Name: In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination (Video) | JoVE Journal
Uploaded: 2026-01-02T12:50:00.000+00:00
Duration: 7 min 58 s
Description: Learn to reconstitute bacterial ubiquitination and VCP/p97-mediated elimination using Streptococcus pneumoniae and mammalian cell lysate. This method reveals cytosolic defense mechanisms against pathogens.

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5 minutes

We express our gratitude to the Bio-safety Level 2 Facility and Confocal Microscopy Facility at IIT Bombay. Sourav Ghosh. (17/06/2018(i) EU-V), Sumit Rakshit (221610197857) and Udit Kumar Das (24D0061) have received fellowships from CSIR, UGC, Government of India, and IIT Bombay, respectively. Anirban Banerjee has received research funding from the Science and Engineering Research Board, Government of India (Grant Number SPR/2019/000808) and Ignite Life Science Foundation (Grant Number IGNITE/FG-OC/2021/004). The funding bodies played no part in the study&#39;s design, data collection and analysis, decision to publish, or in the preparation of the manuscript.

1. Cell culture and lysate preparation
<ol>
	<li>Thaw the A549 cells and grow them in a T25 flask using complete DMEM with 10% fetal bovine serum (FBS). Incubate at 37 &#176;C in a humidified environment with 5% CO2. Pass the cells through three passages for maintenance, then transfer 2 &#215; 106 cells into a T75 flask. Continue the culture until the cell density reaches 8-10 &#215; 106 cells.</li>
	<li>Scrape the cells from the T75 flask at 4 &#176;C using a reaction buffer that contains 200 mM HEPES, 100 mM MgCl2, 1 M KCl, and 5% glycerol at pH 7.4. Centrifuge the scraped cells at 120 &#215; g for 10 min to pellet them, then resuspend in the reaction buffer to achieve a final volume of approximately 0.6 mL in a 1.5 mL centrifuge tube. 
	NOTE: The entire sample must be processed in ice.</li>
	<li>Sonicate the cell suspension using a narrow probe (diameter tip, 2 mm) at an amplitude of 40 &#956;m with 2 s on/off pulses for a duration of 2-2.5 min, keeping the mixture on ice. Centrifuge the sonicated samples at 21,000 &#215; g for 45 min, collect the resulting supernatant, and add 2-3x of 50x protease inhibitor cocktail. 
	NOTE: Either use the lysate immediately for the ubiquitination reaction on the same day or flash freeze it using liquid nitrogen and store it at -80 &#176;C; however, make sure to use it within 3 days at most for effective ubiquitination. Lysate concentration ranging from 2 to 4 mg/mL is suitable for ubiquitination.</li>
</ol>
2. Bacterial culture
<ol>
	<li>Grow the bacteria, Streptococcus pneumoniae (SPN) R6 strain (Serotype II), overnight at 37 &#176;C with 5% CO2 in a THY medium made up of Todd Hewitt broth powder (3.7 g/100 mL) and yeast extract powder (1.5 g/100 mL). 
	NOTE: Avoid inoculating the bacteria if the medium has changed to a dark brown color. Streptococcus pneumoniae is classified as a Biosafety Level 2 (BSL-2) pathogen; take precautions and follow the correct BSL-2 containment protocols, including the use of a certified biosafety cabinet.</li>
	<li>Next day, inoculate from the overnight culture into fresh THY media (1:10) at 37 &#176;C with 5% CO2 and grow the SPN till the Optical Density at 600 nm (OD600 nm) reaches 0.4. Prepare a glycerol stock of SPN (900 &#181;L of SPN culture and 600 &#181;L of Glycerol (50%) and store it in -80 &#176;C.</li>
	<li>On the day of the ubiquitination experiment, thaw a new stock of SPN from -80 &#176;C and completely inoculate it into a 5 mL of THY medium and allow it to grow until OD600 nm reaches 0.4. 
	NOTE: The bacterial culture was grown to an OD600 nm of 0.4, which corresponds to approximately 108 SPN cells per mL. This correlation was established by enumerating colony-forming units (CFUs), determined by serially diluting the culture in 1x PBS to decrease the bacterial cell concentration so that countable colonies can be obtained and spotting on BHI agar plates, a more enriched medium than THY.</li>
</ol>
3. In-vitro ubiquitination assay from mammalian lysate
<ol>
	<li>Take approximately 107 SPN cells and wash thoroughly at least three times with the reaction buffer (200 mM HEPES, 100 mM MgCl2, 1 M KCl, and 5% glycerol at pH 7.4) by resuspending the bacteria in the following buffer, followed by centrifugation at 21,000 &#215; g for 5 min, finally pelleting them down via centrifugation at 21,000 &#215; g for 5 min at room temperature.</li>
	<li>Resuspend the SPN pellet with A549 mammalian cell lysate along with purified ubiquitin (350 &#181;M) and 1 mM ATP and incubate for 1-2 h at 27 &#176;C. 
	NOTE: Avoid freezing and thawing the mammalian cell lysate. Best if the lysate is prepared on the same day of the ubiquitination assay.</li>
	<li>Following incubation, wash the bacteria with reaction buffer containing 1 M Urea once, followed by two washes with only reaction buffer, followed by fixation with 1% paraformaldehyde (PFA). After fixation, incubate cells with PBS containing glycine (1 mg/mL) for 15 min, followed by two 1x PBS washes. 
	NOTE: A 1 M urea wash is used to mildly disrupt noncovalent interactions, ensuring that only covalently ubiquitinated bacteria remain. Afterward, it is important to wash thoroughly with reaction buffer to remove residual urea, as its presence can interfere with the in-vitro killing assay. 
	As a negative control, prepare the entire reaction mixture and incubate it with bacteria on ice to inhibit enzymatic activity. Right after the incubation, rinse the sample and then fix it using 1% PFA to maintain the reaction&#39;s condition.</li>
</ol>
4. In-vitro ubiquitination assay from the E1-E2-E3 enzyme complex
<ol>
	<li>Prepare a reaction mixture first consisting of ubiquitin (350 &#181;M), UBE1 (400-800 nM), UbE2C (3-5 &#181;M), Rbx1-Skp1-Cul1-Fbxw7 complex (500 nM) and 2 mM ATP in reaction buffer (200 mM HEPES, 100 mM MgCl2, 1 M KCl, and 5% glycerol at pH 7.4), to make the total reaction volume of 100-150 &#181;L. 
	NOTE: Store all enzymes at -80 &#176;C. To avoid losing activity from multiple freeze-thaw cycles, create small aliquots and utilize new ones for each ubiquitination reaction.</li>
	<li>Resuspend the bacteria (107 CFU) in this ubiquitin enzyme-containing reaction mixture and incubate for 2 h at 27 &#176;C. Subsequently, wash the bacteria with reaction buffer containing 1 M urea for at least 3x, followed by fixation with 1% paraformaldehyde (PFA). After fixation, incubate cells with PBS containing glycine (1 mg/mL) for 15 min, followed by two 1x PBS washes. 
	NOTE: If there is excessive cell loss, one can reduce the number of washes.</li>
</ol>
5. Visualization of ubiquitinated bacteria
<ol>
	<li>After fixing the sample with 1% PFA, incubate it in 3% BSA for 1 h at 27 &#176;C, then perform two washes with 1x PBS.</li>
	<li>Resuspend the ubiquitinated bacteria using a primary antibody at concentrations higher than those typically utilized in immunofluorescence assays. Use a bacteria-specific antibody along with an anti-ubiquitin antibody (1:100), diluted in PBS containing 1% BSA, for staining at 27 &#176;C for 1 h. 
	NOTE: For optimal antibody staining, use a low protein-binding centrifuge tube to minimize the antibody loss or use a higher antibody concentration to get the best results.</li>
	<li>Subsequently, wash the bacteria twice with 1x PBS and then resuspend them with the corresponding secondary antibody conjugated with Alexa Fluor 488 and 555, respectively, for 1 h at 27 &#176;C.</li>
	<li>Apply around 10-20 &#181;L of the labeled bacterial sample by drop casting and mix in 5-10 &#181;L of mounting medium with or without DAPI, then place a coverslip over it and use tissue paper to absorb any excess liquid. Finally, observe the sample under a confocal microscope on the same day.</li>
</ol>
6. Protein expression and purification
<ol>
	<li>For the expression of wild-type VCP/p97, transform the VCP-containing pET28a plasmid into E. coli BL21-DE3 strain via a standard E. coli transformation protocol20. Incubate colonies formed after the transformation overnight in Luria broth (LB) at 37 &#176;C. The following day, inoculate 1:50 culture into a well-aerated flask containing LB and allow it to grow until OD600 nm reaches 0.7-0.8. Add 200 &#181;M of IPTG to the culture and incubate for 5 h at 37 &#176;C with shaking at 180 rpm; confirm the protein expression using an SDS-PAGE. 
	NOTE: For the entire bacterial growth and expression step, Kanamycin (50 &#181;g/mL) was used as selection pressure.</li>
	<li>For the expression of wild-type UFD1, transform the UFD1-containing pET41b plasmid into E. coli BL21-Rosetta strain via standard E. coli transformation protocol20. Incubate colonies formed after the transformation overnight in Luria broth (LB) at 37 &#176;C. The following day, inoculate 1:50 culture into a well-aerated flask containing LB and allow it to grow until OD600 nm reaches 0.7-0.8. Add 1 mM of IPTG to the culture and incubate for 5 h at 37 &#176;C with shaking at 180 rpm; confirm the protein expression using an SDS-PAGE.</li>
	<li>To express wild-type NPLOC4, introduce the NPLOC4-containing pET41b plasmid into E. coli BL21-Rosetta strain using the standard E. coli transformation method20. Incubate the colonies that arise from the transformation in Luria broth (LB) overnight at 37 &#176;C. The next day, transfer a 1:50 dilution of the culture into a well-aerated flask filled with LB, and let it grow until the optical density at OD600 nm reaches 0.7-0.8. After that, add 1 mM of IPTG to the culture and continue incubation for 16 h at 18 &#176;C while shaking at 180 rpm; verify the protein expression by performing an SDS-PAGE analysis. 
	NOTE: For the entire bacterial growth and expression step done in E. coli BL21-Rosetta strain, Kanamycin (50 &#181;g/mL) and Chloramphenicol (20 &#181;g/mL) were used as selection pressure. Sequences of pET28a-p97 and pET41b-NPLOC4 is provided in Supplemental File 1.</li>
	<li>After protein expression, centrifuge the bacterial cells at 21,000 &#215; g for 10 min and resuspend the pellet in lysis buffer containing 50 mM Tris-Cl, 300 mM NaCl, 5 mM &#946;-mercaptoethanol, 1 mM PMSF, 5% glycerol, pH 8.</li>
	<li>Sonicate the cells for 1 h using a narrow probe (tip diameter, 6 mm) at an amplitude of 50 &#956;m, and with a pulse of 20 sec on/off at 4 &#176;C. Pellet down the debris using 15,000 &#215; g for 1 h and collect the supernatant. 
	NOTE: Total sonication was carried out for 1 h (30 min ON and 30 min OFF as 20 s pulses), and the process was continued until the turbid cell suspension turned translucent to transparent. High-speed centrifugation is required to ensure that cell debris remains pelleted and does not dislodge during loading of the lysate onto the protein purification column.</li>
	<li>Pass the supernatant through a 0.4 &#181;m filter and load it onto the column packed with Ni-NTA beads for affinity purification, pre-equilibrated with purification buffer (50 mM Tris-Cl, 300 mM NaCl, 5 mM &#946;-mercaptoethanol, 5% glycerol, pH 8).</li>
	<li>Rinse the protein-rich column using wash buffer that contains 50 mM Tris-Cl, 300 mM NaCl, 5 mM &#946;-mercaptoethanol, 5% glycerol, pH 8, and 30-60 mM imidazole of approximately 400 mL, and then elute the protein with an elution buffer that includes 50 mM Tris-Cl, 300 mM NaCl, 5 mM &#946;-mercaptoethanol, 5% glycerol, pH 8, and 250 mM imidazole. 
	NOTE: The concentration of imidazole and the volume of wash may differ based on the specific protein being analyzed. To evaluate purity, SDS-PAGE should be performed. It is essential to wash the column thoroughly until the eluent shows a negative reaction with Bradford reagent. The eluted fraction displays a prominent, concentrated band of the target protein, which should predominantly represent the sample, with only minor traces, if any, of other proteins.</li>
	<li>Gather various fractions, analyze them with SDS PAGE to identify which fractions contain the pure proteins, and then perform a buffer exchange into the reaction buffer (200 mM HEPES, 100 mM MgCl2, 1 M KCl, and 5% glycerol at pH 7.4) utilizing a PD-10 column. 
	NOTE: Measure the protein concentration using Bradford Reagent (Coomassie Brilliant Blue G-250). All the proteins should be flash frozen in liquid nitrogen and stored at -80 &#176;C.</li>
</ol>
7. In-vitro bacterial killing assay
<ol>
	<li>Combine 6 &#181;M of VCP/p97 with 1 &#181;M each of NPLOC4 and UFD1, then fill the volume with reaction buffer to approximately 90 &#181;L. Allow this reaction mixture to incubate on ice for 2 h.</li>
	<li>Add 1-2 mM of ATP into the reaction mixture that includes p97-UFD1-NPLOC4, and then combine it with the ubiquitinated SPN (107 cells) for a duration of 2 h at 37 &#176;C.</li>
	<li>To evaluate bacterial viability, immediately following treatment, take a 1&#8239;&#181;l aliquot of the reaction mixture, carry out a serial dilution (1:1, 1:10, 1:100, and 1:1000), and spot 10-2&#8239;&#181;L of each dilution onto Brain Heart Infusion (BHI) agar plates. Repeat this procedure using a 1&#8239;&#181;L aliquot from the reaction after &#8239;2 h of incubation. 
	NOTE: Spot all the dilutions for better results.</li>
	<li>Assess the colony-forming units (CFUs) between the &#8239;0 h and 2&#8239;h time points. Calculate bacterial viability (%) by dividing the CFU count at 2&#8239;h by the CFU count at 0&#8239;h, then multiplying the result by 100. 
	NOTE: To achieve more reliable results, utilize heat-denatured VCP/p97, VCP/p97 lacking the UFD1-NPLOC4 cofactor complex, or an ATPase-deficient variant of VCP/p97 for comparison as a negative control.</li>
</ol>

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<li>Ciechanover, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteolysis%3A+from+the+lysosome+to+ubiquitin+and+the+proteasome%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Proteolysis: from the lysosome to ubiquitin and the proteasome.</a> Nat Rev Mol Cell Biol. 6 (1), 79-87 (2005).</li>
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<li>Kikkert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+HRD1+is+an+E3+ubiquitin+ligase+involved+in+degradation+of+proteins+from+the+endoplasmic+reticulum%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Human HRD1 is an E3 ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum.</a> J Biol Chem. 279 (5), 3525-3534 (2004).</li>
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<li>Asmat, T. M., Agarwal, V., Saleh, M., Hammerschmidt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endocytosis+of+Streptococcus+pneumoniae+via+the+polymeric+immunoglobulin+receptor+of+epithelial+cells+relies+on+clathrin-+and+caveolin-dependent+mechanisms%5BTitle%5D)+AND+%22Int+J+Med+Microbiol%22%5BJournal%5D)">Endocytosis of Streptococcus pneumoniae via the polymeric immunoglobulin receptor of epithelial cells relies on clathrin- and caveolin-dependent mechanisms.</a> Int J Med Microbiol. 304 (8), 1233-1246 (2014).</li>
<li>Ercoli, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracellular+replication+of+Streptococcus+pneumoniae+inside+splenic+macrophages+serves+as+a+reservoir+for+septicaemia%5BTitle%5D)+AND+%22Nat+Microbiol%22%5BJournal%5D)">Intracellular replication of Streptococcus pneumoniae inside splenic macrophages serves as a reservoir for septicaemia.</a> Nat Microbiol. 3 (5), 600-610 (2018).</li>
<li>Chakravortty, D., Rohde, M., Jäger, L., Deiwick, J., Hensel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Formation+of+a+novel+surface+structure+encoded+by+Salmonella+pathogenicity+island+2%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">Formation of a novel surface structure encoded by Salmonella pathogenicity island 2.</a> EMBO J. 24 (11), 2043-2052 (2005).</li>
<li>Surve, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterogeneity+in+pneumolysin+expression+governs+the+fate+of+Streptococcus+pneumoniae+during+blood-brain+barrier+trafficking%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">Heterogeneity in pneumolysin expression governs the fate of Streptococcus pneumoniae during blood-brain barrier trafficking.</a> PLoS Pathog. 14 (7), e1007168(2018).</li>
<li>Franco, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ubiquitin+ligase+SMURF1+functions+in+selective+autophagy+of+Mycobacterium+tuberculosis+and+anti-tuberculous+host+defense%5BTitle%5D)+AND+%22Cell+Host+Microbe%22%5BJournal%5D)">The ubiquitin ligase SMURF1 functions in selective autophagy of Mycobacterium tuberculosis and anti-tuberculous host defense.</a> Cell Host Microbe. 22 (3), 421-423 (2017).</li>
<li>Manzanillo, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ubiquitin+ligase+Parkin+mediates+resistance+to+intracellular+pathogens%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The ubiquitin ligase Parkin mediates resistance to intracellular pathogens.</a> Nature. 501 (7468), 512-516 (2013).</li>
<li>Polajnar, M., Dietz, M. S., Heilemann, M., Behrends, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expanding+the+host+cell+ubiquitylation+machinery+targeting+cytosolic+Salmonella%5BTitle%5D)+AND+%22EMBO+Rep%22%5BJournal%5D)">Expanding the host cell ubiquitylation machinery targeting cytosolic Salmonella.</a> EMBO Rep. 18 (9), 1572-1585 (2017).</li>
<li>Huett, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+LRR+and+RING+domain+protein+LRSAM1+is+an+E3+ligase+crucial+for+ubiquitin-dependent+autophagy+of+intracellular+Salmonella+typhimurium%5BTitle%5D)+AND+%22Cell+Host+Microbe%22%5BJournal%5D)">The LRR and RING domain protein LRSAM1 is an E3 ligase crucial for ubiquitin-dependent autophagy of intracellular Salmonella typhimurium.</a> Cell Host Microbe. 12 (6), 778-790 (2012).</li>
<li>Otten, E. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ubiquitylation+of+lipopolysaccharide+by+RNF213+during+bacterial+infection%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Ubiquitylation of lipopolysaccharide by RNF213 during bacterial infection.</a> Nature. 594 (7861), 111-116 (2021).</li>
<li>Yamada, A., Hikichi, M., Nozawa, T., Nakagawa, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FBXO2%2FSCF+ubiquitin+ligase+complex+directs+xenophagy+through+recognizing+bacterial+surface+glycan%5BTitle%5D)+AND+%22EMBO+Rep%22%5BJournal%5D)">FBXO2/SCF ubiquitin ligase complex directs xenophagy through recognizing bacterial surface glycan.</a> EMBO Rep. 22 (11), e52584(2021).</li>
<li>Apte, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+innate+pathogen+sensing+strategy+involving+ubiquitination+of+bacterial+surface+proteins%5BTitle%5D)+AND+%22Sci+Adv%22%5BJournal%5D)">An innate pathogen sensing strategy involving ubiquitination of bacterial surface proteins.</a> Sci Adv. 9 (12), eade1851(2023).</li>
<li>Ghosh, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Host+AAA-ATPase+VCP%2Fp97+lyses+ubiquitinated+intracellular+bacteria+as+an+innate+antimicrobial+defence%5BTitle%5D)+AND+%22Nat+Microbiol%22%5BJournal%5D)">Host AAA-ATPase VCP/p97 lyses ubiquitinated intracellular bacteria as an innate antimicrobial defence.</a> Nat Microbiol. , (2025).</li>
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<li>Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studies+on+transformation+of+Escherichia+coli+with+plasmids%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">Studies on transformation of Escherichia coli with plasmids.</a> J Mol Biol. 166 (4), 557-580 (1983).</li>
<li>Blythe, E. E., Olson, K. C., Chau, V., Deshaies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ubiquitin-+and+ATP-dependent+unfoldase+activity+of+p97%2FVCP-NPLoc4-UFD1L+is+enhanced+by+a+mutation+that+causes+multisystem+proteinopathy%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Ubiquitin- and ATP-dependent unfoldase activity of p97/VCP-NPLoc4-UFD1L is enhanced by a mutation that causes multisystem proteinopathy.</a> Proc Natl Acad Sci U S A. 114 (22), E4380-E4388 (2017).</li>
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</ol>

The authors have no conflicts of interest to declare.

While the in-vitro reconstitution system detailed here offers a robust framework to explore the molecular intricacies of bacterial ubiquitination and elimination, several significant limitations must be considered. The reliance on supraphysiological concentrations of purified proteins, comprising the ubiquitination machinery (E1, E2, E3 ligases, ubiquitin) and the host effector complex (VCP/p97, UFD1, NPLOC4), offers key mechanistic insights into pathogen ubiquitination and clearance, but may not truly reflect t...

Ubiquitin (Ub) is a small 8.6 kDa protein that modifies target substrates by attaching covalently to them at its C-terminal glycine, creating an isopeptide bond with the &#949;-amino group of lysine residues in a process called ubiquitination1. This phenomenon is governed by a series of orchestrated steps involving an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase1,2,3. Ub can be attached as a monomer or as chains of different lengths through its seven lysine residues (Linear/M1, K6, K11, K27, K29, K33, K48, and K63)4. These unique Ub chains act as molecular markers for various cellular functions, such as proteasomal degradation, cellular trafficking, DNA repair, and selective autophagy3.
While the canonical role of ubiquitination has long been associated with maintaining cellular homeostasis, such as degrading misfolded proteins via proteasomes or directing specific cargoes towards selective autophagy5,6, it is now evident that ubiquitin also functions as a crucial sensing mechanism of the cytosolic immune system against bacterial infections. Intracellular bacteria frequently infiltrate host cells to avoid detection by the extracellular immune response and reproduce within a safeguarded environment7,8. Quite a few pathogens employ secretory systems and toxins to irreparably damage the vacuoles carrying them, enabling escape into the cytosol to obtain nutrients9,10. Once in the cytosol, these bacteria are decorated with polyubiquitin chains by specific E3 ligases, acting as a molecular &#34;death tag&#34;, which directs them towards selective elimination. For example, the E3 ligase SMURF1 engages Mycobacterium tuberculosis by initiating K48-linked polyubiquitination directly on its surface, which leads to its degradation via the proteasome11. Parkin accumulates in vacuoles containing bacteria and forms K63-linked ubiquitin chains12, while ARIH1 and LRSAM1 modify the surfaces of bacteria with K48 and K6 chains, respectively, thereby inhibiting bacterial growth13,14. While the majority of E3 ligases primarily target host protein substrates, some ligases display notable specificity for microbial components. RNF213 identifies and ubiquitinates lipopolysaccharides (LPS) found on Salmonella enterica serovar Typhimurium15, and the SCFFBXO2 complex recognizes bacterial glycans present in Group A Streptococcus as a substrate for ubiquitination16, facilitating xenophagy. SCFFBXW7 complex, on the other hand, recognizes degron motifs within surface proteins of Streptococcus pneumoniae (SPN). These include &#946;-galactosidase A (BgaA) and Pneumococcal surface protein A (PspA), which, following tagging with K48-linked polyubiquitin chains, were confirmed using K48-linked Ub-specific antibody, presumably attract the proteasomal system, allowing for effective bacterial elimination17.
Although the process of labelling these cytosolic invaders with ubiquitin is well understood, the subsequent mechanism by which the proteasomal system removes such large microbial entities remains doubtful. Recent research has revealed that the host AAA+ ATPase, VCP/p97 (Valosin-containing protein), plays a crucial role in this process. In collaboration with cofactors like UFD1 and NPLOC4, VCP can identify bacteria marked with polyubiquitin and physically extracts ubiquitinated proteins from the bacterial membrane, thus rupturing them and aiding in their elimination from the cytosolic milieu without depending on the proteasomal machinery18.
The host cytosol contains a complex network of immune pathways that function in parallel and in a fail-safe manner to guarantee the removal of pathogens. This redundancy is crucial for a strong defense, but it complicates efforts to identify and examine the role of individual immune factors19. In this regard, in-vitro reconstitution systems offer a useful platform to analyze specific pathways under controlled conditions. Here, we introduce a cell-free system that recapitulates the process of ubiquitination and loss of viability of SPN using mammalian cell lysates or E1-E2-E3-Ub enzymes along with purified host effector complexes. In this two-step protocol, we first facilitate the conjugation of polyubiquitin chains on the bacteria, followed by assessing their elimination by VCP/p97 in association with UFD1 and NPLOC4. By reconstituting this pathway in-vitro, we bypass cellular complexities and specifically investigate VCP&#39;s ability to neutralize the pathogen. This precise setup allows for a detailed exploration of substrate recognition, chain specificity, effector&#39;s mode of action, and the essential components needed to initiate an effective response.

<table><tbody><tr><td>50X Protease inhibitor cocktail</td><td>Promega&amp;nbsp;</td><td>G6521</td><td></td></tr><tr><td>6x-His-Ubiquitin</td><td>UBPBio</td><td>E1100</td><td></td></tr><tr><td>A549 cell line&amp;nbsp;</td><td>ATCC</td><td>ATCC CCL-185</td><td></td></tr><tr><td>Alexa Fluor 488</td><td>Invitrogen</td><td>A21202&amp;nbsp;</td><td></td></tr><tr><td>Alexa Fluor 555</td><td>Invitrogen</td><td>A-31572&amp;nbsp; &amp;nbsp;</td><td></td></tr><tr><td>Anti-Ubiquitin antibody</td><td>Enzo</td><td>BML-PW8810-0100</td><td></td></tr><tr><td>Anti-Ubiquitin K48-Specific, clone Apu2 antibody</td><td>Sigma Aldrich</td><td>ZRB2150</td><td></td></tr><tr><td>ATP</td><td>Sigma Aldrich</td><td>&lt;a href=&quot;https://www.sigmaaldrich.com/IN/en/product/sigma/a6419&quot;&gt;A6419&lt;/a&gt;</td><td></td></tr><tr><td>BHI</td><td>HiMedia&amp;nbsp;</td><td>M210</td><td></td></tr><tr><td>DMEM (high glucose)</td><td>HiMedia&amp;nbsp;</td><td>AL251</td><td></td></tr><tr><td>FBS</td><td>Gibco (US origin)</td><td>38220090</td><td></td></tr><tr><td>Glycerol</td><td>PureSynth&amp;nbsp;</td><td>PSR37325</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H4034-100G</td><td></td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P3911</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>TC006</td><td></td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Sigma Aldrich</td><td>E6148</td><td></td></tr><tr><td>pET28a-p97 plasmid</td><td>Our lab</td><td></td><td>Plasmid available upon request</td></tr><tr><td>pET41b-NPLOC4 plasmid</td><td>Our lab</td><td></td><td>Plasmid available upon request</td></tr><tr><td>pET41b-UFD1 plasmid</td><td>Addgene</td><td>Plasmid #117107</td><td></td></tr><tr><td>Rbx1-Skp1-Cul1-Fbxw7&amp;nbsp;</td><td>Sigma Aldrich</td><td>23-030</td><td></td></tr><tr><td>Streptococcus pneumoniae R6 strain</td><td>ATCC</td><td>ATCC BAA-255</td><td>Risk Group -2 pathogen, to be handled in BSL-2 facility</td></tr><tr><td>Todd Hewitt broth powder</td><td>HiMedia&amp;nbsp;</td><td>M313</td><td></td></tr><tr><td>UBE1</td><td>UBPBio</td><td>B1100</td><td></td></tr><tr><td>UbE2C</td><td>UBPBio</td><td>C1300</td><td></td></tr><tr><td>Urea</td><td>HiMedia&amp;nbsp;</td><td>MB032</td><td></td></tr><tr><td>Yeast extract powder&amp;nbsp;</td><td>HiMedia&amp;nbsp;</td><td>RM027</td><td></td></tr></tbody></table>

in-vitro reconstitution of bacterial ubiquitination and vcp/p97-mediated elimination

In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination

Ubiquitination is a versatile post-translational modification that plays a critical role in cytosolic immunity. Upon invasion of the host cells, pathogenic bacteria are initially enclosed within an endosome, from which they break free and escape into the cytosol to avoid lysosomal degradation. Once in the cytosol, bacteria are rapidly tagged with ubiquitin chains by host E3 ligases, marking them for clearance via multiple effector pathways, including autophagy and the proteasome. To delineate the contribution of individual pathways in bacterial elimination, in-vitro reconstitution offers a controlled and unambiguous approach. Here, using Streptococcus pneumoniae (SPN) as a model bacterial pathogen, we present a detailed protocol for its ubiquitination using mammalian cell lysates or pure ubiquitin enzyme complex, followed by treatment with purified VCP/p97-UFD1-NPLOC4 complex to assess its bacteriolytic activity independent of other cellular factors. Overall, this procedure holds the potential to illuminate the function of immunity-linked ubiquitin ligases and effectors in a cell-free context, where dissecting such mechanisms can be challenging in intact cells.

Our protocol details the utilization of mammalian A549 cells to produce a cell lysate that is rich in the necessary components for initiating a ubiquitination process (Figure 1A). This lysate, whether in a concentrated state or substituted with purified components such as UBE1 (the E1 ubiquitin-activating enzyme), UbE2C (an E2 ubiquitin-conjugating enzyme), and the Rbx1-Skp1-Cul1-Fbxw7 complex (the E3 ubiquitin ligase), is mixed with purified ubiquitin and AT...

This protocol describes a method to ubiquitinate Streptococcus pneumoniae using mammalian cell lysate or pure ubiquitin enzyme complex, followed by treatment with purified VCP/p97-UFD1-NPLOC4 protein complex to assess its bacteriolytic activity, enabling the study of ubiquitin-dependent immune effectors.

Ubiquitination is a versatile post-translational modification that plays a critical role in cytosolic immunity. Upon invasion of the host cells, ...

Our work unveils how VCP or p97 acts as a cytosolic defender against multiple different pathogens. This unlocks the potential of VCP or p97 for therapeutic applications and bio-engineering strategies. In addition to routine biochemistry, super resolution microscopy, cryo EM, cryo ET, are utilized to figure out how these host effectors organize on pathogen to destroy them.To begin, grow the streptococcus pneumonia are six strain serotype two overnight at 37 degrees Celsius with 5%carbon dioxide in Todd Hewitt yeast medium. Inoculate one part of the overnight culture into 10 parts of fresh Todd Hewitt yeast medium, and incubate at 37 degrees Celsius with 5%carbon dioxide until the optical density at 600 nanometers reaches 0.4. Then mix 900 microliters of the streptococcus pneumonia culture with 600 microliters of 50%glycerol and store the glycerol stock at minus 80 degrees Celsius.Then thaw a fresh stock of streptococcus pneumonia from minus 80 degrees Celsius and completely inoculate it into five milliliters of Todd Hewitt yeast medium. Take approximately 10 to the power of seven streptococcus pneumonia cells. To wash the cells thoroughly, re-suspend the bacteria in the reaction buffer and centrifuge at 21, 000 G for five minutes at room temperature.Re-suspend the pellet with a 549 mammalian cell lysate. Then add purified ubiquitin and one millimolar adenosine triphosphate and incubate the mixture for one to two hours at 27 degrees Celsius. Then wash the bacteria once with reaction buffer containing one molar urea, followed by two additional washes with only the reaction buffer.Fix the bacteria with 1%paraformaldehyde. After fixation, incubate the cells with PBS, containing one milligram per milliliter glycine for 15 minutes. Then wash twice with PBS.Prepare a reaction mixture by combining ubiquitin with other required components in the reaction buffer. Adjust the total volume of the reaction mixture to 100 to 150 microliters. Re-suspend the bacteria in the ubiquitin enzyme containing reaction mixture, and incubate the suspension for two hours at 27 degrees Celsius.After washing the bacteria three times with reaction buffer, containing urea, fix them with 1%paraformaldehyde. After fixation, incubate the cells with PBS, containing one milligram per milliliter glycine for 15 minutes. Then incubate the sample in 3%BSA for one hour at 27 degrees Celsius, and wash the sample twice with PBS.Re-suspend the ubiquitinated bacteria in a primary antibody solution, prepared at higher concentrations than typically used in immunofluorescence assays. Then incubate for one hour at 27 degrees Celsius. After washing the bacteria twice with PBS, re-suspend them in the corresponding secondary antibody conjugated with Alexa Fluor 488 and Alexa Fluor 555.Now drop cast approximately 10 to 20 microliters of the labeled bacterial sample onto a glass slide and mix in five to 10 microliters of mounting medium with or without DAPI. Place a cover slip over the sample and use tissue paper to blot any excess liquid. Combine six micromolar VCP p97 with one micromolar NPLOC4 and one micromolar UFD-1.Adjust the volume of the reaction mixture to approximately 90 microliters, using reaction buffer, and incubate on ice for two hours. Next, add one to two millimolar adenosine triphosphate to the reaction mixture containing p97 UFD-1, NPLOC4. Then combine this with 10 to the power of seven ubiquitinated streptococcus pneumonia cells, and incubate for two hours at 37 degrees Celsius.Immediately after treatment, take a 10 microliter aliquot from the reaction mixture and perform a serial dilution. Spot 10 to 20 microliters of each dilution onto brain heart infusion auger plates. Assess colony forming units between the zero hour and two hour time points.Calculate bacterial viability percentage by dividing the two hour CFU count by the zero hour CFU count and multiplying by 100. Significant ubiquitination of streptococcus pneumoniae was observed when treated with either mammalian cell lysate or a reconstituted E-1, E-2, E-3 enzyme system. Ubiquitination signal was absent in the control and markedly diminished when ubiquitinated streptococcus pneumoniae was exposed to OTUB1, confirming K48 linked poly ubiquitin modification.A line profile analysis showed full overlap between ubiquitin and bacterial signals confirming their spatial co-localization. Three-dimensional reconstruction confirmed the spatial overlap of ubiquitin and streptococcus pneumoniae signals. Wild type p97 when combined with its co-factors, UFD1 and NPLOC4, reduced bacterial viability to 10 to 20%after two hours, regardless of whether ubiquitination was performed with cell lysate or with purified enzyme system.No reduction in bacterial viability was observed when ubiquitination was omitted, when p97 was heat inactivated, or when using the catalytically inactive p97, E57 8Q mutant, confirming that both ubiquitination and active p97 are essential for bacterial killing. Our study opens the door about how host E3 ubiquitin ligases modify different pathogens and investigate the effector mechanisms to clear the infectious threats. We next aim to explore how p97 or VCP is regulated, and farther engineer it to increase its antibacterial activity.

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Research

JoVE Journal

Immunology and Infection

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

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In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

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In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

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Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs

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Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

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Ubiquitin Chain Analysis by Parallel Reaction Monitoring

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In Vitro Analysis of E3 Ubiquitin Ligase Function

Ubiquitination is a critical protein post-translational modification, dysregulation of which has been implicated in numerous human diseases. This protocol details how phage display can be utilized to isolate novel ubiquitin variants that can bind and modulate the activity of E3 ligases that control the specificity, efficiency, and patterns of ubiquitination.

Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases

We provide a detailed protocol for a ubiquitylation assay of a specific substrate and an E3 ubiquitin-ligase in mammalian cells. HEK293T cell lines were used for protein overexpression, the polyubiquitylated substrate was purified from cell lysates by immunoprecipitation, and resolved in SDS-PAGE. Immunoblotting was used to visualize this post-translational modification.

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

This protocol explains how to measure the effect of genetic variation associated with neurodevelopmental disorders on deubiquitylating enzyme activity by combining recombinant protein purification with ubiquitin chain cleavage assays.

Measuring Enzymatic Activity of Neurodevelopmental Disorder-Associated Deubiquitylating Enzymes via an In Vitro Ubiquitin Chain Cleavage Assay

Learn to reconstitute bacterial ubiquitination and VCP/p97-mediated elimination using Streptococcus pneumoniae and mammalian cell lysate. This method reveals cytosolic defense mechanisms against pathogens.

In-vitro Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination

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