Supporting Information

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1 Supporting Information Wiley-VCH Weinheim, Germany Tuning the Intracellular Bacterial Targeting of Peptidic Vectors** Eric K. Lei, Mark P. Pereira, and Shana O. Kelley* anie_ _sm_miscellaneous_information.pdf

2 Supporting information 1. Materials and Methods General Cell Culture and Bacterial Growth Conditions HeLa cells were cultured in MEM alpha (Invitrogen, Carlsbads CA) supplemented with 10% fetal bovine serum at 37 C with 5% CO 2. L. monocytogenes was grown in Bacto Brain Heart Infusion media (BD, Franklin Lakes NJ) at 37 C. Peptide Synthesis and Characterization Solid phase synthesis was performed on Rink amide or Rink amide MBHA resin (Novabiochem, UK) using a Prelude Protein Technologies peptide synthesizer as described previously [1]. Peptides were synthesized on a 50 µmol scale. Methotrexate conjugated peptides were synthesized as described previously [2]. Fluorescently labeled peptides were synthesized by N-terminal coupling of 5(6)- Carboxytetramethylrhodamine (Anaspec, Freemont, CA) to 16.7 µmol of peptide on resin with O- (benzotriazol-l-yl)-n,n,n,n -tetramethyl-uronium hexafluorophosphate (HBTU, 4 eq, Protein Technologies, Tucson) and N,N-diisopropylethylamine (DIPEA, 8 eq, Sigma-Aldrich, St. Louis), in 1 ml N,N- dimethyl formamide (DMF) for 2 hours at room temperature. Peptides were cleaved from resin using trifluoroacetic acid:triisopropylsilane:water (95:2.5:2.5) and precipitated in ether at -20 C for 1 hour. Peptides were purified using RP-HPLC on a C18 column with a H 2 O/MeCN gradient with 0.1% TFA to >95% purity. Peptide product identities were confirmed by electrospray ionization mass spectroscopy. Methotrexate conjugated peptides were quantified at 302 nm in Dulbecco s phosphate buffered saline (D-PBS, Invitrogen, Carlsbads CA) using the methotrexate extinction coefficient of M -1 cm -1. Carboxytetramethylrhodamine conjugated peptides were quantified at 553 nm in D-PBS using the extinction coefficient of M -1 cm -1. Unlabeled peptides were quantified using a BCA assay (Pierce, Rockford IL). Peptide sequences: NH 2 -Fx-r-Fx-r-Fx-r-CONH 2 ; Expected m/z = , Found = NH 2 -Fx-r-F-r-Fx-r-NH2; Expected m/z = , Found = NH 2 -F-r-Fx-r-F-r-CONH 2 ; Expected m/z = , Found = NH 2 -F-r-F-r-F-r-CONH 2 ; Expected m/z = , Found = NH 2 -Fx-r-F-r-Fx-r-Fx-r-CONH 2 ; Expected m/z = , Found = NH 2 -Fx-r-F-r-Fx-r-F-r-Fx-r- CONH 2 ; Expected m/z = , Found = NH 2 -Fx-r-A-r-Fx-r-A-r-Fx-r-A-r-CONH 2 ; Expected m/z = , Found = TAMRA-Peptide sequences: TAMRA-Fx-r-Fx-r-Fx-r-CONH 2 ; Expected m/z = , Found = TAMRA-Fx-r-F-r-Fx-r-CONH 2 ; Expected m/z = , Found = TAMRA-F-r-Fx-r-F-r-CONH 2 ; Expected m/z = , Found = TAMRA-Fr-F-r-F-r-CONH 2 ; Expected m/z = , Found = TAMRA-Fx-r-F-r-Fx-r-Fx-r-CONH 2 ; Expected m/z = , Found = TAMRA-Fx-r-F-r-Fx-r-F-r-Fx-r-CONH 2 ; Expected m/z =

3 , Found = TAMRA-Fx-r-A-r-Fx-r-A-r-Fx-r-A-r-CONH 2 ; Expected m/z = , Found = TAMRA-Y-r-Y-r-Y-r-CONH 2 ; Expected m/z = , Found = Mtx-Peptide sequences: Mtx-Fx-r-Fx-r-Fx-r-CONH 2 ; Expected m/z = , Found = Mtx- Fx-r-A-r-Fx-r- A-r-Fx-r-A-r-CONH 2 ; Expected m/z = , Found = Fx = L-cyclohexylalanine, r = D- arginine, F = L-phenylalanine, A = L-alanine, Mtx = methotrexate, TAMRA = 5(6)- Carboxytetramethylrhodamine Microscopy Cells were seeded in 8 well µ slides (ibidi, Germany) at a density of or cells per well one day prior to experiments in MEM alpha supplemented with 10% fetal bovine serum. Cells were washed once prior to peptide incubation for 30 minutes in MEM alpha. Cells were then washed twice in MEM alpha and imaged using an inverted Zeiss Observer.Z1 microscope. For intracellular L. monocytogenes localization imaging of the fluorescently labeled modified peptides, the OD 600 of an overnight culture of L. monocytogenes was measured followed by centrifugation of a 1 ml sample at 7,500 g for 3 minutes. The bacterial pellet was washed once with D-PBS followed by incubation in CellTrace Far Red DDAO-SE (Invitrogen, Carlsbads CA, 10 µm) in D-PBS at a final OD 600 of 1.0 for 15 minutes. L. monocytogenes was washed twice in D-PBS following incubation. HeLa cells were infected with a multiplicity of infection (MOI) of 50 or 100 for two and a half hours in MEM alpha at 37 C. Infected HeLa cells were washed three times with MEM alpha followed by peptide incubation for 30 minutes or JC-1 incubation for 20 minutes. JC-1 treatment stocks were spun down prior to treatment at g for 1 minute to remove sediments. For cells treated with Carbonyl cyanide 4- (trifluoromethoxy)phenylhydrazone (FCCP, Sigma-Aldrich, St. Louis) and JC-1, cells were incubated with 20 µm FCCP for 20 minutes prior to JC-1 treatment and all subsequent media used contained 20 µm FCCP. Cells were then washed twice in MEM alpha and imaged. Post infection staining of L. monocytogenes was accomplished through fixation of HeLa cells with 4% formaldehyde/1% glutaraldehyde in D-PBS for 5 minutes at room temperature. Cells were blocked and stained with Rabbit pab to Listeria monocytogenes (Abcam, Toronto, ON) and imaged using Alexa Fluor 350 labeled Goat anti-rabbit IgG. Actin staining was accomplished post fixation (4% formaldehyde/1% glutaraldehyde) and permeabilization (0.1% Triton in D-PBS) using Alexa Fluor 350 phalloidin (Invitrogen, Carlsbad, CA). Flow Cytometry Cells were seeded in 12 well flat bottom tissue culture plates (Corning, Kennebunk, ME) at a density of cells per well one day prior to experiments in MEM alpha supplemented with 10% fetal bovine serum. Cells were washed once with MEM alpha prior to peptide incubation for 30 minutes in MEM alpha. Cells were then washed twice and 500 µl of 0.25% trypsin-edta (Invitrogen, Carlsbad, CA) was added to each well followed by incubation at 37 C for 3 minutes. 1 ml of MEM alpha was added to each well and samples were then centrifuged at 700 g for 5 minutes at 4 C. The samples were decanted and washed once with 4 C D-PBS. Cells were then resuspended in D-PBS with 5 nm SYTOX Red Dead Cell Stain (Invitrogen, Carlsbad, CA) and incubated for 5 minutes at 4 C. Samples were analyzed by on a BD FACSCanto flow cytometer (BD Biosciences) with excitation at 488 nm and emission wavelengths of 564 to 606 collected.

4 Analysis of Toxicity HeLa cell toxicity was analyzed as described previously [1]. HeLas were seeded at 1250 cells per well in 96-well flat bottom tissue culture plates (Starstedt, NC) one day prior to treatment. Cells were washed once with MEM alpha (-nucleosides) prior to treatment with peptide compound for 72 hours in MEM alpha (-nucleosides) supplemented with 10% dialyzed FBS. Cells were then washed with MEM alpha (- nucleosides) followed by determination of cellular viability using CCK-8 viability dye (Dojindo, Rockville, MD) at an absorbance of 450 nm. Minimum inhibitory concentration (MIC) determinations for methotrexate and methotrexate-conjugated peptides against Listeria in vitro were determined as described below. Overnight Listeria cultures were subcultured at 1:60 and grown to and OD 600 of to ensure logarithmic growth. Cells were diluted 1:10000 into fresh media with test compounds. MEM alpha (-nucleosides, Invitrogen, Carlsbad, CA) was used as a defined media for bacterial growth. Bacto Brain Heart Infusion media (BD, Franklin Lakes NJ) was used as an enriched media for bacterial growth. Cells were incubated for 24 hours at 37 C. Growth was measured using absorbance at 600 nm. Minimum inhibitory concentration (MIC) determinations for methotrexate and methotrexate-conjugated peptides against in cellulo Listeria were determined as described below. HeLa cells were seeded on a 24 well plate at cells per well 1 day before infection with Listeria. Overnight Listeria cultures were subcultured at 1:60 and grown to and OD 600 of to ensure logarithmic growth. HeLas were washed twice with MEM alpha (-nucleosides) and incubated in MEM alpha (-nucleosides) for one hour. 500 µl of log phase L. monocytogenes was pelleted at 7,500 g for 3 minutes, washed once with D-PBS and then resuspended in 500 µl D-PBS. HeLa cells were washed once with MEM alpha (-nucleosides), and infected with L. monocytogenes at a multiplicity of infection of 5 for 1 hour at 37 C. Infected HeLas were washed twice with MEM alpha (-nucleosides) and treated with test compounds for 17 hours. Infected HeLas were then washed twice with 500 µl and once with 900 µl MEM alpha (minus nucleotides). HeLas were then lysed for 15 minutes with 200 µl 0.1% Triton X-100 in D-PBS while shaking. 1800uL of D-PBS was then added to each well followed by serial dilution to 1 in 1000 in D- PBS. 20 µl of the 1 in 1000 dilution was spotted on fresh BHI plates and incubated at 37 C overnight. Viable intracellular bacteria were determined by counting individual colonies. Cellular viability of infected HeLa cells after treatment with either Mtx or Mtx-(Fxr) 3 (each at 20 µm) was assessed using differential interference contrast microscopy. HeLa cells were plated in 8 well µ slides (ibidi, Germany) at a density of or cells per well one day prior to experiments in MEM alpha supplemented with 10% fetal bovine serum. Cells were infected with L. monocytogenes and treated with compound as described above. Cellular morphology was observed using differential interference contrast microscopy.

5 2. Supplemental Figures Table S1 Chemical properties of unconjugated peptides Peptide Sequence [a] Z [b] Length (a.a) [c] % Ac Elution [d] Relative Uptake [e] 1 (Fxr) FxrFrFxr FrFxrFr (Fr) FxrFrFxrFxr (FxrFr) 2 Fxr (FxrAr) [a] Shorthand peptide sequence: Fx = L-cyclohexylalanine, r = D-arginine, F = L-phenylalanine, A = L- alanine. [b] Positive charge. [c] Peptide length in amino acid residues. [d] Percent acetonitrile/water elution on a C-18 column (RP-HPLC). [e] Uptake as measured by flow cytometry normalized against peptide 1 Figure S1. Polymerization of actin monomers by intracellular L. monocytogenes indicates motility and viability of L. monocytogenes inside infected HeLa cells. Fluorescently labeled actin is shown in blue. Listeria labeled with commercially available CellTrace Far Red DDAO-SE are shown in red.

6 Figure S2. Differential interference contrast images of L. monocytogenes in infected HeLas.

7 Figure S3. Fluorescence histograms of TAMRA labeled peptide uptake in HeLa cells. Relative fluorescence units are shown on the X-axis while cell counts are shown on the Y-axis. All cells were treated with 5 µm of the corresponding TAMRA-peptide for 30 minutes.

8 Figure S4. Mitochondrial localization of TAMRA labeled peptides in HeLa cells. TAMRA fluorescence is shown in green, while respective differential interference contrast images are shown below. HeLas were treated for 30 minutes at the concentrations indicated; 1: 10 µm treatment, 2: 20 µm treatment, 3:70 µm treatment, 4: 90 µm treatment, 5: 6 µm treatment, 6: 8 µm treatment, 7: 8 µm treatment

9 Figure S5. Localization of TAMRA, TAMRA-Lysine, and TAMRA-(Yr) 3 in HeLa cells. TAMRA fluorescence is shown in green, while respective differential interference contrast images are shown below. HeLas were treated for 30 minutes at the concentrations indicated; TAMRA: 20µM, TAMRA- Lysine: 20µM, TAMRA(Yr) 3 : 80 µm.

10 Figure S6. Depolarization of L. monocytogenes and mitochondrial membrane potential by 20 µm FCCP eliminates J-aggregate formation. Monomeric JC-1 fluorescence is shown in green. Fluorescence from J- aggregates indicative of membrane potential are shown in red. L. monocytogenes labeled with CellTrace Far Red are shown in grey.

11 Figure S7. Toxicity of Mtx-peptide 7 against HeLa cells over 72 hours. At the highest concentration tested, perturbation of mitochondrial function (as measured by mitochondrial membrane potential) is not observed (see Figure S9). Enriched Media Defined Media Enriched Media Defined Media Figure S8. Efficacy of Mtx-peptides are reduced upon treatment in enriched media. shown on the left, while Mtx-peptide 7 is shown on the right. Mtx-peptide 1 is

12 Figure S9. Analysis of mitochondrial toxicity of compound 7. Mitochondrial membrane potential was monitored using the mitochondria potential sensitive dye JC-1. HeLa cells were treated for 24 hours with 24 µm compound in MEM alpha media + 10% dialyzed FBS. FCCP was added at 10 µm for 6 minutes prior to analysis to disrupt the mitochondrial membrane potential. The mitochondrial membrane potential sensitive dye JC-1 was added at 5 µg/ml for 10 minutes at 37 o C. Samples were subsequently analyzed by flow cytometry with excitation at 488 nm and emission wavelengths of 530 nm and 585 nm collected. The ratio of these two emission wavelengths was used to measure changes in mitochondrial membrane potential. Treatment of HeLa cells with compound 1, compound 7, and Mtx was found to not perturb the mitochondrial membrane potential. The mitochondrial membrane depolarizer FCCP resulted in a significant decrease membrane potential.

13 References [1] K. L. Horton, K. M. Stewart, S. B. Fonseca, Q. Guo, S. O. Kelley, Chem. Biol. 2008, 15, [2] M. P. Pereira, S. O. Kelley, J. Am. Chem. Soc. 2011, 133,