Establishment of an effective TLC bioautographic method for the detection of Mycobacterium tuberculosis H37Ra phosphoglucose isomerase inhibition by phosphoenolpyruvate

Abstract

A bioautographic assay based on thin layer chromatography was developed for phosphoenolpyruvate (PEP) detecting as a known but rarely studied inhibitor of phosphoglucose isomerase (PGI). The protocol with NADP⁺/NBT/PMS (β-nicotinamide adenine dinucleotide phosphate/nitrotetrazolium blue chloride/phenazine methosulfate) staining was capable of detecting Mycobacterium tuberculosis H₃₇Ra PGI inhibition using PEP. According to this method, visibly brighter spots (zones) against purple background are observed in the area of inhibition of the above-mentioned enzyme activity. The detection limit for PEP as an inhibitor of Mycobacterium tuberculosis H₃₇Ra PGI was 226 μg per spot/zone. Noteworthy is that we are the first authors to have successfully used a bioautographic assay to detect Mycobacterium tuberculosis H₃₇Ra PGI inhibition by PEP.

• Antimycobacterial activity
• autography
• glycolysis
• mycobacterial cell wall
• potential drug target

Introduction

Mycobacterium tuberculosis (Myc. tbc), the causative agent of tuberculosis, is still a major burden to human health¹. The World Health Organisation reported 5.7 million new TB cases in 2013². Phosphoglucose isomerase (PGI) (d-glucose-6-phosphate aldose-ketose isomerase, PGI, EC 5.3.1.9) is an enzyme that catalyses reversible isomerisation of glucose-6-phosphate to fructose-6-phosphate. PGI plays a key role in the metabolic pathways of phosphorylated sugars, such as glycolysis, phosphate pentose cycle, Entner–Doudoroff cycle, gluconeogenesis, glycogenogenesis and glycogenolysis. Due to this, PGI is sometimes described as a “workhorse” or “housekeeping” enzyme of sugar metabolism^3,4. Apart from its enzymatic function, this protein has a number of other “moonlighting” functions⁵. In mycobacteria, PGI is engaged through glucose-6P with l-rhamnose and is involved in the biosynthesis of the galactan residue of arabinogalactan, the components of the cell wall of these microorganisms^6–8.

The major application of bioautography is screening of the antimicrobial and antifungal properties of various plant samples with Thin Layer Chromatography (TLC)-plates⁹. The term “autography” is used to detect other biological processes, e.g. enzyme inhibition¹⁰. However, the definition of the bioautographic method is often broadened and covers the determination of enzyme inhibition, antioxidant agents as well as oestrogenic compounds^9,11. In our opinion, there is a clear trend towards the use of the TLC bioautography technique as a method for screening active compounds against microorganisms rather than a method for the detection of enzyme activities or inhibitors. The use of TLC (bio)autography was reported for the screening of inhibitors of acetylcholinesterase^12–26, butyrylcholinesterase^{12,16,22,23,27}, dipeptidyl peptidase IV²⁸, glucose-6-phosphate dehydrogenase²⁹, α- and β-glucosidases^30–34, lipase^35–37, monoamine oxidases A and B^38,39, DNA topoisomerase I⁴⁰, tyrosinase^41–43 and xanthine oxidase⁴⁴. To date, the use of the TLC-based autographic method for the detection of PGI inhibition has only been developed for a biocatalyst isolated from the reference E. coli strain ATCC (American Type Culture Collection) 25922⁴⁵.

The mechanism of the bioautographic assay for determining the PGI activity is shown in Figure 1. When PGI is not inhibited by phosphoenolpyruvate (PEP) or other inhibitors, the product of the reaction, glucose-6-phosphate, is converted into gluconolactone-6-phosphate by glucose-6-phosphate dehydrogenase (used as an auxiliary enzyme); the simultaneously generated NADPH (β-nicotinamide adenine dinucleotide phosphate (β-NADP⁺), reduced form) participates in a cycling reaction that employs the tetrazolium salt NBT (nitrotetrazolium blue chloride) to produce a purple coloured formazan with PMS (phenazine methosulfate) as an electron carrier.

Figure 1. The mechanism of bioautographic assay for the detection of phosphoglucose isomerase activity. Abbreviations used: NADP⁺, nicotinamide adenine dinucleotide phosphate; NADPH, nicotinamide adenine dinucleotide phosphate, reduced form; PMS, phenazine methosulfate; NBT, nitrotetrazolium blue chloride.

In this paper, a TLC bioautographic assay was established to detect PEP inhibition of PGI isolated from Mycobacterium tuberculosis (Myc. tbc) H₃₇Ra strain (the commonly used surrogate for virulent Myc. tbc H₃₇Rv strain). Noteworthy is that we are the first authors to have successfully used this method to detect mycobacterial PGI inhibition by PEP.

Materials and methods

Microbial cultures

Myc. tbc H₃₇Ra bacteria (ATCC 25177) were maintained as a Löwenstein slant culture. The cells were also grown in mineral salt medium containing: glutamic acid (1% w/v); citric acid (0.2% w/v), KH₂PO₄ (0.05% w/v); MgSO₄×7H₂O (0.05% w/v); ferric ammonium citrate (0.005% w/v); ZnSO₄×7H₂O (0.0025% w/v) and the carbohydrate source – glucose (2.5% w/v). The pH of the medium was adjusted to 5.5 with KOH before autoclaving. The bacterial strain was cultured in 1 l Roux flasks containing 0.2 l medium at 37 °C for eight days. The cells were harvested by centrifugation, washed with sterile deionised water and stored as a paste at −18 °C until use.

Reagents and materials

Bovine serum albumin (BSA), β-NADP⁺, β-nicotinamide adenine dinucleotide (β-NAD⁺), dithiothreitol (DTT), fructose 6-phosphate (fru-6P), glucose, glucose 6-phosphate (glc-6P), glutamic acid, deoxyribonuclease I (DNase I) from bovine pancreas, glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides (L. mesenteroides), NBT, PMS, PEP and Tris-(hydroxymethyl)aminomethane (TRIS, TRIZMA® base) were all purchased from Sigma-Aldrich (St. Louis, MO). The components of the liquid medium for culturing mycobacterial cells (except glutamic acid and glucose), an organic solvent – ethanol (EtOH), and magnesium chloride hexahydrate were obtained from Avantor Performance Materials (Gliwice, Poland). All reagents were of analytical quality and were used without purification. Dialysis tubing cellulose membrane was purchased from Sigma-Aldrich (St. Louis, MO). Diaflo YM-100 (cutoff: 100 kDa) ultrafiltration membranes were obtained from Millipore Corp. (Bedford, MA). DEAE-Macro® Prep Support was obtained from BIO-RAD (Hercules, CA). Cellulose and silica gel 60 F₂₅₄ plates were purchased from Merck (Darmstadt, Germany).

Purification of the PGI from Myc. tbc H₃₇Ra

The enzyme used for TLC bioautography was partially purified using the steps outlined below. All purification steps were performed at 4 °C unless otherwise specified. The cells (30 g wet weight) were suspended in 150 ml of ice-cold 50 mM TRIS/HCl buffer with 2 mM DTT (pH 7.6) (buffer A), disrupted by sonic oscillation (3 × 5 min, 22 kHz, 180 W) and centrifuged at 3944×g for 30 min (Sigma 3-16K Centrifuge, Germany). Afterwards, DNase I (EC 3.1.21.1) was added (1 U/1 ml) to the supernatant and the sample was incubated overnight. The obtained liquid, designated as “crude extract”, was subjected to (NH₄)₂SO₄ fractionation. The fraction precipitated at 50–70% saturation with (NH₄)₂SO₄ was collected via centrifugation at 1753×g for 45 min (Sigma 3-16K Centrifuge, Germany), dissolved in a minimal volume of buffer A and dialysed against the same buffer overnight (AS-fraction). The AS-fraction was removed from the dialysis tubing and loaded onto the ion-exchange column Macro-Prep® DEAE Support (1.4 × 17 cm) previously equilibrated with buffer A. The column was washed thoroughly with the same buffer (150 ml) and the wash that did not exhibit the activity of PGI was discarded. Myc. tbc H₃₇Ra PGI was eluted from the column with 0.2 M NaCl in buffer A (200 ml). Other proteins were eluted with 0.5 M NaCl in buffer A (150 ml). Fractions containing the active enzyme were pooled and concentrated on the Amicon filtration apparatus (Millipore, Billerica, MA) using a YM-100 membrane.

Enzyme assay

The determination of Myc. tbc H₃₇Ra PGI activity was conducted via a coupled assay with glucose-6-phosphate dehydrogenase from L. mesenteroides (EC 1.1.1.363) as an auxiliary enzyme according to Fraenkel and Levisohn method⁴⁶. The reduction of β-NADP⁺ intermediate with glc-6P dehydrogenase was followed by the increase in A340 nm. The standard assay mixture (1 ml) contained 50 mM TRIS/HCl buffer (pH 7.6), 1 mM d-fructose-6P, 2.5 mM MgCl₂×6H₂O, 1 mM NADP⁺ and 1 U of glc-6P dehydrogenase. After 1 min, the reaction was initiated by the addition of the PGI sample and it was followed by 2 min. The activity of the characterized biocatalyst was estimated using a spectrophotometer (double beam spectrophotometer Evolution 300 UV-VIS, Thermo Fischer Scientific, Madison, WI) operating on the kinetic mode at room temperature (RT). One unit (U) of Myc. tbc H₃₇Ra PGI activity was defined as the amount of enzyme catalysing the reduction of 1 μmol of NADP⁺ per min in the above conditions.

Protein assays

Protein concentration was measured using the method by Lowry et al.⁴⁷ with BSA as a standard, or spectrophotometrically at 280 nm.

Bioautographic assay

Pure diluent sample (EtOH:H₂O deionised 1:2 v/v) as well as PEP dissolved in that diluent were applied onto a TLC cellulose plate (5 × 20 cm) with an aerosol applicator (Automatic TLC Sampler 4, Camag, Muttenz, Switzerland) under the control of WinCATS-4 software. The plate was left to air dry and then sprayed with Myc. tbc H₃₇Ra PGI enzyme solution (2.5 ml, concentration 6 U/ml). Subsequently, the plate was incubated at RT covered with aluminium foil to protect against plate drying which could adversely affect the reaction of the enzyme with the inhibitor. After 40 min, the plate was sprayed with approx. 3 ml NADP⁺/NBT/PMS staining solution. The final reagent concentrations in the staining solution were as follows: 200 mM TRIS/HCl buffer (pH 7.6), 1.67 mM d-fructose-6P, 33 mM MgCl₂×6H₂O, 6.1 mM NADP⁺, 35 U of glc-6P dehydrogenase, 3.26 mM NBT and 0.78 mM PMS. After application of the solution, the plate covered with aluminium foil was placed at RT for 15 min. Visibly brighter spots against a dark purple background indicated lack of characterized PGI activity.

In order to optimize the staining solution for the detection of Myc. tbc H₃₇Ra PGI activity, TLC bioautography was conducted in parallel on two separate plates using NADP⁺/NBT/PMS and NAD⁺/NBT/PMS solutions. The only difference between the compositions of the solutions was the coenzyme used.

In order to confirm the lack of L. mesenteroides glc-6P dehydrogenase inhibition by PEP, additional TLC bioautography was performed. Briefly, diluent sample and PEP samples dissolved in diluent (EtOH:H₂O deionised 1:2 v/v) were applied onto a 2.5 × 20 cm TLC cellulose plate. Next the dried TLC plate was sprayed solution with compounds as follows: TRIS/HCl buffer (pH 7.6), d-glucose-6P, MgCl₂×6H₂O, NADP⁺, L. mesenteroides glc-6P dehydrogenase, NBT and PMS in a total volume of 1.5 ml.

The results were viewed and documented by means of photography taken under white light with an ATS-4 visualizer (Camag, Muttenz, Switzerland) and processed using the WinCATS-4 software.

Results and discussion

TLC hyphenation with bioautography for the detection of enzyme inhibition is a technique with a great potential for implementation in the light of increasing drug resistance of microorganisms. It is worth noting that there is a vast variety of known microorganism enzymes that could be considered as potential drug targets⁴⁸. Thus, there is a significant need to develop new techniques that would allow detection of microbial enzyme inhibition and fast screening of compounds with potential antimicrobial activity, in particular antimycobacterial one.

First of all, during Myc. tbc infection in macrophages, the bacteria harvest approx. 70% of energy from anaerobic glycolysis^5,49. From this aspect, PGI, an enzyme essential for the survival of Myc. tbc, may be treated as a potential target of compounds with antituberculosis activity^5,7. Secondly, as mentioned in the Introduction section, the characterized biocatalyst is known to be responsible for the formation of arabinogalactan, a key component of mycobacterial cell wall. Considering both above-mentioned reasons, studies regarding the usefulness of the TLC bioautographic method for the determination of PGI inhibition are fully justified.

The main problem with enzyme bioautographic assays is their high sensitivity to organic solvents. In this study, the inhibitory influence of pure compound (PEP) on enzyme activity (PGI from Myc. tbc H₃₇Ra) was analysed. Therefore, an analogous test to TLC-direct bioautography “dot-blot”⁹ in which compounds to be detected are not separated on the TLC plate was performed.

The best results were obtained when the TLC bioautographic technique was performed on a cellulose plate using NADP⁺/NBT/PMS staining solution. The lowest amount of PEP inhibitor (μg) that produced visibly brighter zones was 226 μg per zone (detection limit). Thus, PEP inhibited Myc. tbc H₃₇Ra PGI at amount above 226 μg (Figure 2, lane 4). Noteworthy are the visibly brighter inhibition spots (zones) in a purple background reflecting Myc. tbc H₃₇Ra PGI inhibition by PEP were visible after only 5 min upon the use of NADP⁺/NBT/PMS staining solution.

Figure 2. Bioautographic thin layer chromatogram showing the inhibition of Mycobacterium tuberculosis H₃₇Ra phosphoglucose isomerase (Myc. tbc H₃₇Ra PGI) activity by phosphoenolpyruvate using NADP⁺/NBT/PMS staining. Lanes 2, 3, 4, 5, 7, 8, 9 and 10 were spotted with 113, 170, 226, 339, 452, 565, 678 and 904 μg of phosphoenolpyruvate, respectively. Lanes 1 and 6 were spotted with the solvent of phosphoenolpyruvate. The experiment was performed with 15 U of Myc. tbc H₃₇Ra PGI activity.

The initial attempts to measure the Myc. tbc H₃₇Ra PGI activity directly on the glass-backed TLC layer of silica gel 60 F₂₅₄ were unsuccessful (results are not presented). From this aspect, we wanted to check the possibility of using cellulose plates to determine the characterized enzymatic activity. In preliminary studies, we estimated Myc. tbc H₃₇Ra PGI activity in order to obtain the background of cellulose plates with clearly purple colour visible with a naked eye. In our opinion, the activity was approx. 12 U (over a 5 × 20 cm TLC plate), results are not included. It should be mentioned that the activity of the enzyme applied on TLC cellulose plates to optimize the composition of the staining solution and the inhibition of Myc. tbc H₃₇Ra by PEP (Materials and methods, Bioautographic assay) was similar and amounted to 15 U.

In the light of the literature available, glc-6P dehydrogenase from L. mesenteroides was approx. equally effective in using NAD⁺ and NADP⁺ as coenzymes^50,51. Nevertheless, TLC bioautography experiments employing both compounds were performed. The results of TLC bioautography of Myc. tbc H₃₇Ra PGI with the use of NAD⁺/NBT/PMS and NADP⁺/NBT/PMS staining solutions are presented in Figure 3. It was observed that the purple colour in the background of the cellulose plate was darker after the use of NADP⁺ as a coenzyme of L. mesenteroides glc-6P dehydrogenase (Figure 3B) in comparison with the reaction conducted in the presence of NAD⁺ (Figure 3A). Thus, NADP⁺ appeared to be a more suitable component of the staining solution than NAD⁺ in the TLC bioautographic assay of Myc. tbc H₃₇Ra PGI activity.

Figure 3. Comparison of bioautographic thin layer chromatograms obtained for Mycobacterium tuberculosis H₃₇Ra phosphoglucose isomerase (Myc. tbc H₃₇Ra PGI) with the use of staining solutions containing different coenzymes of the auxiliary enzyme Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase. A – reaction with NAD⁺; B – reaction with NADP⁺. The experiments were performed with 15 U of Myc. tbc H₃₇Ra PGI activity. Staining was performed in darkness for 5 min at room temperature.

The glc-6P dehydrogenase derived from L. mesenteroides was expected not to be inhibited by PEP. In the literature survey, the presented kinetic results demonstrated that PEP was an allosteric inhibitor of glc-6P dehydrogenases from various species, e.g. Zymomonas mobilis⁵², but not of that from L. mesenteroides^53,54. However, additional TLC bioautography, in which glucose-6-phosphate was used instead of fructose-6-phosphate, was conducted as described in Materials and methods, bioautography assay, in order to confirm the lack of inhibition of glc-6P dehydrogenase from L. mesenteroides by PEP. This compound did not exhibit any inhibitory activity against glc-6P dehydrogenase (Figure 4).

Figure 4. Bioautographic thin layer chromatogram showing the lack of inhibition of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides by phosphoenolpyruvate using NADP⁺/NBT/PMS staining. Lane 1 was spotted with the solvent of phosphoenolpyruvate. Lanes 2, 3 and 4 were spotted with 226, 339 and 452 μg of phosphoenolpyruvate, respectively.

To our best knowledge, in contrast to 6-phosphogluconate, which has been listed as one of the most important inhibitors of the PGI activity^4,7, the influence of PEP on the activity of mycobacterial PGI had never been examined before. PEP was reported to be an effective inhibitor of PGIs from Bacillus caldotenax⁵⁵ and E. coli⁵⁶ Ogawa et al.⁵⁶ found that PEP exhibited a competitive type of inhibition of E. coli PGI with respect to fru-6P.

Conclusions

In conclusion, the study resulted in a successful implementation of the TLC bioautographic assay as a simple and effective technique for the detection of inhibition of Myc. tbc H₃₇Ra PGI, a key enzyme in the metabolism of phosphorylated sugars in mycobacteria. However, in order to fully confirm the applicability of this technique, further studies regarding the use of the mobile phase are needed. Nevertheless, the authors believe that this method will be extremely helpful in the identification of novel compounds (e.g. plant-derived) with antimycobacterial activity.

Acknowledgements

The authors would like to thank Dr. Agata Przekora from Department of Biochemistry and Biotechnology, Medical University of Lublin, Poland, for providing valuable suggestions on preparing the manuscript.

Declaration of interest

The authors declare no conflict of interest. Financial assistance was provided by Ministry of Science and Higher Education in Poland within DS2/15 project of Medical University of Lublin. This study was conducted using the equipment purchased within the agreement No. POPW.01.03.00-06-010/09-00 Operational Program Development of Eastern Poland 2007–2013, Priority Axis I, Modern Economy, Operations 1.3. Innovations Promotion.