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UBC Theses and Dissertations

Characterization of the ESX-1 (Snm) secretion system in mycobacteria Lalani, Shifana


Tuberculosis is a common and deadly disease caused by the Gram-positive bacteria Mycobacterium tuberculosis. Due to the unique nature of the extracytoplasmic localization of proteins in mycobacteria, specialized secretion systems are essential. Recent studies have identified a novel secretion pathway termed the ESX-1 (or Snm) secretion system. This system is known to secrete two virulence factors, ESAT-6 and CFP-10. The ESX-1 system is comprised of at least three core-components, Rv3870, Rv3871 and Rv3877. Rv3877 is an integral membrane protein, and possibly creates a translocation channel within the membrane. Rv3870 and Rv3871 have sequence homology to AAA-ATPases. Together, Rv3870, Rv3871 and Rv3877 may work to propel ESAT-6 and CFP-10 across the bacterial membrane. These predictions are primarily based on the primary sequences of the proteins, and no other biochemical information exists. Thus, it is important to determine how the secretion system components interact with one another. To investigate these interactions, homologues of Rv3870 (Snm1), Rv3871 (Snm2), and Rv3877 (Snm4) were cloned from the Mycobacterium smegmatis genome and expressed in bacterial expression systems. All three components were successfully expressed, and Snm2 was purified in the presence of adenosine tri-phosphate. Investigation of purified Snm2 revealed the presence of a dimer complex in solution. Snm2 was tested for binding interaction with the CFP-10 and EAST-6 complex using gel filtration chromatography, native gel analysis, radiolabeling techniques, and crosslinking experiments. Through the assistance of a crosslink, CFP-10 was shown to interact with Snm2. This is the first biochemical characterization of the ESX-1 components. The data from these results will provide additional information on the specialized secretion systems of mycobacteria.

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