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Method Article

Preparation of Mycobacterium tuberculosis Culture Filtrate to Understand TB Pathogenesis

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DOI:

10.3791/67540

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March 28th, 2025

 , 
1CSIR-Centre for Cellular and Molecular Biology (CSIR-CCMB), 2Academy of Scientific and Innovative Research (AcSIR), 3National Institute of Immunology

In This Article

Summary

Here, we demonstrate the preparation of Mtb culture filtrate (CF), which is the bacterial growth media that consists of proteins secreted by the bacteria. Mtb secretes proteins to modulate host signaling and immune response, which are crucial for pathogenesis. The CFs prepared are analyzed by mass spectrometry to identify all the secretory factors.

Abstract

Mycobacterium tuberculosis (Mtb) secretes many proteins into the host to modulate various cellular processes, such as host signaling, immune response, apoptosis, etc., to enhance survival. While the function of a few secretory factors has been elucidated, many are yet to be characterized. Hence, investigating the Mtb secretory protein is an essential area of research. Mtb secretes proteins into the extracytoplasmic space with the help of three different secretion systems: general, twin-arginine, and specialized Type VII secretion system. One has to prepare the secretome to identify novel secretory molecules and functionally characterize the known factors. Here, we outline the method for preparing the secretome and sample preparation for mass spectrometry. Secretome preparation requires the utmost care to avoid contamination of cytosolic proteins, which can be monitored by probing with antibodies against well-characterized membrane, secretory, and cytosolic proteins. The culture filtrate thus prepared can be used for different purposes, which include investigating host immunomodulation properties.

Introduction

Mycobacterium tuberculosis (Mtb), one of the deadliest pathogens worldwide, infects healthy individuals through aerosols transmitted from an infected person while sneezing or coughing1. Once it enters the lungs, it is engulfed by the alveolar macrophages, hijacking the host immune system to survive. Among various host modulatory mechanisms that Mtb has for survival, secretory virulence factors are one of them. The proteins secreted into the culture media by the bacilli during its growth are termed Culture Filtrate (CF) proteins2. These culture filtrate proteins help bacteria to survive in harsh conditions inside the host cells. Mtb is equipped with three different kinds of secretion systems: a specialized Type VII secretion system (T7SS), a general Sec secretion system (Sec system), and a twin-arginine secretion system (Tat system). T7SS is the most exclusive secretion system in actinobacteria, unlike type I-VI in Gram-negative bacteria3. The secretion of unfolded protein across the membrane is majorly involved in the Sec system, and the proteins are recognized by an N-terminal signal sequence4,5. Folded or partially folded proteins are transported through the Tat system, identified by an N-terminal signal sequence with a twin-arginine motif6,7. How the Sec and Tat system regulates protein secretion has yet to be determined.

Some known culture filtrate proteins in Mtb that help withstand and survive the host oxidative onslaught upon infection are enzymes such as KatG and SodA8,9, necessary for degrading reactive oxygen species. In addition, CFP10-ESAT6, a heterodimeric protein complex, plays a vital role in regulating cytokine responses, bacterial escape, ROS production, etc.10. RD1 locus is involved in modulating the Type VII secretion system, which includes secretion of CFP10-ESAT6, and the genetic deletion of cpf10-esat6 compromises the survival of bacteria in ex vivo and in vivo infection models. Mycobacterium bovis BCG vaccine strain lacks RD1 locus responsible for the secretion of virulence factors and hence can be used as the live attenuated vaccine11. Different unexplored cellular processes tightly regulate the secretion process. The culture filtrate proteins contain resuscitation-promoting factors (RpFs) that help the bacteria regrow from the latent state. Resuscitation-promoting factors (Rpfs) are peptidoglycan hydrolyzing enzymes that are known to play a role in dormancy. Mtb encodes for five rpf-like genes (rpf A-E) that help the bacteria regrow from dormancy either solo or by interacting with other proteins12. Rpfs are found in bacterial culture filtrate and are known to be involved in Mtb virulence and host immunity13,14. The widely studied rpf genes rpfE and rpfB show protective immunity in vitro13 and enhance immune responses in vivo. Hence, rpfE and rpfB can be used as Mtb vaccine candidates.

Reversible phosphorylation15 is a post-translational modification that alters various cellular processes, cell homeostasis, cell division, etc. For example, phosphorylation of CFP10 plays an important regulatory role, where both phosphoablative and mimetic mutants of CFP10 fail to restore the observed phenotype in the CFP10-ESAT6 deletion strain16. SecA2, on the other hand, is a secretory protein involved in DNA damage of the host cell17. The secretome of the SecA2 mutant is sufficient to change the host immune response and signaling cascade18,19. Thus, it is essential and exciting to investigate the secretome of H37Rv and the Mtb mutants, where the genes of the secretory proteins are deleted (unpublished data), to gain insights into how they impact host immunomodulation and survival.

This report focuses on three aspects: the steps involved in preparing Mtb culture filtrate, processing the samples for mass spectrometry, and performing ex vivo experiments with culture filtrates to examine immunomodulatory functions. Earlier studies showed the identification of culture filtrate proteins by 2D gel electrophoresis followed by Mass spectrometric analysis8. Culture filtrate proteins from drug-sensitive and resistant strains were identified using mass spectrometry to gain insights into the secretome changes and identify virulence factors that may play a role in drug resistance20. We have previously investigated Mtb's secretome and phosphosecretome with the help of mass spectrometry16. Techniques such as 2D gel electrophoresis are low throughput and are not used to investigate global changes in the proteome or secretome. The culture filtrate proteins would vary from bacterial strains, stages of the growing cells, and even in various stress conditions. Depending on different situations, the change in the secretory protein pools inside the cells would offer to explore the whole secretome holistically and provide more information about the bacterial secretion system for therapeutic interventions. It would be even more attractive to look at the secretome in the knockouts or depletion strains of secretory systems to gain further insights into the pathways and targets of each secretory pathway. Treating cells with culture filtrate proteins instead of bacteria changes cytokine levels and other immunomodulatory factors17. With the help of the secretome study, one can identify new virulence factors and their role in virulence.

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Protocol

NOTE: Here, we use the H37Rv pathogenic laboratory strain of Mtb. One should perform the steps required to grow the bacterial culture and isolate the culture supernatant in Biosafety Laboratory level 3 (BSL-3). From the revival of the culture to filtrate preparation, the BSL-3 facility is used.

1. Inoculation of Mtb culture

  1. Preparation of work area in BSL-3
    1. Switch on the UV light of the biosafety cabinet for 10 min before use. Clean the biosafety cabinet with 75% ethanol before and after use. Dispose of the materials used in appropriate waste bins following biosafety guidelines.
  2. Preparation of primary and secondary Mtb H37Rv cultures
    1. Take the glycerol stock from -80 °C and thaw the stock at room temperature. Use four independent stocks for biological quadruplets.
    2. Take 10 mL of 7H9 medium (Table 1) containing 10% ADC (9 mL of 7H9 medium and 1 mL of ADC) in a 100 mL autoclaved conical flask with a screw cap.
      NOTE: 7H9 + ADC: 7H9 is a nutrient-rich media obtained commercially, to which we add ADC (ADC: Albumin, Dextrose, and Catalase; Table 2). Both ADC and OADC can be used to grow the culture. Conical flasks provide more aeration to the culture; however, one can also use a tissue culture flask to grow the bacterial cells.
    3. Inoculate the entire glycerol stock to the media to revive the culture, which becomes the primary culture. Take A600~0.8 growing Mtb culture for making glycerol stocks. Mix 15% glycerol and the growing Mtb culture in a cryovial and store at -80 °C.
    4. Incubate the culture flasks at 37 °C in an incubator shaker for 3-4 days at 100 rpm.
    5. On the 5th day, measure absorbance at 600 nm (A600). When the cultures reach A600~0.8-1, use the culture for inoculating secondary culture at A600 ~0.2 in 100 mL of 7H9 +10% ADC media in a 500 mL flask. Perform the same steps for the replicates.
      1. Measure the A600 of the primary culture. If the A600 is ~ 0.8, pellet down the culture by centrifugation at 2100 x g for 10 min. Discard the supernatant, resuspend the pellet in 5 mL of 7H9 + 10% ADC media, and measure the A600. Calculate how much inoculum will be added into 100 mL of secondary culture of A600 ~ 0.2. For example, if the A600 ~ 4 is used, then 5 mL of that culture needs to be added to 100 mL of fresh 7H9 + 10% ADC secondary culture of A600 ~ 0.2.
    6. Grow them at 37 °C in an incubator shaker at 100 rpm for 2-3 days till OD A600 ~0.8.
    7. Withdraw 10 µL using a pipette man fitted with a filter tip and spot the culture on an LB Agar plate that does not contain antibiotics in the bacterial hood. Incubate the plate at 37 °C for 12-16 h to visualize the bacterial contamination (if any). Discard the cultures and revive the experiment's new cultures if any bacterial contamination is detected.
  3. Bacterial growth in Sauton's media
    1. At the A600~0.8, inoculate bacteria at A600 ~0.2 in 100 mL of Sauton's media in a 500 mL flask (Table 3) and grow for 3-4 days after the OD reaches ~0.8.
    2. Use cultures grown till A600 ~0.8 in Sauton's media (Sauton's primary culture) for inoculation of cultures to be used for culture filtrate preparation.
      NOTE: 7H9 + ADC is a nutrient-rich media that contains albumin. Albumin cannot be used in secretome analysis as albumin can mask the culture filtrate proteins. Hence, Sauton's media is used to prepare culture filtrate. Sauton's media is prepared by reconstituting the components mentioned in Table 3.

2. Preparation of Mtb culture filtrate proteins

  1. Centrifugation for culture supernatant
    1. Use Sauton's primary culture for inoculating 300 mL of Sauton's secondary culture at A600 ~0.2. Grow cultures till OD A600 is ~0.4-0.5. Use actively growing early log phase cultures to avoid contamination from dead cells.
      1. Measure the OD of the secondary culture. If the OD reaches A600 ~0.8, pellet down the culture by centrifugation at 2,100 x g for 10 min. Discard the supernatant, resuspend the pellet in 10 mL of Sauton's media, and retake the OD. Calculate how much inoculum will be added into a 300 mL culture of 0.2 OD from the measured OD. For example, if the OD is 8, 7.5 mL of that culture must be added to 300 mL of fresh Sauton's media of 0.2 OD.
        NOTE: Stick to the OD mentioned to reduce cell lysis and GroEL contamination. GroEL is a chaperon and is constitutively expressed in the bacterial cells. Some of the literature showed the presence of GroEL in culture filtrates because of stress during growth. Here, we are using Sauton's media for culture filtrate preparation, which provides mild stress to the cells; hence, GroEL may present in the culture filtrate. Additionally, if the culture OD is more than 1, cell lysis would occur, leading to the GroEL contamination in culture filtrate.
    2. Pellet down 300 mL of growing cultures in 6, 50 mL tubes at 2,100 x g for 10 min at 4 °C (Figure 1A).
    3. Withdraw 45 mL of supernatant, leaving the pellet untouched, with a 25 mL serological pipette to a new tube in each case. Keep the pellet aside for whole cell lysate (WCL) preparation.
    4. Repeat the centrifugation at 2,100 x g for 10 min at 4 °C. Withdraw 40 mL of supernatant (leaving 5 mL of culture untouched) with a 25 mL serological pipette to a new tube and repeat the centrifugation step.
    5. Withdraw 35 mL of the supernatant into a fresh tube and filter the culture supernatant using a 0.2 µm syringe filter. Filter into a fresh tube to eliminate pathogenic bacterial contamination. From 300 mL of culture, 210 mL of culture filtrate per replicate is recovered.
      NOTE: The filter that has been used here to filter culture filtrate is a sterile syringe filter of 0.22 µm (type SY25PL-S).
    6. After filtration of the culture filtrate, inoculate 1 mL aliquot of the filtered culture filtrate onto an agar plate and incubate in the 37 °C incubator for 4 weeks to ensure the presence of other pathogenic bacterial contamination in the culture filtrate. Take 1 mL aliquot from the Whole Cell Lysate (WCL) and follow the same safety measures.
    7. Wipe the sample tubes with 75% ethanol and take them out of BSL-3 through a pass box.
  2. Preparation of Whole Cell Lysate (WCL)
    1. Resuspend the pellet in 1 mL of 1x PBSG containing phenylmethylsulfonyl fluoride (PMSF), 1x protease inhibitor cocktail, and transfer the lysate into 2 mL screwcap bead beating tubes containing 1/3rd volume of 0.1 mm Zirconia beads.
      NOTE: The 1x PBSG is made up of 1x PBS + 5% glycerol. Take Pellet-to-buffer weight ratio of 1:3.
    2. Lyse the samples by bead beating them in a mini bead beater: 1 min of bead beating followed by 2 min of incubation on ice. Repeat this 8x.
    3. Centrifuge the tubes at 18,000 x g for 15 min at 4 °C. Collect the supernatant in a fresh 1.5 mL microcentrifuge tube and keep it on ice.
    4. Repeat the centrifugation step. Pass the supernatant through a 0.2 µm syringe filter to eliminate pathogenic bacterial contamination.
      NOTE: The filter used here to filter culture filtrate is a sterile syringe filter of 0.22 mm (type SY25PL-S).
    5. Wipe samples with 75% ethanol and take them out of BSL-3 through a pass box. Check the concentrations of lysates using the BCA assay kit, as mentioned below. Store the lysates at -80 °C.
  3. Concentrate the culture filtrate proteins using Centrifugal Filter Units.
    1. Concentrate 210 mL of Mtb culture filtrate proteins with the help of centrifugal filter units. Use 3 kDa 15 mL centrifugal filter unit tubes (centricons) to concentrate the culture filtrate into ~1 mL (Figure 1A). The 3 kDa centrifugal filter unit tubes retain proteins of the same size or larger than 3 kDa.
    2. Add approximately 15 mL of each replicate of culture filtrate in one centricon tube and centrifuge at 2,100 x g for 1 h at 4 °C.
    3. Discard the flow-through as per norms, and repeat step 2.3.2 for 15x–16x until 210 mL of the culture filtrate concentrate to ~1 mL.
    4. Aliquot 100  µL of the concentrated culture filtrate proteins into ten low protein binding microcentrifuge tubes and estimate the protein concentration using a BCA kit.
  4. Estimation of concentrated proteins
    1. Estimate the protein concentration with respect to BSA as the standard. Use 1 µg, 2 µg, 3 µg, and 4 µg of BSA in 25 µL volume in duplicates. If the sample was undiluted, use a 5x diluted (in PBS) sample in 25 µL volume in duplicates for the estimation. The estimations are performed in 96 well plates. Use a no-sample control (Blank) containing 25 µL of PBS.
    2. Add 175 µL of mixed reagent as per the manufacturer's instructions. Mix samples with the help of a multichannel pipette and incubate at 37 °C for 1 h. Measure the readings at 562 nm using an ELISA reader. Deduct the values from the blank values to obtain values for BSA and the samples.
    3. Fit the standard curve to the linear regression curve and estimate the samples with respect to the standard. Follow the BCA kit for the protein estimation manual.

3. Confirmation of culture filtrate proteins using different markers

  1. Detection of proteins by Coomassie staining
    1. To confirm the presence of the proteins in culture filtrate, take 30 µg of protein and resolve on 10% Sodium Dodecyl Sulphate –Polyacrylamide Gel Electrophoresis (SDS-PAGE; Table 4).
      NOTE: Follow the table description to make the SDS-PAGE, Coomassie staining, and destaining solutions buffers.
    2. Add 20 mL of Coomassie staining solution (Table 5) to the gel and incubate for 30 min on rockers at room temperature. Discard staining solution.
    3. Add 30 mL of destaining solution (Table 6) to the gel and incubate for 30 min on a rocker at room temperature. Discard the destaining solution and add a fresh destaining solution. Repeat until the gel gets destained properly.
    4. Check the protein streak on the trans illuminator (Figure 1B). Since the culture filtrate protein contains several proteins, a streak of protein bands can be seen in the gel after destaining (Figure 1B).
  2. Western blot confirmation for secreted protein marker
    1. Use the antibody of the secreted protein to check the presence of the secreted protein in culture filtrate. Ag85B (ab43019) is a well-known Mtb secreted protein in culture filtrate and is present in the cytosol; the Ag85B (concentration 5 mg/mL) antibody (1:5000 dilution in 5% BSA) is used as a positive control for the secreted protein.
    2. Load a WCL with the culture filtrate proteins to differentiate between the cytosolic/membrane and secreted protein fractions. Ag85B is cytosolic and secreted, so this should be present in both fractions; on the other hand, PknB is known to be localized to the membrane and is not present in the culture filtrate.
    3. Determine the concentration of proteins in WCL/ culture filtrate with the help of a BCA kit. Mix 30 µg of WCL/ culture filtrate with 2x-lamelli buffer in a 30 mL volume (Table 7). Resolve the samples in 10% SDS-PAGE for PknB and 12% SDS-PAGE for Ag85B till the dye front reaches the bottom using the SDS gel runnimg buffer (Table 8). Do not let the dye front runoff from the gel.
      NOTE: The size of the Ag85B is ~35 kDa, so 12 % SDS-PAGE is used.
    4. Cut 9 cm x 6 cm 0.2 µm nitrocellulose membrane for the transfer (Table 9) of the protein from the gel onto the membrane. Setup the transfer using a transfer cassette, soaking the cassettes, foam pads, blotting paper, gel, and membrane in 1x ice-cold transfer buffer.
    5. Place the foam pad at one side of the cassette, then place a blotting paper, place the gel, put nitrocellulose membrane on top of the gel, place a blotting paper again, and lastly, place another foam pad and close the cassette holder. Place the cassette holder in the transfer tank so the membrane side faces toward the anode and the gel side faces toward the cathode.
    6. Connect the cathode and anode lead to the tank to close the setup and set it at 120 mA current for 120 min. Start running the transfer in a cold room.
    7. Place the membrane in a small plastic box after the transfer is over. Block the membrane using 10 mL of 5% BSA solution made in 1x PBST20 to reduce non-specific binding for 1 h at room temperature on the rocker.
    8. Remove the blocking solution, add 5-10 mL of primary antibody, which is anti-mouse Ag85B antibody (1:5,000 dilutions in 5% BSA solution), and incubate at 4 °C overnight in a rocker. Store the antibody at -20 °C and reuse up to 3x.
    9. Remove the primary antibody, add 5-10 mL of 1x PBST20, and wash the membrane for 8 min at room temperature by vigorous shaking. Remove the first wash, add 5-10 mL of 1x PBST20 again, and wash. Repeat this 2x.
    10. Add 5-10 mL of 1x PBST20 containing secondary antibody, which is anti-mouse Horse redox peroxidase (HRP) conjugated antibody (1:10,000 dilution), and incubate for 2 h at room temperature on a rocker.
    11. Wash the membrane after the secondary antibody following step 3.2.9. Add an equal amount of HRP substrate and enhancer to develop the membrane under a chemiluminometer (Figure 1C).
      NOTE: To confirm the absence of cytosolic protein contamination, we probed the Western blot with an anti-PknB antibody (Figure 1C). The antibody was raised at the National Institute of Immunology, an animal facility, against the purified extracytoplasmic domain of PknB. One can observe the PknB band in the WCL, not in the culture filtrates, and PknB is solely cytosolic and membrane-bound and should not be present in the culture filtrate.

4. Preparation of samples for mass spectrometry

  1. Perform in-solution protein digestion by Trypsin.
    1. Take 100 µg of culture filtrate protein, add 6x volume of ice-cold acetone, and keep it at -20 °C overnight.
      NOTE: Use glass wares to prepare all the reagents for the mass spectrometry sample. Use low-protein binding microcentrifuge tubes and low-protein binding tips. Do not use plastic material to avoid polymer contamination in mass spectrometry analysis.
    2. Precipitate the proteins by centrifugation at 18,000 x g at 4 °C for 30 min. Discard the supernatant.
    3. Wash the pellet with 500 µL of acetone, pellet down at 18,000 x g for 10 min at 4 °C. Discard the supernatant. Remove the residual acetone with the help of a vacuum concentrator.
    4. Add 20 µL of 8 M urea dissolved in 25 mM ammonium bicarbonate (2 M stock prepared in MS grade water). Vortex the samples and incubate for 30 min at room temperature.
    5. Ad 60 µL of 25 mM ammonium bicarbonate to dilute 8 M urea into 2 M. Incubate at room temperature for 1 h. Add 4 µL of TCEP (10 mM final concentration made in 25 mM ammonium bicarbonate) and incubate at room temperature for 30 min, shaking at 1,800 x g.
    6. Add 4 µL of IAA (25 mM final concentration made in 25 mM ammonium bicarbonate) and incubate at room temperature for 30 min, shaking at 1,800 x g in the dark. Add 110 µL of 25 mM ammonium bicarbonate.
    7. Add Trypsin (1:20; Trypsin: protein in µg) to the solution and keep it at 37 °C, shaking at 300 rpm, for overnight digestion (Figure 2). Stop the digestion reaction by adding 10% TFA (trifluoro acetic acid). Speed vacuum the samples completely to dry.
  2. Desalting the sample
    1. Add 100 µL of 5% ACN + 0.1% TFA to the digested peptides. Vortex for 30 min at 1,500 rpm at room temperature.
    2. Centrifuge at 18,000 x g for 10 min at room temperature. Take the supernatant (peptide) without touching the pellet into fresh microcentrifuge tubes and proceed with desalting. Make all the washing and elution solutions before the desalting step.
    3. Equilibrate the C-18 column tip with 100 µL of 100% ACN + 0.1% TFA by pipetting the solution and repeating the process 5x.
    4. Add 100 µL of 50% ACN + 0.1% TFA and repeat the process 5x. Add 100 µL of 5% ACN + 0.1% TFA and repeat the process 5x.
    5. Allow the peptide binding to occur in the C-18 column tip by pipetting 30x. Keep the solution at -20 °C until the sample is processed for Mass spectrometry. Now, the peptide is bound to the C-18 column.
    6. Wash the C-18 column with 100 µL of MS grade water + 0.1% TFA by pipetting and repeat the process 5x.
    7. Wash the C-18 column with 100 µL of 5% ACN + 0.1% TFA by pipetting and repeat the process 5x. Elute the peptides from the C-18 column in 100 µL of 50% ACN + 0.1% TFA by pipetting 30x (Figure 2).
      NOTE: After the elution, the C-18 column tip can be stored at 4 °C until the Mass spectrometry is done.
    8. Speed vacuum the eluted peptide entirely and store it at -20 °C until the sample is given for Mass spectrometry.
  3. Injection of the peptide samples into a Mass spectrometry.
    1. Add 20 µL of solution A (provided by the Mass spectrometry facility) to the dried peptide samples. Vortex the samples for 30 min and centrifuge for 90 min at 4 °C.
    2. Inject 20 µL of the sample into the Mass spectrometry machine for a 90 min gradient run.

5. Data analysis for secreted proteins present in culture filtrate

  1. Protein identification
    1. Analyze proteomics samples using the Quadrupole mass spectrometer. Query the obtained spectra against the Mtb H37Rv UniprotKB database.
    2. Set the precursor and fragment mass tolerances at 10 ppm and 0.5 Da. Set the enzyme to Trypsin with a maximum missed cleavage value of two.
    3. Use the proteome software with Sequest as the search algorithm with the carbamidomethylation of cysteine as a fixed modification.
    4. Identify all the peptide-spectrum matches (PSM) at a 1% false discovery rate. Apply default settings for all other parameters.
    5. Map the raw Mass spectrometry files of the peptide sequence with the UniProt ID of the respective Mtb proteins. List all Mtb proteins, their Uniprot IDs, and functional classification using the Mycobrowser database.
    6. For further analysis, map the UniProt IDs of identified proteins to their respective Rv number. Deposit the Mass spectrometry secretome data to the ProteomeXchange Consortium through the PRIDE partner.

6. Treating H37Rv with culture filtrate protein to check the change in the host signaling

  1. Isolation of peritoneal macrophages from mice.
    NOTE: Follow the reference21 for peritoneal macrophage isolation, seeding the cells, and infection.
    1. Inject Balb/c mice (4-6 weeks age) with 1 mL of 4% thioglycolate and isolate peritoneal macrophages from the mouse peritoneum on the 4th day of infection21 (Figure 3).
    2. Collect the macrophage cells by injecting 1x PBS into the mouse peritoneum. Centrifuge the cells at 1,800 x g for 5 min, discard the supernatant, and add 5 mL of 1640-RPMI media supplemented with 10% FBS (Fetal Bovine Serum; complete media) and 1x pen-strep (antibiotics).
    3. Seed 3 x 105 cells per well in 24 well plates and incubate the plates in a 37 °C incubator overnight. The next day, change the media into fresh, complete media, and the cells will be ready for infection or treatment with culture filtrate proteins.  
  2. Treatment of peritoneal macrophages with culture filtrate proteins
    1. Take the macrophage cells to BSL-3 and keep them in the incubator. Treat the cells with 1 mg/mL culture filtrate proteins (Figure 3). Harvest the cells at different time points of 4 h, 24 h, and 48 h post-culture filtrate treatment.
      1. Seed the isolated macrophage cells in 24 well plates in triplicates at each time point. Treat the cells with 1 mg/mL of culture filtrate proteins and incubate at 37 °C incubator in the presence of CO2. Harvest the cells at three time points, 4 h, 24 h, and 48 h post-treatment.
      2. Treat the cells with i) 2x-lamella buffer to make the macrophage cell lysates and check for different host cell markers by performing Western blotting, and ii) different buffers for the immunological assays provided by the manufacturers along with the kits.
    2. Prepare cell lysates of the host and check for different markers, cytokine profiling of the host cells, and various signaling pathway protein expressions upon culture filtrate-treated macrophage cells.
      NOTE: If a lab has a gene replacement mutant of secretory pathway protein, culture filtrates prepared from Mtb H37Rv and the Mtb mutant can be used to evaluate their impact on host immunomodulatory parameters.

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Results

In this protocol, we demonstrated the isolation of culture filtrate proteins from Mycobacteria (Figure 1A). The culture filtrate proteins were then checked for the different markers to confirm the culture filtrate isolation. As mentioned in the protocol, a Coomassie staining was performed to check the protein loading. One can observe a streaked protein band in the WCL fraction (at the last lane Figure 1B), whereas in CF (culture filtrate) lanes, all the...

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Discussion

One of the critical research areas pertains to understanding Mtb pathogenesis and developing effective diagnostics and vaccines22,23. Isolation of culture filtrate proteins from Mtb culture media is a process that enables the identification of the virulence factors secreted by the pathogen. Investigating the role of secretory proteins in modulating Mtb pathogenesis yields mechanistic insights and may also provide candidates for therapeu...

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Disclosures

The authors have nothing to disclose.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.2 µm Syringe filtersmdiSYPL0601MNXX204
0.2µm Nitrocellulose membraneAmersham10600001
37% HClMerckH1758
AcetoneEMPLURA MERCKSL9F690866
AcetonytrileSigma-Aldrich34851-2L
AcrylamideBIORAD1610107
AlbuminBio Basic CanadaCat No: AD0023
Amicon Ultra-15Merck, MilliporeUFC900396
Ammonium biocarbonateSigma-Aldrich09830-500G
Ammonium persulpahteSigma-AldrichA3678-100G
anti-Ag85BAbcamAB43019
anti-mouse HRP conjugated secondary antibodyThermo Fischer62-6520
AsparagineSigma-AldrichA0884-500G
bis-acrylamideSIGMA146072-500G
BSAMerckMB083-500G
C-18 columnThermo FischerSEM SS18V
Catalase Sigma-AldrichC1345
Citric AcidSigma-AldrichC0759-500G
DextroseSigma-AldrichG7021-1KG
Difco Middle brook 7H9 broth  Fisher Scientific271310
Ferric ammonium(III) citrateSigma-AldrichF5879-100G
Formic Acid 98-100%MerckZ0823704246
Glacial acetic acid 100%MerckCD0C700160
GlycineSigma-AldrichG8898-1KG
GlycineSIGMAG8898-1KG
GycerolSigma-AldrichG5516
Immobilion Western Chemiluminescent HRP substrateMerckWBKLS0500
IodoacetamideAmershamRPN6302V
Magnesium sulpahteSigma-AldrichM7506-500G
Mycobroswer
Oleic acidSigmaCat No: O1008
Pierce BCA protein assay KitThermo Fischer23225
Pierce Trypsin ProtaseThermo Fischer90058
Proteome Discoverer 2.0Thermo Fischer Scientific
Q-Exactive HF (Thermo Scientific) (proteomics experiments - peptide sequencing, protein identification and quantification)Thermo Fischer Scientific
Sodium chlorideSigmaCat No: S3014
Sodium ChlorideSigma-AldrichS7773-1KG
Sodium dodecyl sulphateSigma-AldrichL3771-1KG
TCEPSigma-AldrichC4706-25
TemedSigma-AldrichT9281-100ML
Tris BaseBioRad161-0719
Tris-HClAMRESCO0234-500G
Tween 80Sigma-AldrichP1754
UniProt protein data base
UreaMP Bio194857

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