Abstract
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. Analysis of proteins secreted by Mtb has been of interest to the field of tuberculosis research since certain secreted proteins interact with the host to promote virulence, while others may be important antigens or serve as biomarkers of infection. Here, we describe a protocol to prepare whole cell extracts (WCE) and short term culture filtrate (CF) from Mtb or the vaccine strain Mycobacterium bovis- bacillus Calmatte- Guérin (BCG) (Mehra et al., 2013). These are both slow growing mycobacteria, but the same basic procedure can easily be adapted to analyze secreted proteins from rapidly growing mycobacteria, such as Mycobacterium smegmatis (Msmeg), a non-pathogenic species commonly used in the laboratory. The fractions obtained can be analyzed by western blotting to examine proteins of interest or by mass spectrometry if antibodies are not available or to examine the entire secretome. Genetic knockout mutants for the gene of interest serve as a negative control. Additionally, levels of a cytosolic protein such as the chaperone GroEL or the pyruvate dehydrogenase E2 component sucB (Rv2215/dlaT) should be assessed in the CF fraction to rule out the possibility that a positive signal in CF is due to bacterial lysis (see Figure 1). By varying the growth conditions of the strain, this in vitro secretion assay can be used to examine conditions that alter the secretome. We are thankful to Magnus Stiegedal for helpful tips on TCA (trichloroacetic acid) precipitation.
Keywords: Mycobacteria culture, Culture filtrates, Whole cell extracts, TCA precipitation, Bead beating
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.
Equipment
Acronyms
Procedure
Notes
Recipes
Acknowledgments
This protocol was originally used in the published work Mehra et al. (2013). This published work was supported by grants and fellowships from the NIH (R01 AI087682), the Doris Duke Charitable Foundation, the Infectious Disease Society of America, the Michael Saperstein Medical Scholars Research Fund (New York University School of Medicine), Potts Memorial Foundation and the American Society of Microbiology.
References
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