Programmable protein delivery with a bacterial contractile injection system

Plasmid construction

The PVCpnf structural and accessory region (pvc1-16) and payload and regulatory region (Pdp1, Pnf and regulatory genes PAU_RS16570-RS24015) were synthesized de novo (GenScript) and cloned into pAWP78 and pBR322 backbones, respectively. All manipulations involving payload and regulatory plasmids (pPayload) involved standard PCR amplification with Phusion Flash 2x Master Mix (ThermoFisher), assembly with either Gibson Assembly Master Mix (NEB E2611L) or Golden Gate Assembly with AarI and T4 DNA Ligase (ThermoFisher ER1582; NEB M0202), and transformation into chemically competent Stbl3 cells. PVC structural and accessory plasmids (pPVC) were amplified with KOD Xtreme Hot Start DNA Polymerase (Sigma-Aldrich 71975) with several modifications to the manufacturer’s protocol: 100 ng template DNA, 16 cycles and 30 min extension time. These plasmids were then assembled using Gibson Assembly Master Mix with 2–4 h incubation periods at 50 °C and electroporated into EPI300 electrocompetent cells (Lucigen EC300110). A summary of plasmids generated during this work can be found in Supplementary Table 7; annotated plasmid sequences can be found in Supplementary Data 1.

PVC purification

For each PVC condition, one variant each of pPVC and pPayload were electroporated into EPI300 cells and PVC particles were purified using a modified version of a method used previously9. Colonies were inoculated into 2 ml Terrific Broth (US Biological T2810) and shaken at 37 °C for 16 h before being inoculated (at 1:1,000) into 500 ml TB medium and shaken at 30 °C for an additional 24 h. Cultures were then spun for 30 min at 4,000g and resuspended in 28 ml lysis buffer (25 mM Tris-HCl pH 7.5 (ThermoFisher 15567027), 140 mM NaCl (AmericanBio AB01915), 3 mM KCl (Sigma-Aldrich P9541), 5 mM MgCl2 (Sigma-Aldrich M4880), 200 μg ml−1 lysozyme (ThermoFisher 89833), 50 μg ml−1 DNase I (Sigma-Aldrich DN25), 0.5% Triton X-100 (Sigma-Aldrich 93443), and 1 × Protease Inhibitor Cocktail (MedChem Express HY-K0010)) and were subsequently shaken at 37 °C for 90 min to promote lysis. Lysates were then pelleted at 4,000g for 30 min at 4 °C to remove bulk cell lysate. Supernatants were then extracted and spun in an ultracentrifuge at 120,000g for 2 h at 4 °C to pellet PVC protein complexes. Pellets were resuspended in 1 ml PBS (Life Technologies 10010049) and spun at 16,000g for 15 min at 4 °C to remove residual solid lysate. Supernatants were then applied to 28 ml cold PBS before repeating the ultracentrifuge spin (120,000g for 2 h) and clarification spin (16,000g for 15 min) another 2 times. Final pellets were resuspended in 50 µl PBS and PVC yield was quantified by A280 measurement on a NanoDrop instrument (ThermoFisher). For mouse experiments, lipopolysaccharide was then removed from the final PVC samples using a detergent-based method46; in brief, samples were diluted into 1 ml cold PBS and 20 µl of Triton X-114 (Sigma-Aldrich X-114) was added. Samples were then incubated at 4 °C in a tube turner for 30 min, transferred to 37 °C for 10 min to allow the detergent to come out of solution, and spun at 20,000g for 20 min at 37 °C to separate the protein and detergent phases. The upper phase (containing the protein) was extracted and the procedure was repeated 2 more times (that is, Triton X-114 was added 3 times in total) and the final protein phase was incubated with 300 mg Bio-Beads SM-2 (Bio-Rad 1523920) at 4 °C in a tube turner overnight. Protein samples were then extracted from the beads, passed through a 0.2-μm sterile filter (Pall 4612), and concentrated down to 50 µl PBS with a final ultracentrifuge spin; endotoxin levels were then quantified using a Pierce Chromogenic Endotoxin Quant Kit (ThermoFisher A39552). All PVC samples were stored in PBS at 4 °C for a maximum of 1 week prior to use.

Purification of PVC payloads

To determine whether endogenous PVC payloads (Pdp1 and Pnf) produced cytotoxicity independent of the PVC complex, we purified each of these proteins in isolation. Each payload was tagged with an affinity and solubility tag (6×His–Strep–SUMO) and was transformed into E. coli BL21 (DE3) competent cells (Sigma-Aldrich CMC0016). Colonies were inoculated into 5 ml TB medium and shaken at 37 °C for 16 h before being inoculated (at 1:200) into 1 l additional TB. These cultures were then shaken at 37 °C until they reached an A600 of 0.6–0.8, whereupon they were induced with 0.5 mM IPTG (Goldbio I2481C) and shaken at 37 °C for an additional 4 h. Cultures were then spun at 4,000g for 30 min and resuspended in 50 ml cold lysis buffer (50 mM Tris-HCl pH 7.5 (ThermoFisher 15567027), 280 mM NaCl (AmericanBio AB01915), 3 mM KCl (Sigma-Aldrich P9541), 5 mM MgCl2 (Sigma-Aldrich M4880), 1 µl benzonase (Sigma-Aldrich E1014) per 50 ml of buffer, and 1 tablet cOmplete (Sigma-Aldrich 11836170001) per 50 ml of buffer); resuspended cells were stirred for 30 min to ensure a homogenous mixture, and were then twice passed through a Microfluidics M110P microfluidizer. Lysates were then spun at 9,000g for 30 min at 4 °C and supernatants were applied to 2.5 ml of a 50% slurry (in lysis buffer) of Strep-Tactin Superflow Plus resin (Qiagen 30004) and stirred at 4 °C for 30 min. The resin was then pelleted at 2,000 g for 3 min at 4 °C, twice washed with 40 ml lysis buffer, and finally applied to a column (ThermoFisher 29922) and allowed to drain. With the column capped, we next added 12.5 ml of cold elution buffer (25 mM Tris-HCl pH 7.5 (ThermoFisher 15567027), 140 mM NaCl (AmericanBio AB01915), 3 mM KCl (Sigma-Aldrich P9541), 5 mM MgCl2 (Sigma-Aldrich M4880), and 100 µl per column of SUMO protease (a gift from J. Strecker)) and incubated the column overnight at 4 °C to liberate the protein from the resin. The purified protein was then concentrated using a 10 kDa Amicon Ultra filter (Sigma-Aldrich UFC901024), quantified by A280 measurement on a NanoDrop instrument (ThermoFisher), and verified for proper expression and purification by SDS–PAGE followed by Coomassie stain. Raw, uncropped versions of all protein gels can be found in Supplementary Fig. 1.

Payload loading assays

To determine whether a protein was loaded into PVCs, we exploited the tendency of our PVC purification procedure to preferentially purify large molecular weight complexes over free proteins (Extended Data Fig. 3a). Payload proteins (cloned into pPayload) were tagged with C-terminal HiBiT tags and PVC particles containing the tagged payloads were purified. The baseplate of the PVC (encoded by pvc12) was also tagged with HiBiT to serve as a loading control for the western blot. Twenty micrograms of the resulting PVCs (containing loaded payloads) was then mixed with NuPAGE LDS Sample Buffer (ThermoFisher NP0008) and NuPAGE Sample Reducing Agent (ThermoFisher NP0009), both to a final concentration of 1×, and were subsequently boiled at 95 °C for 10 min. The denatured PVC payload samples were then run on NuPAGE Bis-Tris 1–12% protein gels (ThermoFisher NP0321) for 30 min at 200 V in 1× MOPS buffer (ThermoFisher NP000102) and were blotted onto PVDF membranes using an iBlot 2 instrument (ThermoFisher). To visualize low molecular weight payloads (as was done in Extended Data Fig. 3c,d), we instead ran the denatured protein samples on NuPAGE Bis-Tris 12% protein gels (ThermoFisher NP0342) in 1× MES buffer (ThermoFisher B0002). Finally, payload bands were visualized using the Nano-Glo HiBiT blotting system (Promega N2410) and images were captured with a Bio-Rad ChemiDoc instrument. A representative amino acid sequence of a non-native protein loaded via a PVC packaging domain can be found in Supplementary Table 5.

Cell culture

A list of cell lines used in this study can be found in Supplementary Table 8. Cell lines were not authenticated or tested for Mycoplasma prior to use as they were primarily obtained from commercial sources. Unless otherwise stated, mammalian cells were maintained in T75 flasks (ThermoFisher 156499) at 37 °C with 5% CO2 in either DMEM-GlutaMAX (ThermoFisher 10569044) or RPMI-GlutaMAX (ThermoFisher 61870127), and insect cells were gently shaken in 125-ml shaker flasks (Sigma-Aldrich CLS431143) at 28 °C in ESF921 (VWR 100000-000). All media were supplemented with 10 µg ml−1 gentamicin (Sigma-Aldrich G1397) and 1× penicillin-streptomycin (ThermoFisher 15140122); mammalian media were also supplemented with 10% FBS (VWR 97068-085). For growth of primary splenocytes, the medium was also supplemented with mouse IL-2 (Peprotech 212-12) and 50 µM 2-mercaptoethanol (ThermoFisher 21985023).

In vitro PVC delivery experiments

To detect PVC-mediated protein delivery in vitro, target cells were seeded into 96-well clear-bottom 96-well plates (VWR 89091-012) and allowed to grow to about 80% confluence. PVCs were then added to a final concentration of 150 ng µl−1 in 50 µl of cell culture medium per well. For assays involving co-transfection of a Cre reporter plasmid or a guide RNA plasmid, DNA was transfected immediately after adding PVCs using GeneJuice Transfection Reagent (Sigma-Aldrich 70967) for human cells or Insect GeneJuice Transfection Reagent (Sigma-Aldrich 71259) for Sf9 cells. For assays involving transfection of a target receptor (for example, EGFR or surface-displayed anti-epitope tag antibodies in Fig. 3), this was done 24 h prior to addition of PVCs. For toxin delivery experiments, cytotoxicity was assessed using CellTiter-Glo 2.0 Cell Viability Assay (Promega G9241) and/or staining with viability stain (8 ng µl−1 FDA (Sigma-Aldrich F7378) + 20 ng µl−1 PI (Sigma-Aldrich P4170)) and imaging under a Zeiss Observer D1 microscope; these analyses were carried out at t = 24 h for mammalian cells and t = 2 days (CellTiter-Glo)/4 days (FDA/PI stain and imaging) for Sf9 cells. For CellTiter-Glo assays, any wells exhibiting higher luminescence than the control well (PBS) were assigned a cytotoxicity value of 0% to avoid negative cytotoxicity. For assays involving Cre-driven GFP expression, cells were incubated for four days and were then imaged with a Leica DMi8 confocal microscope and analysed with flow cytometry (see ‘Flow cytometry analysis for in vitro PVC experiments’). For gene editing experiments, cells were incubated for 4 days, genomic DNA was extracted with 50 µl QuickExtract DNA Extraction Solution (Lucigen QE09050), and indels or base substitutions were quantified with NGS (see ‘Deep sequencing’). All numerical data from PVC experiments were plotted with Prism (9.3.1) and figures were graphically assembled in Adobe Illustrator (25.2.3).

In silico protein structure prediction

To predict the structure of novel PVC tail fibre designs, we leveraged ColabFold, a Google Colab-based implementation of AlphaFold235,36,37. For all tail fibre designs, sequences were queried as trimers in AlphaFold2_mmseqs2 (v1.2) with default model/MSA settings and num_recycles set to 12. Runs were supported by Google Cloud virtual machines running NVIDIA Tesla A100 GPUs. The resulting structures were visualized and recoloured with PyMOL (2.5.2).

Electron microscopy

Routine negative-stain TEM analysis of purified PVC particles was performed either at the Koch Institute Nanotechnology Materials Core Facility or the MIT Materials Research Laboratory. In brief, 5–10 µl of each PVC sample (diluted to 100–500 ng µl−1) were applied to a glow discharged 200-mesh carbon-coated copper TEM grid (VWR 100489-722) for 60 s before removing excess liquid with a Kimwipe. Grids were then twice treated with 10 µl of 2% uranyl acetate stain (dabbing away the first immediately and the second after 30 s) or 5 times treated with 2% uranyl formate stain (incubating with gentle agitation for 5s, 5 s, 10 s, 30 s and 30 s) and allowed to dry at room temperature. Grids were then imaged in either a (1) JEOL 2100 FEG microscope at 200 kV equipped with a Gatan 2k × 2k UltraScan CCD camera, or a (2) FEI Tecnai (G2 Spirit TWIN) microscope at 120 kV equipped with a Gatan Orius SC1000B camera.

To determine whether PVC particles bind to target cells, we used a modified negative-stain TEM method. A549 cells were allowed to adhere at high density to glow discharged 200-mesh carbon-coated gold TEM grids (VWR 76499-704) in 24-well plates before being exposed to a high dose of PVC sample (1.8 µg µl−1 final concentration) for 3 h. The cells were then fixed for 10 min with 4% paraformaldehyde (Electron Microscopy Sciences 1574), washed once with PBS, 5× stained with 2% uranyl formate (via the same method as above), and allowed to dry at room temperature. The cells were then imaged with a FEI Tecnai (G2 Spirit TWIN) microscope at 120 kV equipped with a Gatan Orius SC1000B camera.

High-resolution imaging of PVC-treated human cells was conducted using scanning electron microscopy (SEM). A549 cells were grown to 80–90% confluence on 12-mm glass coverslips (VWR 354087) in 24-well plates before being exposed to a moderate dose of PVC sample (500 ng µl−1) for 3 h. The cells were then fixed for 1 h with 2.5% glutaraldehyde/2% paraformaldehyde/100 mM sodium cacodylate at 4 °C, rinsed twice with 100 mM sodium cacodylate (each for 5 min at 4 °C), treated with 1% osmium tetroxide/sodium cacodylate for 30 min at 4 °C, rinsed 3–4 times (10 min each) with distilled water, dehydrated with ethanol, treated with 50% TMS/50% ethanol for 15 min, treated with 80% TMS/20% ethanol for 15 min, twice treated with 100% TMS for 5 min each, and allowed to air dry before sputter coating and imaging in a Zeiss Crossbeam 540 SEM/focused ion beam.

Immunofluorescence

To determine whether PVC particles bound to target cells, we tagged an external PVC protein (Pvc2) with an N-terminal Flag tag and exposed the resulting PVC particles (at 300 ng µl−1) to target cells for 3 h at 37 °C. The cells were then fixed for 10 min with 4% paraformaldehyde (Electron Microscopy Sciences 1574), blocked for 1 h with blocking buffer (10% goat serum (Sigma-Aldrich G9023) and 0.1% Triton X-100 (Sigma-Aldrich 93443) diluted in PBS), stained for 1 h with M2 anti-Flag antibody (Sigma-Aldrich F1804; diluted 1:500 in blocking buffer), stained for 1 hr with an Alexa Fluor 488-conjugated secondary antibody (ThermoFisher A11001; diluted 1:1,000 in blocking buffer), stained for 10 min with 1 µg ml−1 DAPI (ThermoFisher D1306; diluted in PBS), and imaged using a Leica DMi8 confocal microscope. An amino acid sequence depicting the position of the Flag tag on Pvc2 can be found in Supplementary Table 6.

We also used immunofluorescence to examine the effect of PVCs on the cytoskeleton (Extended Data Fig. 6a). Target cells were first seeded into 96-well plates and allowed to grow to about 80% confluence before being exposed to PVCs (150 ng µl−1 final concentration) for 24 h. The cells were then fixed for 10 min with 4% paraformaldehyde, blocked for 1 h with blocking buffer, stained for 1 h with rhodamine phalloidin (ThermoFisher R415; diluted to 1× final concentration in blocking buffer), stained for 10 min with 1 µg ml−1 DAPI (ThermoFisher D1306; diluted in PBS), and imaged using a Leica DMi8 confocal microscope.

Flow cytometry analysis for in vitro PVC experiments

For experiments involving PVC-mediated delivery of Cre, we measured delivery efficiency using flow cytometry. Cells were first harvested by incubation with TrypLE Express dissociation reagent (ThermoFisher 12604), pelleted at 300g for 3 min, and resuspended in 100 µl of flow cytometry buffer (PBS supplemented with 2% EDTA (Life Technologies 15575020) and 5% FBS (VWR 97068-085)). Samples were run on a Beckman Coulter Cytoflex S flow cytometer, and analysis was performed using CytExpert (2.3.1.22) and FlowJo (10.8.2). A representative scheme for gating and threshold setting is shown in Extended Data Fig. 4c.

Deep sequencing

To detect PVC-induced genomic edits in target cells, we first amplified the target region out of each genomic DNA extract (see ‘In vitro PVC delivery experiments’) with NEBNext High-Fidelity 2× PCR Master Mix (NEB M0541). Target regions were then barcoded with indexed Illumina P5 and P7 NGS primers. Libraries were purified with a Qiagen PCR Purification Kit (Qiagen 28104), quantified on a NanoDrop instrument (ThermoFisher), and sequenced on an Illumina MiSeq instrument (with read length set to 300 bp). Indels and base substitutions were then quantified with Geneious Prime (2020.0.5). Primers used for deep sequencing can be found in Supplementary Table 9.

Quantitative PCR

To assess the effect of regulatory genes on PVC gene expression, we used quantitative reverse transcription PCR (RT-qPCR). E. coli EPI300 cells were electroporated with one variant each of pPVC and pPayload (as described in ‘PVC purification’) and colonies were shaken in 5 ml TB at 37 °C for 16 h. The cultures were then spun for 5 min at 4,000g, resuspended in 750 µl TRI reagent (Zymo R2073), incubated at room temperature for 5 min, and mechanically lysed by vortexing (1 min) with 250 µl of 0.5 mm Zirconia beads (Fisher NC0450473). We then added 200 µl chloroform, incubated at room temperature for 3 min, spun for 15 min at 12,000g (4 °C), and extracted the aqueous phase for RNA extraction via a Zymo Direct-zol RNA Miniprep Kit (Zymo R2073) with the optional DNAse step. We then generated cDNA from these bulk RNA extracts using ProtoScript II Reverse Transcriptase (NEB M0368) and random primers (NEB S1330) with the manufacturer’s protocol. Finally, we ran qPCR on the resulting cDNAs using Fast SYBR Green Master Mix (ThermoFisher 4385612) in a Bio-Rad CFX Opus 384 qPCR instrument. Delta-delta Ct values were computed against the housekeeping gene gapA47. Primers used for qPCR can be found in Supplementary Table 10.

Mass spectrometry

PVCs were diluted to about 36 μg µl−1 in PBS before being sent to the Koch Institute Biopolymers and Proteomics Facility for analysis by mass spectrometry. In brief, proteins were reduced with 10 mM dithiothreitol (Sigma-Aldrich 11583786001) for 10 min at 95 °C and then alkylated with 20 mM iodoacetamide (Sigma-Aldrich I5161) for 30 min at 25 °C in the dark. Proteins were then digested with trypsin on S-Trap micro columns (ProtiFi C02-micro-80) per the manufacturer’s protocol. The tryptic peptides were separated by reverse-phase HPLC (Thermo UltiMate 3000) using a PepMap RSLC C18 column and a 2 μm EASY-Spray tip (ThermoFisher ES903) over a 90-min gradient before being subjected to nano-electrospray using an Exploris mass spectrometer (Thermo). The resulting mapped peptide hits can be found in Supplementary Data 2.

Intracranial injections

All mouse experiments conformed to guidelines established by the National Institutes of Health and were conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Broad Institute of MIT and Harvard. Animals were chosen randomly for treatment with either control or experimental conditions without blinding. Female Ai9 mice (aged 8–12 weeks) were obtained from the Jackson Laboratory (strain 007909). All mice were maintained on a 12-h light:dark cycle with ad libitum access to food and water. Mice were anaesthetized using isoflurane (2–3%) and prepared for stereotaxic surgery; fur was shaved, and mice were placed in a stereotaxic frame (Kopf Instruments). A heating pad was placed under the mice to prevent hypothermia. Isoflurane (1–2%) was delivered via a nose cone throughout the surgery. Ophthalmic ointment was used to protect the eyes. Buprenorphine -SR (1 mg kg−1, subcutaneous) was given before the start of surgery. Bupivacaine (1 mg kg−1) was injected intradermally along the incision line as a form of local anaesthetic. Meloxicam (2 mg kg−1) was also administered subcutaneously prior to surgery. The scalp was disinfected with betadine scrub and 70% ethanol. An incision was made using a scalpel along the scalp midline. The exposed skull was thoroughly cleaned, and a craniotomy was made above the hippocampus. PVC proteins were targeted to the hippocampus (−2.3 AP, 1.25 ML, −3 & −1.5 DV), and slowly pressure-injected (100 nl min−1) using a 10 µl Hamilton syringe (700 Series Microliter Syringes, Hamilton, Model 701 N Syringe) and a micro-syringe pump controller (Micro 4; WPI). After injection, the needle was left in place for 2 min and then slowly withdrawn. A total of 1,000 nl (Fig. 4c; 500 nl at −2.0 DV and 500 nl at −1.5 DV) at 7.5 µg µl−1 or 3,000 nl (Fig. 4d–f and Extended Data Fig. 8b,e,f; 1,500 nl at −3.0 DV and 1,500 nl at −1.5 DV) at 1.2 µg µl−1 of PVC sample was injected per mouse. After injection, the skin was sealed with a simple, continuous suture pattern with 4-0 Ethilon nylon sutures. Incisions were swabbed clean with 0.9% sterile saline and sterile cotton tip applicators. Mice were postoperatively hydrated with saline and housed in a temperature-controlled environment until achieving an ambulatory recovery. To relieve post-operative pain, meloxicam (2 mg kg−1) was administered subcutaneously every 24 h up to a minimum of 72 h post-surgery.

Imaging of mouse brain sections

At t = 12 days post-injection, mice were deeply anaesthetized with Fatal-Plus at a dose of 90 mg kg−1 and transcardially perfused with 20 ml of PBS, followed by 20 ml of 4% paraformaldehyde solution. Brains were quickly extracted and stored in 4% paraformaldehyde solution at 4 °C for 24 h, and were then transferred to 30% sucrose in PBS solution and allowed to equilibrate for 2 days. Brains were then mounted on a cryostat using OCT and sectioned coronally (50 µm). The floating sections were washed in PBS and stained for neurons using anti-NeuN antibody (Sigma-Aldrich MAB377; 1:500) and Alexa 488 secondary antibody (ThermoFisher A11001; 1:1,000). The sections were mounted on slides with PVA-DABCO. Images were acquired using a Leica DMi8 confocal microscope with a 10× and 20× air objective.

Isolation and flow cytometry of PVC-injected neurons

Animals were deeply anaesthetized after t = 1, 3 and 7 days with CO2 and transcardially perfused with 20 ml of PBS. Brains of PVC- or mock-injected mice were extracted, and targeted hemispheres were cut into pieces using scalpels and digested with 50 µg ml−1 liberase (Sigma-Aldrich 05401119001) at 37 °C for 30 min. Single-cell suspensions were generated using slow repetitive pipetting. Myelin was manually removed using Myelin Removal Beads II, human, mouse, rat (Miltenyi Biotec 130-096-733) and LS columns (Miltenyi Biotec 130-042-401) followed by enrichment of neuronal cells using the adult neuron isolation kit (Miltenyi Biotec 130-126-603) and LS columns. Enriched cell populations were fixed using Cytofix Fixation Buffer (BD 554655) at 4 °C for 30 min and blocked with 1:50 TruStain FcX (anti-mouse CD16/32) reagent (BioLegend 101320) prior to antibody staining for flow cytometry; antibodies and dilutions can be found in Supplementary Table 12.

Isolation and culture of mouse primary neurons for in vitro PVC targeting

Ninety-six-well plates were coated with 0.05 mg ml−1 poly-d-lysine (BD 354210) one day prior to isolation. A dissection solution was made using HBSS (ThermoFisher 14025092) supplemented with 10 mM HEPES (ThermoFisher 15630080), 33 mM d-glucose (Sigma-Aldrich G8270) and 43 mM sucrose (Sigma-Aldrich S0389). Timed-pregnant female C57BL/6J mice (aged 12 weeks) were killed according to the standard operating procedures of the Institutional Animal Care and Use Committees (IACUC) of the Broad Institute of MIT and Harvard. Brains were extracted from embryos at embryonic day 16.5 and dissected in dissection solution. Pan-cortex tissue was used for downstream neuron isolation. Tissues were digested using TrypLE Select (ThermoFisher 12563011) for 30 min and washed twice in dissection solution supplemented with trypsin inhibitor (Sigma-Aldrich T9253) and BSA (Sigma-Aldrich A9418). Single-cell suspension was prepared by repetitive trituration and cells were cultured in Neurobasal-A Medium (ThermoFisher 10888022) supplemented with B-27 Plus Supplement (ThermoFisher A3582801).

Assessment of in vivo CNS inflammation

Isolation of CNS-infiltrating myeloid and T cells was performed as previously described48. In brief, mice were deeply anaesthetized after t = 1, 3 and 7 days with CO2 and transcardially perfused with 20 ml of PBS. Brains of PVC- or mock-injected mice were extracted, and targeted hemispheres were cut into pieces using scalpels and digested with 50 µg ml−1 liberase (Sigma-Aldrich 05401119001) at 37 °C for 30 min and subsequently mashed through 100-µm and 70-µm cell strainers (Greiner One-Bio 542000 and 542070). Myelin was removed using a 30% continuous Percoll (Sigma-Aldrich GE17-0891-01) gradient and density centrifugation at 2,700 rpm. Following myelin removal, single-cell suspension of brain-infiltrating immune cells were prepared in PBS and blocked with 1:50 TruStain FcX (anti-mouse CD16/32) reagent (BioLegend 101320) prior to antibody staining for flow cytometry. DAPI staining solution (Miltenyi Biotec 130-111-570) was added at 1:100 dilution immediately prior to flow cytometry analysis to discriminate live cells. Interstitial fluid surrounding the parenchymal cells of the brain was isolated by washout of minced brain tissue at indicated post-injection timepoints in PBS and centrifugation at 500g. Cytokine ELISAs for interleukin-1β (IL-1β), interleukin-6 (IL-6), interferon-γ (IFN-γ) and tumour necrosis factor (TNF) were performed according to the manufacturer’s protocol (Invitrogen 88-7013-22, 88-7064-22, 88-7314-22 and 88-7324-22, respectively) and absorbance at 450–570 nm was measured. Cytokine concentrations were calculated corresponding to diluted standards as per the manufacturer’s protocol.

In vivo PVC clearance assay

To study the persistence of PVCs in the mouse brain, interstitial fluid was isolated from brain homogenates. In brief, PVC-treated mice were euthanized and transcardiac perfusion with PBS was performed prior to extraction of full intact brains. Brain tissue was mechanically dissociated using sterile scalpels followed by dounce homogenization into single-cell suspensions. These single-cell suspensions were centrifuged at 500 g for 5 min, and the clarified supernatants were diluted into 28 ml PBS and ultracentrifuged at 120,000g for 2 h at 4 °C to pellet any intact PVC protein complexes. Pellets were resuspended in 50 µl PBS and spun at 16,000g for 15 min at 4 °C to remove residual bulk homogenate. Finally, we analysed the resuspensions with negative-stain TEM to detect intact PVC complexes; see ‘Electron microscopy’.

Statistics and reproducibility

All statistical analyses were performed in Prism (9.3.1). Quantitative data are presented as mean ± s.d. with n = 2–4 biological replicates per condition; the number of replicates presented are listed in the figure legends. Unless otherwise stated, biological replicates represent independent treatments in separate culture wells or mice. All micrographs, gels, and blots are representative images from at least n = 3 independent experiments. Statistical significance was computed using one-way or two-way ANOVA followed by Bonferroni post hoc tests (to correct for multiple comparisons), as indicated in the figure legends. P values below 0.05 were considered statistically significant; the results of all statistical tests (including P values) are included in the Source Data alongside the associated source data for each figure panel.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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