Difluoromethylornithine (DFMO) and AMXT 1501 inhibit capsule biosynthesis in pneumococci
Characterization of the impact of α-difluoromethylornithine (DFMO) on the growth of pneumococci
S.pneumoniae serotypes 4 (TIGR4), 2 (D39), 19F (EF3030) and 3 (WU2) were cultured in Todd Hewitt broth supplemented with yeast extract (THY). DFMO supplementation ranging from 1.1 to 549 mM was performed in triplicate in a 96-well plate and incubated at 37°C and 5% CO2 in a Cytation™ 5 Multi-Mode Cell Imaging Reader (BioTek, Winooski, VT). The change in optical density at 600 nm (OD600) was measured hourly for 24 h and used to determine the minimum inhibitory concentration (MIC) of DFMO. Optical density data were analyzed using GrowthRates 4.0 software25. To determine whether DFMO at sub-MIC is bacteriostatic or lethal to pneumococcal cells, all strains were cultured in the presence or absence of 1/2 MIC DFMO. OD600 was measured hourly and CFUs were counted on a blood agar plate (BAP) every 2 h over an 8 h incubation period.
Impact of DFMO and AMXT 1501 on the pneumococcal capsule
S.pneumoniae strains were cultured with DFMO and AMXT 1501 at different final concentrations of 1/2, 1/4 and 1/8 MIC to OD600 0.2 to 0.3 individually or together to estimate any additive or synergistic effect. Capsular polysaccharides (CPS) were extracted and quantified in triplicate, as previously described26 with a slight modification. Briefly, CPS was extracted in lysis buffer (4% deoxycholate, 50 µg/mL DNase I and 50 µg/mL RNase A) at 37°C for 10 min and centrifuged at 18,000 ×g for 10 mins. The supernatant (2 μL) was deposited on a nitrocellulose membrane with 0.2 μm pores (Thermo Fisher Scientific, Waltham, MA, USA) and dried in the oven at 60° C. for 15 min. The membranes were blocked and incubated with different types of antisera for the detection of capsules: type 4 (for TIGR4), 2 (for D39), polyclonal (for EF3030) and 3 (for WU2) antisera (Cedarlane, Burlington, NC, USA). Conjugated polyclonal goat anti-rabbit IgG-HRP (Agilent Technologies, Santa Clara, CA, USA) was used for enhanced chemiluminescence (ECL) detection in all strains (Thermo Fisher Scientific, Waltham, MA, USA) and scanned using a ChemiDoc XRS+ with Image Lab software (Bio-Rad, Hercules, CA, USA). Densitometry analysis of the immunoblot was performed with NIH ImageJ software27.
Agmatine supplementation of pneumococci treated with DFMO
We determined the impact of different concentrations of DFMO (1/8 to 1/2 MIC) on CPS in all serotypes (2, 4, 19F and 3) used in this study. However, agmatine supplementation (1/4 MIC, 20 mM) with potential to restore capsule loss has been achieved with serotype 2 (D39) which is more sensitive to DFMO (1/8 MIC, 34 mM) . D39 was treated with 1/8 MIC DFMO alone, 1/4 MIC agmatine (Agm) alone, a combination of 1/8 MIC DFMO and 1/4 MIC Agm, and a control without DFMO or Agm. CPS was extracted as described in the previous section and immunoblot analysis with type 2 antisera was performed to detect CPS semi-quantitatively.
Metabolomics of pneumococci treated with DFMO
S.pneumoniae TIGR4 (10 mL, n=5) was grown in the absence or presence of DFMO (137 mM, 1/2 MIC) at OD600 from 0.4 to 0.5. This was done to directly compare possible changes in intracellular polyamine and capsule precursor concentrations following DFMO inhibition of polyamine synthesis with the previous report where polyamine synthesis was affected by suppression of the gene in TIGR4.24. D39 was cultured in the absence or presence of impacting concentrations of DFMO (1/8 MIC, 34 mM), AMXT 1501 (1/2 MIC, 7.4 µM), agmatine (1/4 MIC, 20 mM), and combination of DFMO or AMXT 1501 and agmatine at OD600 0.4–0.5. Cells were harvested (5000×g10 min, 4°C), resuspended in extraction solvent [40% acetonitrile, 40% methanol, 20% water with formic acid (0.1 M)] and transferred to bead tubes (MP Biomedicals, Irvine, CA, USA). Cells were lysed using a FastPrep-24™ Classic (45 sec, 6.5 m/s, RT, 3 times) (MP Biomedicals, Irvine, CA, USA) and incubated on ice ( 5 min after each centrifugation). TIGR4 lysates were clarified (6000×g5 min, 4°C), and metabolite differentiation and detection were performed using published protocols28.29. A metabolomics analysis based on liquid chromatography and mass spectrometry (LC-MS) was performed with an Agilent Accurate Mass 6230 TOF coupled to an Agilent 1290 LC system using a Cogent Diamond Hydride Type C column. Briefly, the mobile phase consisted of the following elements: solvent A (ddH2O with 0.2% formic acid) and solvent B (acetonitrile with 0.2% formic acid). The gradient used was as follows: 0–2 min, 85% B; 3–5 mins, 80% B; 6–7 mins, 75% B; 8–9 mins, 70% B; 10–11.1 min, 50% B; 11.1–14 mins 20% B; 14.1–24 min 5% B followed by a 10 min re-equilibration period at 85% B at a flow rate of 0.4 mL/min. Mass axis dynamics were calibrated by continuous infusion of a reference mass solution using an isocratic pump. This setup achieved mass errors of 5 ppm, mass resolution of 10,000–25,000 (on m/z 121–955 atomic mass units), and dynamic range of 5 × log10. The identities of the metabolites were searched using a mass tolerance of 30. Data were normalized (quantile), log-transformed, and a significant fold change between WT and DFMO-treated WT was identified by Student’s you-test (p ≤ 0.05).
To measure polyamines, glucose and glucuronic acid in D39, the TSQ Quantum Access triple quadrupole tandem mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with the acquity UPLC system (Waters, Milford, MA, USA ) was used in positive and negative ion mode. The chromatographic separation was carried out on a Cogent Diamond Hydride HPLC column (2.1 mm × 150 mm, 4 μm) coupled to a Congent Diamond Hydride HPLC Guard column (2.0 mm × 10 mm, 4 μm). The flow rate was 500 µL/min, the sample injection volume was 10 µL, and the column temperature was 40°C. Mobile phase A was composed of water with 0.1% v/v formic acid, and mobile phase B was composed of acetonitrile with 0.1% v/v formic acid. The gradient condition was 0 min (5% A, 95% B), 1 min (5% A, 95% B), 4.0 min (30% A, 70% B), 6 min (95% A , 5% B ), 6.5 min (95% A, 5% B), 7 min (5% A, 95% B) and 10 min (5% A, 95% B). The total run time was 10 min and the column eluate was directed into the mass spectrometer using an electrospray ionization interface in positive and negative modes. The MS conditions were defined as follows: spray voltage = 3500 V, vaporizer temperature = 350°C, sheath gas = 25 psi, auxiliary gas = 10 psi and capillary temperature = 350°C. The samples were analyzed in Selected Reaction Monitoring (SRM) mode and the MS/MS parameters optimized for ionization are shown in Table 1. Both spermidine-d8 and Glucose-dseven were used as internal standard (IS) in both positive and negative modes. The optimal collision energy and S-lens conditions were determined for each compound using automatic tuning software for each analyte by post-column infusion of the individual compounds in a 50% A/50% B mixture of the mobile phase. pumped at a rate of 0.5 mL/min. Xcalibur software was used for data acquisition and processing.
Inhibition of decarboxylase activity by DFMO and DFMA
SP_0916 in TIGR4 encodes an ADC that has minimal ODC and lysine decarboxylase (LDC) activities18. We determined the impact of DFMO (ODC/ADC inhibitor) and α-difluoromethylarginine (DFMA, an ADC inhibitor) on the decarboxylase activities of SP_0916. BL21(DE3) cells expressing the recombinant His tag SP_0916 were lysed with B-PER reagent buffer (Thermo Fisher Scientific, Waltham, MA, USA), the lysate was separated by loading onto a HisPur Cobalt column Spin (Thermo Fisher Scientific, Waltham, MA, USA), desalted using Sephadex G-25 PD-10 column (GE Healthcare, Chicago, IL, USA) and estimated protein concentration at using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).
Protein encoded by SP_0916 (0.926 µmol L−1) was mixed with a 10 mM substrate (arginine, lysine or ornithine) in the reaction mixture (500 µL reaction volume, 50 mM Tris-HCl, pH 8.0) which contains 2.5 mM MgSO4 and 0.6 mM PLP. DFMO was diluted in 3-900 mM water and added to the reaction mixture after 5 min preincubation at 37°C in the dark. DFMA stock was made in the same way but was diluted in water to 0.3–1000 µM. The enzyme and inhibitor were incubated at 37°C for 15 min in the dark and the reaction was terminated with 0.3 M perchloric acid. After incubation on ice for 15 min, the samples were were neutralized with 25 µL of 10 N KOH and extracted with 1 mL of 1-butanol. The organic layer was dried under nitrogen gas and reconstituted with 100 µL of water containing spermidine-d8 and Glucose-dseven as an internal standard. Extract analysis was performed on a Surveyor LC-MS system. 5 μL of reconstitution were injected into the LC-MS system for the detection of products (agmatine, cadaverine or putrescine). We used internal standards to normalize and determine the maximum intensity (area ratio) for each detected metabolite. Enzyme activity without inhibitor is called control activity. All experiments were performed in triplicate. The concentration of inhibitor that inhibited 50% of the control activity (IC50 value) was determined by varying the concentration of the indicated DFMO or DFMA and measuring the decarboxylase activity. Sigma Plot c. 12 was used to fit the curve through the points and IC50 values were interpolated from the fitted curve.