Oral Presentation Melbourne Protein Group Student Symposium 2013

Structural & Functional Characterisation of a Novel Phospholipid Binding Protein in Mycobacterium smegmatis Implemented in Macrophage Survivability (#4)

Adam Shahine 1 , Phooi Chan 1 , Rajini Brammananth 1 , Paul Crellin 1 , Ross Coppel 1 , Jamie Rossjohn 1 , Travis Beddoe 1
  1. Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia

The Corynebactereae suborder of bacteria includes significant human pathogens such as mycobacterium tuberculosis & mycobacterium leprae. The emergence of drug resistance in mycobacteria has resulted in an urgent need for the development of new treatments. Mycobacteria proliferate within the phagosomes of host macrophages, requiring a vast array of essential proteins for survival. The understanding of mycobacterial survivability within macrophages would allow for the development of new antimycobacterial therapeutics.

We’ve recently identified a novel gene in M. smegmatis that, when inactivated, leads to rapidly accelerated cell death in host macrophages. It was found that cell death was not a result of oxidative stresses or acidic pH’s typical of the macrophage phagosome1 . The identified gene product, denoted as MSMEG_5817, encodes a 13.5kDa protein of unknown function, and is conserved within multiple mycobacterial species. A potential role of MSMEG_5817 is maintaining cell membrane integrity due to the mutant’s increased ability to uptake dyes from growth media.

To gain insight into its function, the crystal structure of MSMEG_5817 was solved to 2.1Å by 3-wavelength MAD data collected on a selenomethionine derivative2 . The crystal structure revealed a highly hydrophobic binding pocket, potentially involved in lipid binding & mediation to aid in survivability within the host. Structural homology searches have revealed that MSMEG_5817 shares similarity to sterol carrier proteins (SCP), which play roles in non-specific lipid transfer across membranes. Lipid binding of MSMEG_5817 been confirmed by ELISA & found to bind a broad range of phospholipids. Differences in binding specificity was also observed upon comparison with the Human SCP.

MSMEG_5817 is a previously uncharacterised protein vital for survivability within host macrophages, with a potential role in maintaining cell membrane integrity. Interference with the function of MSMEG_5817 may provide a novel therapeutic approach for control of pathogenic mycobacteria.

  1. Pelosi, A., D. Smith, et al. (2012). "Identification of a novel gene product that promotes survival of mycobacterium smegmatis in macrophages." PLoS ONE 7(2)
  2. Shahine A., et al. (2013) “Cloning, expression, purification and preliminary X-ray diffraction studies of a mycobacterial protein implicated in bacterial survival in macrophages”, Acta Crystallographica Section F: Structural Biology and Crystallisation F69, 566-569