoro-anthranilates in Mtb, and to better understand the utility of the tryptophan biosynthesis pathway as a target for new anti-TB therapeutics, we set out to analyse the metabolic profile of M. tuberculosis cells treated with fluoroanthranilates in vitro and extracted targeted data from the LC-MS experiments. The results of these experiments led to more detailed growth inhibition and competition studies.

Results Metabolic profiling 4 ACS Paragon Plus Environment

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In order to understand the metabolic fate of fluoro-anthranilate in mycobacterial tryptophan biosynthesis, we cultured M. tuberculosis mc2 6230 cells in Sauton’s media containing various fluoroanthranilate isomers. In initial experiments, the concentrations that caused a 50% reduction in bacterial growth (GIC50) in a resazurin microtitre assay (REMA) were determined to be 12 ± 8.0, 17 ± 11 and 250 ± 200 µM for 4-FA, 5-FA and 6-FA, respectively. Thus, in our hands 4-FA and 5-FA are more potent inhibitors of bacterial growth than 6-FA. The discrepancy the between the GIC50 for 6-FA presented here and the MIC reported by Zhang and Rubin3 is unclear; however it should be noted that different media were used and the GIC50 value is a rate change based on fluorescence changes, whereas the MIC reported previously is based on a visual endpoint. Subsequently, bacilli were grown to an OD600 of 0.4, 8× the GIC50 (final concentration) of 4-FA, 5-FA or 6-FA was added to the culture medium and the cells were incubated for a further six days. Metabolites were extracted from harvested cells using 70% methanol and analysed by LC-TOF-MS with the aim of detecting fluorinated metabolites downstream of anthranilate in the tryptophan biosynthesis pathway. Data were extracted for ions having m/z values of 368.0541 +/- 20 ppm, (fluoro-PRA; C12H15FNO9P), 368.0541

+/-

20

ppm,

(fluoro-1-(2-carboxy)phenylamino-1′-deoxyribulose-5′-phosphate;

C12H15FNO9P), 306.0537 +/- 20 ppm (fluoro-indole-3-glycerol phosphate; C11H13FNO6P), 136.0557 +/- 20 ppm (fluoro-indole; C8H6FN) or 223.0877 +/- 20 ppm (fluorotryptophan; C11H11FN2O2) representing the fluorinated products of TrpD, F, C, A, and B respectively. The structures of the nonfluorinated compounds are shown in Fig. 1. In no case was an ion that could be attributed to a fluorinated intermediate in tryptophan synthesis seen; the limits of detection for 5-fluoro- and 6fluoro-indole, based on a signal to noise ration > 3, were 66 pmol and 105 pmol, respectively. These results suggest that either fluoro-anthranilate was not metabolized, in contrast to results of our earlier in vitro work with TrpD5 or that the fluorinated compounds rapidly moved through the pathway to form fluorotryptophan. When the data were extracted for ions having m/z values of 223.0877 +/- 20 ppm, corresponding to protonated fluorotryptophan, a clear peak was seen in all cases. This peak was identified as fluorotryptophan based on accurate mass (m/z value) and retention time when compared with commercially available standard compounds. Representative chromatograms are presented in Fig. 2. Other fluorotryptophan isomers exhibited slightly different retention times with similar m/z values (± 20 ppm). The variations in retention time are due to the variations in the 5 ACS Paragon Plus Environment


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expected position of the fluorine in the aromatic ring of tryptophan derived from the fluoroanthranilate isomers included in the cultures (Table 1). In all cases non-fluorinated tryptophan was also observed; the ratio of fluorotryptophan to tryptophan ranged from a low of 0.86 when the cells were treated with 4-FA to a high of 6.2 when treated with 5-FA. Taken together, these data suggested that fluoro-anthranilate was rapidly converted to fluorotryptophan, which then accumulated in the bacilli.

Mechanism of action The hypothesis that fluorotryptophan was responsible for the cytotoxic effects was first tested by determining GIC50 values for 5- and 6-fluorotryptophan using a rapid, kinetic method to assess the in vitro effect of compounds against M. tuberculosis growth that correlates well with MIC.8 In those initial experiments it was observed that the GIC50 values were variable and appeared to correlate with incubation time. Therefore, kinetic analyses were undertaken with data collected every 24 hours for five days. A rapid decrease in GIC50 was seen for 4-FA between 24 and 48 hours of exposure (Table 2), suggesting that the 4-FA is not cytotoxic per se, but that there is a timedependent build-up of toxic material. This, combined with the observation that no fluorinated intermediates between anthranilate and tryptophan could be identified, implies that cytotoxicity is dependent on the formation of fluorotryptophan. In support of this, the inhibitory effects of the FA were completely abrogated by the addition of 2 mM tryptophan to the growth medium. Similar patterns of growth inhibition were observed when the bacteria were exposed to 6-, 5or 4-fluoro-indole (Table 2). 6- and 5-fluoro-indole displayed GIC50 values that were initially low but increased with time.

Interestingly, 4-fluoro-indole (corresponding to 6-FA biosynthetically)

demonstrated the highest GIC50 of the 3 indoles tested, a value that also increase more rapidly than that of the other indoles. Next, the GIC50 values for fluorotryptophans were determined kinetically (Table 2). The GIC50 for 4-fluoro-DL-tryptophan was > 100 µM (the highest concentration of the racemic compound tested). For 5- and 6-L-fluorotryptophan the GIC50 values were lowest at 24 hours of exposure, and were similar to the values for the corresponding fluoro-anthranilate (i.e. 4-FA for 6-L-fluorotryptophan and 5-FA for 5-L-fluorotryptophan, Table 1). These values steadily increased with time until they 6 ACS Paragon Plus Environment

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exceeded the highest concentrations tested, suggesting that the fluorotryptophan is utilized over the duration of the experiment, presumably incorporated into protein as mycobacteria have been reported not to catabolize tryptophan.9 The results are also consistent with feedback inhibition such that, as time passes, the fluorotryptophan levels in the medium are depleted, allowing increased tryptophan synthesis, thus reducing the apparent GIC50 at the longer time points. Interestingly, a similar trend was seen for the GIC50 values for 4-FA and 5-FA. In the case of 4-FA, the measured GIC50 was high at 24 hours, decreased to a minimum at 48 hours, and then steadily increased to exceed the highest concentration tested by 120 hours of exposure. This result is consistent with the hypothesis that 4-FA is not toxic per se, but that it is converted to a toxic product, fluorotryptophan, which requires more than 24 hours to reach a concentration that inhibits bacterial growth and is subsequently depleted. The increase in GIC50 over time, seen in all experiments (Table 2) could, potentially, also be explained by a rapid and consistent emergence of resistance. In order to differentiate between the depletion of fluorotryptophan and the emergence of resistance, bacilli were incubated in medium containing either no compound or concentrations of 1× or 5× GIC50 of 5- or 6-L-fluorotryptophan observed at 24 hours of exposure. The used medium was replaced with fresh medium containing the indicated concentration of fluorotryptophan every 24 hours. The growth rate of bacteria cultured in medium with no added fluorotryptophan increased steadily for 96 hours before entering stationary phase (Fig. 3). Bacteria treated with 1× GIC50 grew at a rate approximating 50% of the untreated cells over the entire time course and the cells treated with 5× GIC50 failed to grow, as expected. Thus, there was no evidence for development of resistance, and the daily decrease in GIC50 seen in Table 2 can be attributed to a depletion of fluorotryptophan, relieving feedback inhibition through utilization, as regular replacement of medium containing fluorotryptophan abolished the changes in GIC50. Further, the incorporation of 6-L-fluorotryptophan was demonstrated in Mycobacterium smegmatis expressing recombinant protein and M. tuberculosis mc2 6230. M. smegmatis was tranformed with an expression plasmid carrying the pruA gene. PruA expression was induced by addition of acetamide to the medium and 6-fluorotryptophan was immediately added.

The

recombinant protein was purified, digested with trypsin and subjected to selected reaction monitoring 7 ACS Paragon Plus Environment


mass spectrometry.

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As demonstrated by the data presented in Figure S1 (Supplemental

Information) the 6-fluorotryptophan is clearly incorporated into the PruA. To demonstrate that fluorotryptophan was incorporated into protein in M. tuberculosis mc2 6230 we took advantage of the fact that, unlike tryptophan, 4-fluorotryptophan does not fluoresce10 (Fig. 4). Exposure of bacilli to 4-DL-fluorotryptophan or 6-FA for 24 hours, resulting in the formation of 4-L-fluorotryptophan, reduced the fluorescence of purified recombinant protein and cytosolic protein that could be attributed to the presence of tryptophan in M. smegmatis and M. tuberculosis mc2 6230, respectively. After 48 hours of treatment the fluorescence of the protein from treated bacilli returned to that of the untreated bacilli.

These results are consistent with the common practice of incorporation of

fluorinated aromatic amino acids into recombinant proteins in E. coli for structural analyses.11,12

Discussion In summary, the results presented indicate that the growth inhibition caused by exposure to fluoroanthranilate are due to production of fluorinated tryptophan, as has previously been observed in E. coli6 and cultured mouse fibroblasts,7 rather than due to tryptophan starvation induced by inhibition of tryptophan biosynthesis downstream of TrpD by a fluorinated intermediate or due to accumulation of a toxic fluorinated intermediate. The results presented here are also consistent with feedback regulation of tryptophan synthesis. These results provide insight into the mechanism of inhibition of bacterial growth by the fluoroanthanilates. As indicated earlier, genetic experiments indicate M. tuberculosis mutants auxotrophic for tryptophan fail to cause disease and treatment of infected mice with an ester of 6-FA or 6-FA reduced bacterial growth in the spleen and lungs. This observation has been attributed to an innate immune response where host cells limit amino acid availability, starving the pathogen. In the case of tryptophan depletion this effect is often attributed to the activity of indolamine 2,3dioxygenase (IDO) activity.13 Consequently, it has been suggested that tryptophan synthesis is an attractive drug target in M. tuberculosis,13–15 albeit in conditions of limited exogenous tryptophan availability. However, tryptophan is an essential amino acid in the human diet and circulating serum levels of tryptophan determine tissue levels of this compound.16 Studies of pulmonary tuberculosis patients indicate that these individuals have reduced serum tryptophan levels, but only by about 30% 8 ACS Paragon Plus Environment

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(50 µM vs 70 µM), and that low serum tryptophan levels/high IDO activity is a significant independent predictor of patient death.17 Mycobacteria constitutively express amino acid biosynthesis genes,18–22 and the tryptophan biosynthetic locus is expressed even in the presence of exogenous tryptophan;3,20 thus, in mycobacteria tryptophan synthesis is not controlled transcriptionally in response to amino acid availability, as is seen in many bacteria. These observations led to the suggestion that M. tuberculosis synthesizes amino acids regardless of environmental availability. However, this does not take into consideration post-translational regulation; it has long been known that tryptophan inhibits tryptophan biosynthesis in mycobacteria via feedback inhibition of chorismate synthesis, a precursor of tryptophan synthesis23 (Fig. 1) and more recently it was reported that ~200 nM tryptophan, inhibited 50% of TrpE activity.3 This latter tryptophan concentration is at least an order of magnitude lower than those reported in lungs of mice infected with Toxoplasma gondii,24 xenographs of human tumors in interferon-γ-treated mice25 or interferon-γ-stimulated human or nonhuman primate macrophages,26 consistent with the 20 µM KM of the high affinity member of the two IDO isoforms typically found in mammals.27 Thus, based on these observations, and the results presented here, it appears that there should be sufficient tryptophan available to inhibit mycobacterial synthesis of this amino-acid during pathogenesis; however, ample data indicate that M. tuberculosis strains auxotrophic for tryptophan fail to establish infections3,14 and inhibitors of tryptophan synthase (TrpAB) show moderate (1.4 – 1.5 log reduction of colony forming units) in vivo efficacy in both zebrafish14 and mouse15 disease models. Thus, the precise mechanism of bacterial growth inhibition by defects in tryptophan biosynthesis in vivo remains unclear. Given the diversity and complexity of tubercule lesions, as well as the diverse immunopathology of granulomas and cavities, which generate a plethora of microenvironments in which the bacilli reside,28 further validation of tryptophan biosynthesis as a drug target in mycobacteria will likely require extensive genetic and infection treatment experiments. In particular, there is a clear need to evaluate a conditional knock-down of tryptophan biosynthesis after disease has been established. In addition, understanding the results of these experiments calls for focused studies to determine levels of bacterial exposure to host tryptophan, determination of relative rates of uptake of exogenous tryptophan by host and pathogen cells, and the relative affinities of competing transporters and 9 ACS Paragon Plus Environment


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metabolic enzymes of these cells in the various physiological compartments associated with disease progression.


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Methods

M. tuberculosis cell growth methods. The avirulent M. tuberculosis strain mc26230,29,30 a generous gift from Dr. William Jacobs, was stored frozen in glycerol stocks at -80ºC and grown in fully defined Sauton’s medium containing 24 µg/mL D-pantothenate (supplemented Sauton’s medium) at 37° C for two passages prior to use. Fluorinated anthranilate isomers were purchased from Fluorochem. LC-MS grade water, methanol and acetonitrile were from Burdick and Jackson (Muskegon, MI, USA). Mass spectrometry grade formic acid, 5-fluoro-L-tryptophan, 6-fluoro-L-tryptophan, D-pantothenate and resazurin were purchased from Sigma-Aldrich (St. Louis, MO, USA). 4-Fluoro-DL-tryptophan was from ChemCruz. All other reagents were of at least reagent grade and purchased from standard sources.

Resazurin microtiter plate assay (REMA) growth inhibition assay M. tuberculosis mc26230 was grown to an optical density at 600 nm (OD600) of 0.6-0.8, cells were diluted to an OD600 of 0.05 in supplemented Sauton’s media and aliquots were transferred to sterile 96-well plates. Stock solutions of fluoro-anthranilates were prepared in dimethyl sulfoxide and added to the bacterial cultures at 4 - 2000 µM in a final culture volume of 100 µl. The inoculated plates were placed in Ziplock bags containing a moist paper towel, sealed to avoid evaporation of media and incubated at 37ºC for 5 days. Subsequently, 10 µl of sterile 0.01% resazurin were added to all wells and the plates were incubated for another 24 hours. Fluorescence, excitation at 530 nm and emission at 590 nm, was determined using a BioTek Synergy HT plate reader. Data, averages of triplicates, were processed using GraphIT version 4.0 to determine GIC50.

OD based growth inhibition assay M. tuberculosis mc26230 cells were cultured to an OD600 of 0.6-0.8 as described above. The cells were harvested, washed twice in fresh medium, and diluted to an OD600 of 0.2 with fresh supplemented Sauton’s medium. Microtiter plates were loaded with fluorinated anthranilate or tryptophan derivatives and serially diluted to cover a range of concentrations for appropriate GIC50 11 ACS Paragon Plus Environment


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determination. Bacteria were then added to the plate to a final volume of 100 µl, the plates were sealed in Ziplock bags as described above and incubated at 37°C with constant agitation for 5 days. OD600 was measured at time 0 and at 24h intervals for 120 hours. Growth rate was calculated as the slope of the growth curves (OD600 vs time). The GIC50 was calculated at each time point from averages of four replicates, using SigmaPlot (v.11). Subsequently, M. tuberculosis mc2 6230 cells were grown, harvested and washed twice as described above. Stocks of supplemented Sauton’s medium containing 0, 5.0 and 25 µM of 5fluorotryptophan and 10 and 50 µM GIC50 of 6-fluorotryptophan were prepared. Cells were diluted into the appropriate medium, loaded into 16 mm glass culture tubes, and the OD600 was measured. At 24-hour intervals, the OD600 was recorded, followed by centrifugation at 3000 rpm. The used medium from each tube was discarded and fresh medium from the stocks described above was added to appropriate tubes over a period of 120 hours. Growth rates were calculated as the slope of the growth curves and averaged from four independent replicates.

Metabolite extraction and analysis Cultures, 50 mL, of M. tuberculosis mc2 6230 strain cells were grown in supplemented Sauton’s medium in Erlenmeyer flasks. The flasks were incubated on a rotary incubator at 37ºC and allowed to grow to an OD600 of 0.4. Cells were then treated with 4-, 5- and 6-fluoro-anthranilates at concentrations of 1× GIC50 and 8× GIC50 for 6 days. Bacterial cells were centrifuged and washed twice with deionized water to remove residual media and salts. These cells were frozen until used for metabolite extraction. Cells were thawed and suspended in LC-MS grade water, and lysed on ice using a sonicator (Ultrasonic homogenizer 4710 series Cole-Palmer instrument Co) with a fine probe cycling 60 seconds on and 30 seconds off. Finally, LC-MS grade methanol was added to the lysed cells and adjusted to a final concentration of 70% to serve as an extraction solvent. The samples were vortexed for 5 min to facilitate the extraction of metabolites and centrifuged at 14,000 rpm for 10 min at room temperature; supernatants were transferred to LC-MS vials and subjected to metabolite analysis by LC-MS. The extracted metabolites were analyzed using a 1290 series high performance liquid chromatograph coupled with a 6230 Time-of-Flight mass spectrometer (LC-TOF-MS) equipped with 12 ACS Paragon Plus Environment

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electrospray ionization source (Agilent Technologies, Palo Alto, CA). Metabolites were chromatographically separated employing a C18 stationary phase column (150 x 2.1 mm, 3.5 µm, XBridge, Waters). Autosampler and column compartment temperatures were set to 4ºC and 35ºC, respectively, for the entire analysis period. Aliquots of the extract were injected and metabolites were eluted from the column with a gradient program of solvent A (LC-MS grade water containing 0.1% formic acid) and solvent B (LC-MS grade acetonitrile containing 0.1% formic acid). The gradient program was initiated at flow rate 0.3 mL/min, with solvent composition A-B (98:2) for 3 min, linearly changing to 50:50 at 15 min, and finally to 0:100 at 28 min, with the program terminating at 32 min. The solvent was returned to the initial condition at 33 min followed by an 8 min re-equilibration prior to the next sample analysis. Eluted metabolites were directly introduced into a TOF mass spectrometer equipped with an electrospray ionization system. The mass spectrometer was operated in positive mode for data acquisition, at a scan rate of 1 spectra/sec, in a scan range of 100 - 1500 m/z. Purified nitrogen was used as nebulizing agent with a flow rate 9 mL/min at 325ºC, and nebulizer pressure was set at 45 psi. In this experiment, the fragmenter voltage was set to 120, whereas the capillary voltage was 3500. MassHunter software was employed for LC-TOF-MS control and data analysis.

Isolation of cytosolic protein Cultures (100 mL Sauton’s medium) of M. tuberculosis mc2 6230 cells were grown to an OD600 of 0.3 on an incubator shaker at 37ºC. Cells were treated with 4-fluoro-DL-tryptophan (100 μM) or 6-FA (150 μM) for 24 hours, harvested by centrifugation and washed thrice with 20 mM potassium phosphate buffer to remove residual media and salts. Isolation of cytosolic protein was performed essentially as described previously31 with minor modification. Briefly, bacteria were harvested by centrifugation, pellets were re-suspended in 20 mM potassium phosphate buffer (pH 7.2) and bacteria were broken by sonication. Cell debris and unbroken cells were removed by centrifugation at 2000 × g for 5 min at 4 °C. The resultant supernatant was then centrifuged at 27000 × g for 15 min at 4 °C the pellet was discarded and the supernatant was subjected to ultracentrifugation at 100,000 × g for 60 min at 4 °C to separate the membrane enriched fraction (pellet) from the soluble cytosolic fraction. The buffer was exchanged with fresh 20 mM potassium 13 ACS Paragon Plus Environment


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phosphate buffer (pH 7.2) by ultrafiltration six times and the concentration of the cytosolic protein was determined using a BCA Protein Assay Kit. Equal concentrations (150 µg/mL) of all fractions were used to record the fluorescence spectra.

Cloning, expression and purification of PruA Full length M. tuberculosis pruA (Rv1187) was amplified from genomic DNA using the forward primer 5’ ATT CAT ATG GAC GCG ATC ACC CAG GTG CCG ’3 and reverse primer 5’ TAT AAG CTT TCA GTC GAC CGC CAT GTG CGG ’3 and cloned into the E. coli pET28a+ expression vector between NdeI and HindIII restriction sites (underlined), subsequently the gene was subcloned the gene into pMyNT,32,33 a mycobacterial expression vector. The fidelity of clone was verified by restriction digestion and sequencing. The resulting plasmid was used to transform M. smegmatis. The transformant was used to inoculate 10 ml Middlebrook 7H9 broth supplemented with 0.2% (v/v) glycerol, 10% ADC, 0.05% Tween-80, and 50 µg/mL hygromycin (Calbiochem) and incubated at 37 °C

for 36 hrs. The culture was then used to inoculate two flasks containing 100 mL of Middlebrook

7H9, which were incubated with shaking at 37 °C. At mid-log phase, protein expression was induced with 0.2% acetamide (w/v).

Ten μg/mL of 4-fluoro-DL-tryptophan or 6-fluorotryptophan was

immediately added to one of the cultures and the cultures were then incubated for a further 24 hours at 37 °C. After 24 hours of growth, cells were harvested via centrifugation, and suspended in 20 mM potassium phosphate (pH 7.2) containing 1 mM phenyl methyl sulfonyl fluoride. Bacteria were sonicated, debris was removed by centrifugation and soluble, His-tagged, recombinant PruA was isolated from the soluble fraction on a Ni-NTA affinity column. Imidazole was removed from the eluted protein by buffer exchange on a PD-10 column and the purified protein was concentrated by ultra-filtration. The final concentration of protein was determined using a BCA Protein Assay Kit.

Fluorescence spectroscopy Fluorescence of protein samples was determined using a FluoroLog 3 (Horiba Scientific) spectrofluorometer. The fluorescence spectra between 300 and 420 nm with excitation at 280 nm of PruA or PruA isolated from bacilli treated with 4-fluoro-DL-tryptophan were recorded. Similarly, the fluorescence spectra of cytosolic proteins from M. tuberculosis mc2 6230 and M. tuberculosis 14 ACS Paragon Plus Environment

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mc2 6230 treated with either 4-fluoro-DL-tryptophan or 6-FA were recorded. All comparisons were done at equal amino acid or protein concentrations.

Acknowledgements Funded by NIH/NIAID grants AI049151 and AI119567.

Supporting Information Methods – trypsin digestion of PruA, Selected reaction monitoring mass spectrometry (SRM-MS); PruA amino acid sequence; theoretical trypsin digest of PruA; SRM-MS of PruA peptides; references.

References (1)

World Health Organization. Global Tuberculosis Report 2017; 2017; pp 1–262.

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Page 20 of 27

Table 1. Predicted structures of fluorotryptophan isomers biosynthetically derived from fluoro-anthranilate isomers

Fluoro-anthranilate

Structure

Fluorotryptophan

4-Fluoro-anthranilate (4-FA)

6-Fluorotryptophan


5-Fluorotryptophan


4-Fluorotryptophan


Structure

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Table 2. Compound concentration required to inhibit 50% of M. tuberculosis mc2 6230 growth (GIC50). All values presented are averaged from four independent replicates.

GIC50 (µM)

Anthranilate 4-Fluoro-anthranilate 4-Fluoro-anthranilate + 2 mM tryptophan 5-Fluoro-anthranilate 6-Fluoro-anthranilate Indole 6-Fluoro-indole 5-Fluoro-indole 4-Fluoro-indole 6-Fluorotryptophan 5-Fluorotryptophan

24h

48h

72h

96h

120h

>500 >500

>500 8.1 ± 0.12

>500 120 ± 12

>500 180 ± 14

>500 >500

>500

>500

>500

>500

>500

130 ± 8.7 130 ± 12 >500 27 ± 10 64 ± 57 96 ± 42 10 ± 1.8 4.4 ± 0.19

>500 >500 >500 11 ± 1.5 5.1 ± 0.86 >500 22 ± 2.4 13 ± 0.98

>500 >500 >500 20 ± 2.2 9.5 ±1.3 >500 >500 >250

>500 >500 >500 >500 >500 >500 >500 >250

>500 >500 >500 >500 >500 >500 >500 >250



Page 22 of 27

Figure Legends

Figure 1. Schematic of the tryptophan biosynthetic pathway. Enzyme names and corresponding Rv numbers derived from the M. tuberculosis genome34 are indicated.

Figure 2. Representative extracted ion chromatograms of 6-fluorotryptophan standard (Panel A) and 6-fluorotryptophan extracted from bacterial cells treated with 4-fluoroanthranilate (Panel B). Data from total ion chromatograms were extracted for protonated ions with m/z values of 223.0877 +/20 ppm (C11H11FN2O2). Spectra of the observed ions are inset into each panel.

Figure 3. Effect of daily treatment of 5- and 6-fluorotryptophan on bacterial growth rate. Used medium was removed every 24 hours and replaced with fresh medium containing the indicated concentrations 5-fluorotryptophan (Panel A) or 6-fluorotryptophan (Panel B). Concentrations of each fluorotryptophan corresponded to approximately 1× or 5× of the GIC50 observed at 24 hours of exposure in Table 2. Values presented are averaged from three independent replicates.

Figure 4. Effect of incorporation of 4-fluorotryptophan on protein fluorescence. Panel A - Relative fluorescence of tryptophan (W) and 4-fluoro-DL-tryptophan (4f-DL-W).

Panel B - Relative

fluorescence of recombinant PruA and recombinant PruA isolated from M. smegmatis treated with 10 µM 4-fluoro-DL-tryptophan (4f-DL-W PruA). Panel C - Relative fluorescence of cytosolic protein isolated from M. smegmatis (Cytosol) and M. smegmatis treated with 100 µM of 4-fluoro-DLtryptophan for 24 hours (4f-DL-W Cytosol). Panel D - Relative fluorescence of cytosolic protein isolated from M. tuberculosis mc2 6230 (Cytosol) and M. tuberculosis mc2 6230 treated with 6-FA for 24 hours (6-FA Cytosol). In each panel equal concentrations of amino acid or protein were utilized. Results are representative of multiple independent experiments.


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Figure 1.



Figure 2.

A

4x106

2x10

6

223.0874

Detector Response (counts)

3x106

4x106 3x106 2x106 206.0613

106 0

1x106

180 190 200 210 220 230 240

m/z 0

8x104

6x104

4x104

0



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 27

2

4

6

8

10

8

10

223.0879 X Data

8x10

4

6x104 4x104 206.0613

2x104 0

2x104

180 190 200 210 220 230 240

m/z 0 0

2

4

6

Time (min)


B

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Figure 3.



-2

A

5x103 4x103

W

6x103 4f-DL-W PruA

3x103

4x103

2x103 4f-DL-W

2x103

Buffer

3

1x10

0

0 C

25x103

D Cytosol

20x103

Cytosol

15x103

25x103 20x103 15x103

6-FA Cytosol

10x103 5x103

10x103 Buffer

4f-DL-W Cytosol Buffer

5x103

0

0 300 320 340 360 380 400 420 300 320 340 360 380 400 420 Wavelength (nM)


Relative Fluorescence Units

Relative Fluorescence Units

B 8x103

PruA

-2

Relative Fluorescence Units (X10 )

Figure 4.

Relative Fluorescence Units (X10 )

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Table of contents/abstract graphic:


Mechanism of Fluorinated Anthranilate-Induced Growth Inhibition in

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