Analysis of Mycobacterial Protein Secretion

Figure 1. Western analysis of secretion of EsxH by BCG. BCG containing an empty vector control and EsxG-EsxH-FLAG expression construct (FLAG tag at C terminal of EsxH) were analyzed for presence EsxH by anti-FLAG western in WCE and CF prepared as described in the protocol. DlaT was used as a loading control to indicate the degree of bacterial lysis.

Materials and Reagents

Note: All work with live Mtb must be performed in a Biosafety Level 3 (BSL3) facility.

1. Middlebrook 7H9 Broth (Difco, catalog number: 271310 )
   
2. Tween-80 (Sigma-Aldrich, catalog number: P4780 )
   
3. Glycerol (Sigma-Aldrich, catalog number: G5516 )
   
4. Albumin-dextrose-catalase (ADC) (BD, catalog number: 212352 )
   
5. Oleic-albumin-dextrose-catalase (OADC) (BD, catalog number: 212351 )
   
6. Potassium phosphate (monobasic) (KH₂PO₄) (Sigma-Aldrich, catalog number: P9791 )
   
7. L-asparagine monohydrate (Sigma-Aldrich, catalog number: A8381 )
   
8. Citric acid monohydrate (Sigma-Aldrich catalog number: C1909 )
   
9. Ferric ammonium citrate (Sigma-Aldrich catalog number: F5879 )
   
10. Zinc sulfate monohydrate (ZnSO₄^.H₂O) (Sigma-Aldrich, catalog number: 96495 )
    
11. Magnesium sulfate heptahydrate (MgSO₄^.7H₂O) (Sigma-Aldrich, catalog number: 230391 )
    
12. Chelex 100 resin (Bio-Rad Laboratories, catalog number: 142-2822 )
    
13. 100x Halt Protease Inhibitor single use cocktail (Pierce, catalog number: 1860932 )
    
14. 100% trichloroacetic acid (TCA) (pre-chilled prior to use) (Sigma-Aldrich, catalog number: 49010 )
    
15. Acetone (pre-chilled prior to use) (Sigma-Aldrich, catalog number: 32201 )
    
16. Phosphate buffered saline (PBS) (Life Technologies, Gibco^®, catalog number: 10010-023 )
    
17. Bromophenol Blue, sodium salt (US Biological, catalog number: 12370 )
    
18. Tris (MP Biomedicals, catalog number: 02194855 )
    
19. Sodium Dodecyl Sulfate (SDS) (US Biological, catalog number: 18220 )
    
20. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
    
21. β-mercaptoethanol (2-ME) (Sigma-Aldrich, catalog number: M6250 )
    
22. 7H9 complete media (see Recipes)
    
23. Sauton’s media (see Recipes)
    
24. Chelated sauton’s media (an alternative minimal media for mycobacterial growth) (see Recipes)
    
25. Protein extraction buffer (see Recipes)
    
26. 5x SDS-PAGE sample buffer (see Recipes)
    

Equipment

1. Autoclave
   
2. Steriflip-GV filter units (0.22 µM pore size) (Millipore, catalog number: SE1M179M6 )
   
3. 20 ml syringes (BD, catalog number: 302830 )
   
4. 0. 22 µM syringe filter units (33 mm) (Millipore, catalog number: SLGV033RS )
   
5. Disposable Sterile Filter system (1L, 0.22 µm pore size) (Corning, catalog number: 09761104 )
   
6. 0.1 mm zirconia/silica beads (Bio Spec Products, catalog number: 11079101z )
   
7. 30 ml square media bottles (Nalgene^®, catalog number: NE/2019-0030 )
   
8. 125 ml square media  bottles (Nalgene^®, catalog number: NE/2019-0125 )
   
9. 50 ml falcon tubes (Corning, catalog number 430290 )
   
10. 15 ml falcon tubes (Corning, catalog number: 430052 ) with plug seal caps
    Note: These 50 ml tubes are compatible with organic solvents and high speed centrifugation. Falcon tubes with these features can be used from different vendors.
    
11. Microtubes (2 ml screw cap with O rings) (SARSTEDT AG, catalog number: 72.693 )
    
12. Spectrophotometer
    
13. Centrifuge with swinging bucket rotor for spinning down bacterial cultures (for example, Beckman Coulter, model: Allegra X-15R ; bench top centrifuge with SX4750 rotor)
    Notes:
    1. Msmeg and BCG should be handled according to institutional standards of practice for biosafety.
       
    2. Mtb cultures should be handled in biosafety level 3 facilities according to institutional standards of practice.
       
    3. Centrifuging BCG and Mtb requires appropriate aerosol containment.
14. Beckman Aerosolve^® canisters to contain aerosols during centrifugation of mycobacterial cultures (e.g. Beckman Coulter, catalog number: BK359232 )
    
15. 37 °C shaking incubator
    
16. Aerosol containment units for shaking BCG and Mtb liquid cultures in the shaking incubator
    Note: Incubator should be placed in BSL3 facility for Mtb cultures.
    
17. High speed centrifuge for 50 ml polypropylene falcons used for TCA precipitation of CF (e.g. Beckman Coulter centrifuge with JLA16.2 rotor)
    
18. 50 ml falcon adaptors for rotor JLA16.2
    
19. Bead beater (Bio Spec Products, model: Minibead beater 16; http://www.biospec.com/product/34/mini_beadbeater/)
    
20. Standard table top centrifuge with refrigeration
    
21. Heating block for Eppendorf tubes (set to 95 °C)
    
22. pH meter
    

Acronyms

1. Mtb: Mycobacterium tuberculosis
   
2. BCG: Mycobacterium bovis bacillus Calmatte- Guérin
   
3. Msmeg: Mycobacterium smegmatis
   
4. WCE: Whole cell extract
   
5. CF: Culture filtrate
   
6. DlaT: Rv2215/pyruvate dehydrogenase E2 component sucB protein of mycobacteria
   
7. TCA: Trichloroacetic acid
   
8. BSC: Biosafety cabinet
   
9. OD₆₀₀: Absorbance or Optical Density at wavelength of 600 nm
   

Procedure