The following antibodies were obtained from commercial sources: anti-bacteriophage antibody (1:1000, Sigma), anti-caspase-7, anti-caspase-9, anti-atg-5, anti-atg-7, anti-beclin-1 (1:2000, Cell Signaling Technology), anti-HMG-B1 (1:2000, Millipore), anti-actin antibody (1:5000, Novus Biologicals), anti-rabbit Cy3, anti-mouse FITC, anti-goat FITC (1:300, Zymed), anti-mouse HRP (1:5000, Bio-Rad), Alexa Fluor 488 goat anti-rabbit (1:500, Invitrogen), goat and mouse pre-immune sera (1:300, Jackson Immunoresearch). The following fluorescence probes were used: 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories), ProLong Gold antifade reagent, Orange MitoTracker (Invitrogen), and annexin V-FITC (BD Biosciences). Molecular biology reagents included restriction enzymes (New England Biolabs and Roche), T4 DNA ligase (Invitrogen), Taq DNA polymerase (Promega, Stratagene and Invitrogen); gel extraction, PCR purification, and plasmid isolation kits (Qiagen), isopropyl b-D-1-thiogalactopyranoside (IPTG; Fisher Scientific). We used commercial recombinant ribosomal protein L30 (RPL30; Novus). All the synthetic peptides were produced to our specifications (PolyPeptide Laboratories).
Human Kaposi sarcoma (KS1767) cells were maintained in MEM containing 10% FBS, MEM-vitamins, non-essential amino acids, penicillin G (100 units), streptomycin SO4 (100 mg ml−1), and 2.7 mM. L-glutamine (Invitrogen) at 37 °C in a 5% CO2-humidified incubator.
Phage internalization assay
For mammalian cell internalization, KS1767 cells were grown in 8-well tissue-chamber slides and incubated with 109 TU of iPhage, mutant iPhage, or parental insertless phage in MEM containing 1% bovine serum albumin (BSA) at 37 °C. After 24 h of incubation, cell membrane-bound phage was removed by acidic washing (20 mM glycine, pH 2.3). Cells were washed with PBS and fixed with 4% paraformaldehyde in PBS at room temperature for 15 min. After washes with PBS, fixed cells were rendered permeable with PBS containing 0.1% Triton X-100 (Sigma), blocked with PBS containing 1% BSA, and incubated with anti-bacteriophage (1:200 dilution) in PBS containing 1% BSA for 2 h at room temperature. Next, Cy3-conjugated anti-rabbit (1:200 dilution) was added, and incubated at room temperature for 1 h. Finally, cells were washed with PBS, fixed with PBS containing 4% paraformaldehyde, mounted in the presence of DAPI (Invitrogen), and visualized under a fluorescence or confocal microscope.
Generation of the iPhage peptide display library
To generate iPhage constructs, we transformed the M13-derived vector f88-4 (AF218363) into MC1061 E. coli
. Single colonies were selected on Luria-Bertani (LB) agar plates with tetracycline (40 mg ml−1
) and streptomycin (50 mg ml−1
) and cultured overnight. Each plasmid DNA was first isolated by standard plasmid purification kit (Siegen) and was subsequently re-purified through a CsCl gradient. Next, annealed oligonucleotides encoding the penetratin peptide, sense 5′-cacaagctttgccaacgtccctcgacagataaagatttggttccaaaacggcgcatgaagtggaagaagcctgcagcaca-3′; antisense 5′-tgtgc tgcaggcttcttccacttcatgcgccggttttggaaccaaatctttatctgtcgagggacgttggcaaagcttgtg-3′), and f88-4 plasmid were digested with Hind
III and Pst
I restriction endonucleases. DNA fragments were gel-purified (Qiagen) and ligated at various vector: insert molar ratios. Restriction enzyme-digested and sequence-verified individual clones were electroporated into MC1061 E. coli
; iPhage particles were cultured in LB media containing 1 mM IPTG, tetracycline (40 mg ml−1
), and streptomycin (50 mg ml−1
), and were purified by the polyethylene glycol-NaCl method56
. To produce an iPhage library displaying the peptide insert in the minor pIII coat protein (general arrangement X4
), we fused the iPhage construct (described above) and fUSE5 genomes to create a chimeric vector. Both plasmids were digested with BamH
I and Xba
I restriction enzymes, and the products were resolved on a 0.8% agarose gel. A DNA fragment of 3,925 bp from fUSE5 (containing the rpIII) and another 5,402 bp fragment of iPhage (containing the rpVIII) were gel-purified and ligated overnight at 16 °C. Ligated products were precipitated in ethanol and electroporated into MC1061 E. coli
; these bacteria were plated on LB-agar and selected with tetracycline and streptomycin. Next, we performed a CsCl plasmid purification of the iPhage/fUSE5 chimeric vector, which was digested with Sfi
I restriction endonuclease. In parallel, we performed a PCR amplification of the degenerate oligonucleotide (5′-cactcggccgacggggctnnknnknnknnktatnnk nnknnknnkggggccgctggggccgaa-3′) insert sequence with Bgl
I restriction sites at both DNA ends57
. The PCR products were purified on Qiagen columns, digested with Bgl
I, and gel-purified. Sfi
I-linearized iPhage/fUSE5 and double-stranded oligonucleotide inserts were prepared for large-scale ligations. After overnight incubation, ligated products were precipitated in ethanol and reconstituted in sterile water. We performed over 250 electroporations in MC1061 E. coli
and transferred them into super optimal broth with catabolite repression medium at 37 °C for 1 h. Finally, the bacterial culture was amplified in LB medium (4 l) containing 1 mM
IPTG, tetracycline (40 mg ml−1
), and streptomycin (50 mg ml−1
); phage particles were recovered to yield an X4
-iPhage display library.
iPhage library intracellular selection
KS1767 cells were incubated with 5×1011
TU of the X4
iPhage library overnight at 37 °C. The next day, cells were washed extensively with pre-warmed PBS and were subsequently detached with trypsin. Cells were washed with ice-cold PBS, incubated with hypotonic buffer (10 mM NaCl, 1.5 mM MgCl2
, 10 mM Tris–HCl (pH 7.5)) for 15 min, and placed in a standard Dounce homogenizer to disrupt cell membranes. Next, we added stabilization buffer (525 mM mannitol, 175 nM sucrose, 2.5 mM EDTA (pH 7.5), and 12.5 mM Tris–HCl (pH 7.5)). The organelle suspension obtained was centrifuged at 1,300g
for 5 min at 4 °C; the supernate was transferred to a new tube, and centrifuged at 17,000g
for 15 min; this pellet contained the mitochondria-/ER-enriched fraction. The subcellular fraction-bound phage population was recovered through infection of log-phase k91kan E. coli
for 1 h at room temperature. Serial dilutions of the infected bacteria were plated on LB plates containing tetracycline (40 mg ml−1
) and kanamycin (100 mg ml−1
) to determine the recovery phage titre. In addition, infected bacteria were grown overnight in LB containing tetracycline and kanamycin, and phage was recovered by polyethylene glycol/NaCl precipitation56
. After 3 rounds of selection, 96 bacterial colonies from each round were randomly selected for DNA sequencing.
The cells were imaged with an inverted Olympus FV1000 (Olympus, Center Valley, Pennsylvania, USA) laser scanning confocal microscope using a ×60 O3 PLAPO oil objective. The imaging and 3D reconstruction was performed by taking a series of horizontal scans through the cells, arranged as a vertical stack/z-stack by means of image analysis software (FV1000 version 1.6, Olympus Inc). Optical sections were acquired at 0.44 μm intervals, with an imaging stack consisting of 22 optical sections. The image size was an 800×800 pixel matrix per image (132×132 μm) with a colour depth of 12 bits per pixel. In this study, we used the UV LD405 nm laser (Olympus America, Center Valley, Pennsylvania, USA) as well as the Argon ion 488 nm and green HeNe 543 nm lasers (Melles Griot, Albuquerque, New Mexico,USA) and suitable filter sets.
Binding assay and ligand receptor purification
By coupling the selected synthetic peptide to a Sepharose column (Carboxy-link kit, Pierce) and exposing the columns to KS1767 cell-derived lysates, we purified a candidate receptor. After extensive washing, the receptor was eluted with the corresponding competitive peptide at a concentration of 5 mM. Eluted fractions were analysed by absorbance (optical density at 280 nm), dialysed, and concentrated at 4 °C. Equal amounts of protein (5 μg per well in 50 μl PBS) were immobilized on a 96-well plate overnight at 4 °C. Wells were washed twice with PBS, blocked with PBS containing 2% BSA for 2 h at room temperature, and incubated with targeted iPhage or parental insertless iPhage (109 TU each) in 50 μl PBS containing 0.1% BSA. After 2 h at room temperature, wells were washed ten times with PBS, and each phage population was recovered by host bacterial infection.
Cell death assays
KS1767 cells grown in 48-well plates were maintained for 24 h in non-supplemented MEM. The medium was subsequently supplemented with 10% FBS plus each respective test peptide. Cellular viability was assessed by measurement of cellular metabolism by the MTT (Roche), and WST-1 (Roche) assays at 37 °C, according to standard protocols. Staining for annexin V (Clontech) was performed to monitor early stages of apoptosis. KS1767 cells grown in 8-well slide chambers were incubated with the peptides for 6 h. Next, cells were incubated with annexin V-FITC, washed, and analysed under a fluorescence microscope. For flow cytometry analysis, cells were detached and washed twice with cold PBS and then resuspended in binding buffer (Clontech) at a concentration of 106 cells ml−1. We transfer 100 μl of the solution to a 5-ml culture tube and added annexin-V-FITC and incubated for 15 min room temperature. The cells were washed twice with binding buffer and analysed by flow cytometry.
Student's t-tests were used for statistical analysis as indicated.