Protocol for oleuropein-induced autophagy mediating drug tolerance in P. falciparum

Summary

The anti-inflammatory activity of a phytocompound (oleuropein [OLP]) in the lipopolysaccharide (LPS)-mimicked macrophage model of inflammation demonstrates the importance of PI3K-Akt1 signaling in establishing “immune homeostasis.” Here, we present a protocol for the cultivation of in vitro cultures of P. falciparum for carrying out drug sensitivity assays. We describe steps for parasite synchronization, drug treatment, DNA isolation, and starvation-induced autophagy. This protocol provides insights into autophagy and parasite tolerance to drug pressure.

For complete details on the use and execution of this protocol, please refer to Sharma et al.¹

Graphical abstract

Highlights

• •Oleuropein-induced autophagy in macrophages stimulated by LPS
• •Inflammation is regulated by the PI3K-Akt1 signaling pathway
• •P. falciparum-induced autophagy by starvation and oleuropein treatment
• •P. falciparum withstands oleuropein pressure and develops tolerance due to autophagy

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The anti-inflammatory activity of a phytocompound (oleuropein [OLP]) in the lipopolysaccharide (LPS)-mimicked macrophage model of inflammation demonstrates the importance of PI3K-Akt1 signaling in establishing “immune homeostasis.” Here, we present a protocol for the cultivation of in vitro cultures of P. falciparum for carrying out drug sensitivity assays. We describe steps for parasite synchronization, drug treatment, DNA isolation, and starvation-induced autophagy. This protocol provides insights into autophagy and parasite tolerance to drug pressure.

Before you begin

We have developed a humanized mouse model to understand the parasite biology.^2,3,4,5 Also, we deployed our efforts to understand how parasite is gaining resistance by the step-wise experimental induction of resistance to artesunate. The selected resistant P. falciparum was shown to resist the 100-fold increased dose of AST (240 mg/kg) to the clinically approved therapeutic dose (2.4 mg/kg).⁶

In addition, we deployed our efforts to confirm the immunogenicity of PfMSP-1₁₉⁷ and the engineered chimeric fusion protein (PfMSP-Fu₂₄)⁸ when delivered via the elastic liposomes to the wild type mice. Based on our results, chimeric fusion protein could be a very good vaccine candidate as it addressed the limited dominant epitope issue of PfMSP-1_19. Our group is in the process of confirming the vaccine potential of fusion protein ex-vivo in the samples collected on patients’ malaria infection and human RBCs reconstituted NSG mice of P. falciparum infection.

In-vitro culture of asexual blood stage of P. falciparum (3D7, Dd2, and D10-GFP-Atg18)

Timing: ∼24–72 h

This protocol outlines the steps for culturing and maintaining various laboratory strains of P. falciparum. Also, we detailed the sorbitol synchronization of the routine asynchronous culture of P. falciparum required for the drug sensitivity assay.

• 1.Culture the P. falciparum strains (3D7, Dd2 and D10-GFP-Atg18∗) in appropriate culture media.⁹

Note: We used RPMI 1640 medium (containing 2 g/L glucose, 2 g/L sodium bicarbonate, 300 mg/L glutamine, 25 μg/mL gentamicin and 100 μM hypoxanthine) (Thermo Fisher Cat. No. 31800-022)

• 2.Add 0.5% (% w/v) albumax II (Thermo Fisher Cat. No. 11020-021) to the medium.
• 3.Adjust the hematocrit to 2 and 5% using human red blood cells (hRBCs).

∗Add the selection marker (blasticidin) at 1 μg/mL to the medium for the maintenance of D10-GFP-Atg18 strain.

• 4.Incubation conditions:

Incubate^# the culture under a gas mixture comprising: 5% CO₂, 5% O₂ and 90% N₂.

Temperature: 37°C^10,11 (^#initially after overnight incubation observe the culture, followed by continuing/stopping the culture as per the specific requirement).

• 5.Observe the culture at regular intervals (for the first time after overnight incubation-16 h) by the Giemsa staining on drawn smear from the culture with respect to the growth and other contamination issues.

Synchronizing malaria parasites

Timing: ∼30–40 min

The 5% sorbitol method is widely used for synchronizing malaria parasites by selectively lysing mature forms (trophozoites and schizonts) while sparing ring-stage parasites.¹² This synchronization step helps in studying specific stages of the parasite’s life cycle and in conducting experiments with a more homogeneous population of parasites.

• 6.Preparation of sorbitol solution:

Prepare a 5% (% w/v) sorbitol solution by dissolving sorbitol powder in sterile water. Ensure thorough mixing until the sorbitol is completely dissolved.

• 7.
  Harvesting parasites:

  • a.
    Collect the culture of malaria parasites at the desired stage of development, typically when the majority of parasites are in the ring stage.
  • b.
    Pellet down the parasites by centrifugation at a low speed (e.g., 800–1000 × g) for 5 min. Discard the supernatant carefully without disturbing the pellet.
• 8.Resuspending in Sorbitol Solution:

5% sorbitol prepared in water and kept at 37°C and 5 volumes of sorbitol to that of parasite pellet was mixed and keep at 37°C for 5 min. Ensure thorough mixing to achieve a homogenous suspension.

• 9.Incubation with Sorbitol:

Incubate the parasite suspension in the sorbitol solution for a specific duration, typically 10–15 min, at room temperature or 37°C, depending on the protocol.

• 10.
  Centrifugation and Washing:

  • a.
    Centrifuge the parasite suspension at a low speed (e.g., 800–1000 × g) for 5 min to pellet the parasites.
  • b.
    Carefully remove the supernatant and wash the parasite pellet once or twice with appropriate culture medium to remove residual sorbitol.
• 11.Resuming Culture:

Re-suspend the synchronized parasites in fresh culture medium at the desired hematocrit suitable for their growth.

• 12.Monitoring synchronization:

Monitor the synchronized parasite culture over time to confirm the effectiveness of synchronization. This can be done by microscopic examination of thin blood smears stained with Giemsa to observe the different developmental forms of parasites.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
AKT1 (1:1,000)	Cell Signaling Technology (USA)	Cat# C73H10
pAkt (1:1,000)	Cell Signaling Technology (USA)	Cat# S473
NF-κB (1:200) & (1:1,000)	Cell Signaling Technology (USA)	Cat# S536
NF-κBp (1:200) & (1:1,000)	Cell Signaling Technology (USA)	Cat# D14E12
pfAtg8 (1:200) & (1:100)	Raised in rabbit by Dr. Sijwali, CCMB	–
FITC-conjugated anti-rabbit IgG (1:1,000)	Santa Cruz, USA	Cat# sc2357
Beclin-1 (1:1,000)	Cell Signaling Technology (USA)	Cat# D40C5
CD40 (1:1,000)	Cell Signaling Technology (USA)	Cat# D8W3N
Cas-3 (1:1,000)	Cell Signaling Technology (USA)	Cat# 9662S
Cas-8 (1:1,000)	Cell Signaling Technology (USA)	Cat# D35G2
Bim (1:1,000)	Cell Signaling Technology (USA)	Cat# C34C5
IDO1 (1:1,000)	Cell Signaling Technology (USA)	Cat# D5J4E
LC-3 (1:200) & (1:1,000)	Cell Signaling Technology (USA)	Cat# 4108S
Alexa Fluor 488-conjugated anti-mouse antibodies (1:1,000)	Cell Signaling Technology (USA)	Cat# 4408
Bacterial and virus strains
Plasmodium falciparum 3D7, Dd2	Received from NIMR, New Delhi
D10 parasite expressing GFP-Atg18 line of P. falciparum	Dr. Puran S. Sijwali, CSIR-CCMB, Hyderabad, India
P. berghei; ANKA strain	Dr. Agam Singh, NII, New Delhi
Biological samples
Crude extract of oleuropein (OLP) (This was absolutely grinded leaf powder and not sure about the impurities, we used the abstract as such, and now experiments are on course with the oleuropein bought from Sigma, USA)	Prof. B. S. Chandel, Sardarkrushinagar Dantiwada Agricultural University, Gujarat, India
Chemicals, peptides, and recombinant proteins
RPMI-1640 media	Gibco Life Technologies, USA	Cat# 31800-022
Fetal bovine serum (FBS)	Gibco Life Technologies, USA	Cat# 10270106
Antibiotic cocktail (Penicillin + Streptomycin)	Gibco Life Technologies, USA	Cat# 15070063
β-Mercaptoethanol	GCC Biotech	Cat# GPC-009
Phorbol 12-myristate 13-acetate (PMA)	Sigma	Cat# 524400
LPS	Sigma	Cat# L7895
Chloroquine (CQ)	Sigma-Aldrich	Cat# C6628
Artesunate (AST)	Sigma-Aldrich	Cat# A3731
3-Methyl adenine (3-MA)	Sigma-Aldrich	Cat# M9281
Trizol	Invitrogen, US	Cat# 15596026
SYBR Green I	Invitrogen, US	Cat# S7563
HEPES	HiMedia Laboratories Pvt. Ltd., India	Cat# MB016
NaHCO3	HiMedia Laboratories Pvt. Ltd., India	Cat# TC230
Hypoxanthine	Sigma-Aldrich, USA	Cat# H9636-5G
AlbuMAX	Gibco Life Technologies, USA	Cat# 11020-021
Gentamicin	Gibco Life Technologies, USA	Cat# 15750060
Antifade	Thermo Fisher Scientific, USA	Cat# P36930
Blasticidin	Gibco, USA	Cat# R210-01
GENES2ME Green Eye-Ab universal qPCR master mix	Imperial Life Sciences, India	Cat# SMM03; Genes2me
Paraformaldehyde solution	HiMedia	Cat# TCL119
Sorbitol	Sigma	Cat# S1876

Materials and equipment

Dissolve the compounds to be tested (e.g., OLP, artesunate) in appropriate solvents:

OLP: For hTHP-1 macrophage experiments, it was dissolved in 1% DMSO as 3 mg/mL stock solution and then further diluted to make the working solution (30 μg/mL).

OLP was dissolved in 1% DMSO as 0.3 mM stock solution and then further diluted to make the working solution (40–320 μM).

Chloroquine (CQ) was dissolved in sterile water to make a stock solution of 1 mM and further diluted to make a working solution of 500 μM.

Artesunate (AST): hTHP-1 macrophages experiments, it was dissolved in 70% ethanol (v/v) as 3 mg/mL to make the stock solution which was further diluted to make the working solution (30 μg/mL).

For P. falciparum experiment AST was dissolved in 70% ethanol (v/v) to make 0.3 mM stock and which was further diluted to make a working solution of 40–320 μM.

LPS: It was supplied as off-white lyophilized powder (SIGMA) and dissolved in RPMI medium in the biosafety cabinet to make the 1 mg/mL (stock solution). 1 μg/mL was used as a working solution and aliquot in small volumes were prepared and store in 2–8°C for 1 month and stored at −20°C for long term storage (up to 2 years).

Lysis Buffer: 20 mM Tris-Cl, 5 mM EDTA, 0.008% saponin (w/v), 0.08% Triton X-100 (v/v), pH 7.5, SYBR Green-I at the manufacturer’s recommended dilution- https://www.thermofisher.com/order/catalog/product/S7563).

Blasticidin S HCl: Supplied as white solid powder (Note: weigh out Blasticidin and prepare solution in a hood). The stock solution of 5 mg/mL was prepared by dissolving it in the sterile water and solution was filter-sterilized before use. Aliquot in small volumes and store in 4°C for short term (1–2 weeks) and store at −20°C for long-term storage for up to 6–8 weeks.

PMA solution: Supplied as clear colorless invisible film or white foam and dissolved in DMSO in biosafety cabinet by making a stock solution of 1 mg/mL and further diluted to make a working solution of 100 ng/mL. Stored at −20°C for up to 2 years, protected from light.

Step-by-step method details

Development of “macrophage model of inflammation (MMI)”

Timing: ∼90–94 h

This protocol outlines the steps for the stimulation of human monocytes (hTHP-1) line differentiation into macrophages by PMA. The macrophages were then stimulated with the antigen (LPS) to mimic the inflammation and performed the gene expression profiling through RT-PCR following the drug treatment. This gives detailed methodology for investigating the effects of OLP and artesunate (AST), alone and in combination, on gene expression in the treated macrophages.

• 1.
  Cell Culture Preparation:

  • a.
    Procure the hTHP-1 monocytic cell line from ATCC, USA.
  • b.
    Culture the cells in RPMI-1640 medium supplemented with:

    • i.
      10% (v/v) FBS.
    • ii.
      Penicillin and streptomycin (100 U/mL).
    • iii.
      β-mercaptoethanol (3.5 μL/L).
  • c.
    Maintain the cells in a humidified atmosphere of 95% O₂ and 5% CO₂ at 37°C.
• 2.
  Cell Differentiation:

  • a.
    Seed hTHP-1 monocytes at a density of 1 × 10⁶ cells in culture vessels.
  • b.
    Differentiate the cells by treating them with 100 ng/mL PMA for 24 h.
• 3.Stimulation with Antigen (LPS):

The PMA differentiated macrophages were stimulated with LPS at a concentration of 1 μg/mL for another 24 h.

• 4.Drug Treatment:

The stimulated cells were treated with OLP, AST (at a concentration of 30 μg/mL) individually as well as in combination for 24 h.¹³

• 5.mRNA Isolation from the drug treated stimulated macrophages

Isolate total mRNA from hTHP-1 macrophages using Trizol reagent (Thermo Fisher Scientific, Cat No 15596026) according to the manufacturer’s instructions.

• 6.cDNA Synthesis:

Convert approximately 400 ng of total mRNA to cDNA using iScript Reverse Transcription Supermix (Bio-Rad, Cat No 1708890).

• 7.
  RT-PCR Setup:

  • a.
    Use 50 ng of total DNA for RT-PCR analysis.
  • b.
    Set up the RT-PCR reaction on a CFX96 Connect system (Bio-Rad, USA).
  • c.
    Use SYBR Green master mix for expression analysis.
  • d.
    Prepare the reaction mixture containing 10 μM forward and reverse primers, 2× SYBR Green master mix.
  • e.
    Perform RT-PCR with the following cycler conditions:

    • i.
      Initial denaturation at 95°C for 3 min.
    • ii.
      Annealing at 55°C for 30 s.
    • iii.
      Extension at 72°C for 30 s.
    • iv.
      Run for 40 cycles.
• 8.Normalization:

Normalize the expression of P. falciparum-specific genes to the loading and house-keeping controls (β-Tubulin and human-specific genes to GAPDH).¹⁴

• 9.Combination Treatment:

For combination treatment, use 20 nM each of OLP and AST.

Immunofluorescence assay (IFA) of stimulated and drug treated macrophages

Timing: ∼40–42 h

Further qualitative and quantitative protein expression or localization of nuclear and signaling markers in the stimulated and drug (OLP, AST) treated macrophages were confirmed using the immunofluorescence assay (IFA). This section illustrates the preparation and treatment of macrophages for carrying out IFA. This protocol involves differentiation of THP-1 monocytes to macrophages, treatment with the compounds, retaining cellular morphology, and showing an interaction of markers through the specific antibody interactions. By the fluorescence imaging, the qualitative expression of proteins markers and the quantification of the protein markers showing co-localization have been shown.

• 10.Cell Seeding and Differentiation:

Seed hTHP-1 cells onto glass coverslips and differentiate them into macrophages.

• 11.Drug Treatment:

Treat the macrophages with 30 μg/mL of OLP and AST each and incubate for 24 h.

• 12.
  Cell Fixation and Permeabilization:

  • a.
    Aspirate the media and wash the cells with 1× PBS.
  • b.
    Fix the cells with 4% (v/v) paraformaldehyde (PFA) for 5 min at room temperature (RT).
  • c.
    Permeabilize the fixed cells with 0.1% Triton-X 100 (v/v) for 3 min.
• 13.Blocking:

Block non-specific binding sites by incubating the permeabilized cells with 5% BSA (w/v) reconstituted in 1× PBS for 1 h.

• 14.Primary Antibody Incubation

Incubate the blocked cells with primary antibodies (e.g., AKT, pAKT, NF-κB, NF-κBp, pfAtg8) at a dilution of 1:200 for 2-h at RT or overnight at 4°C.

• 15.
  Secondary Antibody Incubation:

  • a.
    Wash the cells with 1× PBS to remove excess primary antibodies.
  • b.
    Incubate the washed cells with FITC-conjugated anti-rabbit IgG (or appropriate secondary antibody) for 1 h at RT.
• 16.
  Mounting:

  • a.
    Wash the antibody-stained cells with 1× PBS to remove unbound secondary antibodies.
  • b.
    Mount the coverslips with the stained cells onto glass slides using an anti-fade mounting medium containing DAPI to counterstain the nuclei.¹⁵
• 17.
  Confocal Microscopy Imaging:

  • a.
    Capture images of the stained cells using a confocal microscope, Nikon confocal microscope.
  • b.
    Use appropriate excitation and emission wavelengths for DAPI (nuclei staining) and FITC (antibody labeling).
• 18.Data Processing and Quantification:

Process and analyze the captured images using image analysis software, such as ImageJ-win64, to quantify fluorescence intensity or colocalization.

Now, the similar experimental conditions will be used for P. falciparum. The culturing and synchronization of malaria parasite are explained in the beginning (refer before you begin).

In vitro antimalarial drug screening using P. falciparum strains

Timing: ∼96–98 h

Timing: ∼24–72 h (for steps 25–28)

Timing: ∼40–42 h (for steps 29–35)

This protocol covers the process of preparing parasite cultures for drug screening experiments and inducing the autophagy following the starvation.^10,16 The drug sensitivity/screening assays confirmed the parasite clearance activity of oleuropein. The starvation induced autophagy has been shown to confirm whether parasite employs this escape mechanism to survive drug pressure.

• 19.Serial Dilution of Compounds:

Prepare serial dilutions of the compound stocks by diluting them two-fold in 50 μL of culture medium across a 96-well tissue culture plate.

• 20.Controls Preparation:

Include controls in the plate: 50 μL 0.5% (v/v) DMSO was used as a positive control and chloroquine (50 nM) as a negative control.

• 21.Addition of Parasite Culture:

Add 50 μL of ring stage parasite culture (1–2% parasitemia at 4% hematocrit) to each well and incubate the plate at 37°C for 50 h for pyrimethamine, chloroquine, and artesunate and 96 h for OLP.

• 22.Replacement of Culture Medium (OLP Assay Only):

Replace the culture medium in the OLP assay plate with fresh medium containing the corresponding concentration of OLP, DMSO, or chloroquine.

• 23.Addition of Lysis Buffer:

After the specified incubation period, add 100 μL of lysis buffer to each well and incubate the plate at 37°C for 30 min.

• 24.Fluorescence Measurement:
  Measure the fluorescence of each well using a microplate reader with excitation at 485 nm and emission at 530 nm.

  • a.
    Background Subtraction:
    Subtract the background fluorescence of the chloroquine control from those of the DMSO and test compound wells.
  • b.
    Normalization:
    Normalize the adjusted values as a percentage of the fluorescence value of the DMSO control.
  • c.
    Data Analysis:
    Plot the percentage inhibition versus concentrations using GraphPad Prism software.
• 25.
  Starvation Induced autophagy in P. falciparum:

  • a.
    Cultivate the parasites in albumax-deficient medium.
  • b.
    Treat the schizont-stage parasites with 3 mM 3-MA for 3 h to induce autophagy. Starve the parasites for another 4 h to confirm the induction of autophagy under physiological stress.
  • c.
    Confirm the induction of autophagy by observing the parasites load under the microscope.¹⁷ Draw thin blood smears following the incubation of parasites treated with drugs.
• 26.
  SYBR Green-I Staining:

  • a.
    Dilute SYBR Green-I in sterilized distilled water (10×). Add 25 μL of SYBR Green-I (10×) to 100 μL of sample.
  • b.
    Incubate the samples for 30 min at 37°C.
• 27.
  Fluorescence Measurement:

  • a.
    Measure the fluorescence at Ex485/Em530 nm using a microplate reader (Biotek Synergy).
  • b.
    Include the following experimental groups in the assay:

    • i.
      P. falciparum specific RPMI-1640 medium (background).
    • ii.
      Uninfected RBCs (cell background).
    • iii.
      Infected RBCs (control).
• 28.Treat the parasites alone and in combination with different concentrations of tested and control drugs (AST and OLP) (40, 80, 160, and 320 nM) for 72 h. The drug treated parasites were harvested and processed for further experimentation.
• 29.
  P. falciparum culture.

  • a.
    Synchronize P. falciparum 3D7 and D10-Atg18 strains at 4–5% parasitemia until the late-trophozoite stage (36 h).
  • b.
    Divide the synchronized cultures into four equal parts:

    • i.
      One part cultured in complete culture medium.
    • ii.
      The second part grown in albumax-deficient culture medium (starvation).
    • iii.
      The third part cultured in complete culture medium containing 320 nM OLP.
    • iv.
      The fourth part cultured in complete culture medium with 3 mM 3-MA.
  • c.
    Allow all cultures to grow for an additional 3 h.
• 30.
  Preparation of slides for IFA.

  • a.
    Wash a 100 μL aliquot of each culture with 1× PBS. Dilute the washed cells in PBS to achieve a 10% hematocrit.
  • b.
    Immobilize the cells on poly-L-lysine-coated slides and wash the slides with 1× PBS to remove non-attached cells.
• 31.Fixation and Quenching:

Fix the cells with a solution of 3% (v/v) paraformaldehyde and 0.01% (v/v) glutaraldehyde in PBS. Quench the fixation reaction by incubating the cells with 0.1 M glycine prepared in 1× PBS.

• 32.
  Permeabilization and Blocking:

  • a.
    Permeabilize the cells using 0.1% Triton X-100 (v/v in 1× PBS).
  • b.
    Block nonspecific binding sites with 3% bovine serum albumin (w/v) (in 1× PBS) for 1 h at RT (25°C).
• 33.
  Antibody Incubation:

  • a.
    Incubate the slides with mouse anti-Atg8 antibodies for 1.5 h at RT (1: 100).
  • b.
    Wash the slides with 1× PBS and incubate the slides with donkey Alexa Fluor 488-conjugated anti-mouse antibodies along with DAPI (10 μg/mL) for 1 h at RT.
  • c.
    Wash the slides with 1× PBS & air dry the slides.
• 34.Mounting and Sealing:

Mount the dried slides with prolong diamond antifade. Cover the slides with glass coverslips and seal the edges.

• 35.Microscopy and Image Analysis:

Observe the slides under a 100× objective of a ZEISS Axioimager. Capture images with Zeiss AxioCam. Analyze the images using Axiovision software.

Expected outcomes

This protocol was developed to validate the induction of autophagy like cell defense mechanism in MMI and response of P. falciparum to oleuropein pressure. Autophagy like escape mechanism employed by the macrophages in MMI and P. falciparum may have implications during the drug resistance. This work is of particular importance as it may answer how P. falciparum gains resistance to the antimalarial drugs that enables develop new drug and drug targets for treating malaria infection.

Limitations

The attestation of the activation of autophagy by the macrophage in response to OLP treatment, and laboratory P. falciparum strains is incomplete unless the autophagy is validated in the human RBCs reconstituted NSG (immunodeficient) mice (humanized mice). Therefore, oleuropein induced autophagy may confirmed in the humanized mice of P. falciparum infection.

Troubleshooting

Problem 1

MMI and induction of autophagy in human macrophages by the oleuropein treatment does not truly mimic the natural inflammation.

Potential solution

An experimental mouse model of rheumatoid arthritis followed by the oleuropein treatment could mimic the inflammation and explain the activation of autophagy.

Potential solution

The experimental mouse model of P. falciparum infection (humanized mouse model) could represent natural inflammation.

Problem 2

Validation of autophagy in MMI and wild-type mouse models.

Potential solution

GFP-LC3-RFP-LC3DG transgenic mice could be a viable option for the attestation of oleuropein treatment driven autophagy.

Potential solution

Rapamycin induced autophagy in wild type mice and inhibition by 3- Methyl adenine (3-MA) could be another solution.

Problem 3

Routine in vitro culture of P. falciparum does not share completely mimic the natural malaria infection. Moreover, rodent model of P. berghei infection used as “surrogate” and needs to be validated.

Potential solution

Human RBCs reconstituted immunodeficient mice (NOD-scid IL2Rg^null; NSG) could be a viable option to confirm autophagy.

Potential solution

huRBCs reconstituted NSG mice could be used to induce autophagy by Rapamycin treatment followed by the inhibition following the 3-MA treatment, could be useful for validation of the autophagy.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Rajeev K Tyagi (rajeevtyagi@imtech.res.in, rajeev.pasteur@gmail.com).

Technical contact

Requests for any further technical help or information can be directed to: prakriti.sh10@gmail.com, nikunj.tandel@nirmauni.ac.in, rajeevtyagi@imtech.res.in.

Materials availability

This study did not generate new unique reagents. Other chemicals, reagents, and instruments used in this study are mentioned in the key resource table.

Data and code availability

• •This study does not report any original code.
• •All unique/stable reagents generated in this study are available from the lead contact without restriction.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request
• •All the data from the studies are presented here, in Sharma et al.¹