Molecular Genetics of Mycobacterial Protein Secretion and Pathogenesis
Patricia A. Champion
Approximately one third of the world’s population is infected with Mycobacterium tuberculosis, and there are roughly 2 million tuberculosis-related deaths annually. Treatment for tuberculosis infection is complicated by long courses of antibiotics and the recent emergence of strains resistant to first and second line defenses. The biology of mycobacterial infection is complex, and poorly understood.
During infection, the pathogen manipulates the host cell to avoid detection by the immune response, and to create a niche that favors replication and persistence. One of the major virulence determinants used by mycobacterial and many Gram-positive pathogens is the ESX-1 (ESAT-6 System-1) protein secretion system. While there are many proteins known to play a role in ESX-1 protein secretion, the way the majority of these proteins function is unclear. My lab is interested in determining how components of this complex secretion machine function to select and transport protein substrates during infection. To do so, we use Mycobacterium marinum, a mycobacterial species closely related to Mycobacterium tuberculosis. M. marinum causes a tuberculosis-like infection in ectotherms, but rarely causes serious disease in humans. The ESX-1 secretion system is conserved and functional in M. marinum, and strains lacking the secretion machine are severely attenuated. This attenuation can be complemented using the paralogous genes from M. tuberculosis, suggesting that the functions of these proteins are conserved in both species.
Additional studies are ongoing to understand the interaction of M. marinum with the environmental amoeba, Acanthamoeba castellanii. My laboratory will use this host-pathogen model to study the ESX-1 secretion system, and to identify additional mycobacterial virulence determinants.
The majority of the experiments in my laboratory combine molecular, genetic and biochemical approaches to better understand mycobacterial pathogenesis. Understanding how mycobacteria cause disease will lead to better anti-tuberculosis vaccine development.
The ESX-1 Secretion System
Figure 1: The ESX-1 secretion machine translocates virulence factors across the mycobacterial cytoplasmic membrane. Rv3877 is a multi-transmembrane protein that likely contributes to the formation of a trans-membrane pore. There are three AAA ATPases associated with ESX-1, including Rv3870, Rv3871 and Rv3868, which likely provide energy for secretion. Substrates include the CFP-10/ESAT-6 pair, EspC, EspF, EspA, EspB, EspR and Mh3864. Our work supports a model in which C-terminal regions of ESX-1substrates function to target them to cognate ATPases, either directly or through protein interaction with other substrates. The CFP-10 signal sequence targets substrates to Rv3871, while the C-terminal amino acids of EspC targets substrates to Rv3868. One possibility is that prior to or after the formation of a multi-substrate complex (likely including CFP-10, ESAT-6, EspF and EspC), engagement of the C-termini by the ESX-1-associated ATPases activates the machine for secretion. EspB likely is indirectly recognized through Rv3879c by Rv3871 (McLaughlin et al., 2007), while EspA (Fortune et. al. 2005) is likely targeted through Rv3868, although the mechanism by which this occurs is unknown thus far. Mh3864 (Carlsson et al., 2009) and EspR (Raghavan et. al.,2008) are also secreted by ESX-1, but the way that these substrates are targeted remains unknown.
Kennedy, G. K., Morisaki, J.K. and P.A. DiGiuseppe Champion. (2012) Conserved mechanisms of Mycobacterium marinum pathogenesis within the environmental amoeba, Acanthamoeba castellanii. Applied and Environmental Microbiology, March; 78(6): 2049-2052
Li,Y., Champion, M.M., Sun, L., DiGiuseppe Champion, P.A., Wojcik, R., and Norman J. Dovichi. (2012) CZE-ESI-MS/MS as an alternative proteomics platform to UPLC-ESI-MS/MS for samples of intermediate complexity. Analytical Chemistry. Feb 7;84(3):1617-22
Shiloh, M.U. and DiGiuseppe Champion, P.A. To catch a killer: What can mycobacterial models teach us about M. tuberculosis? Review, Current Opinions in Microbiology, 2010 Feb; 13(1):86-92.
DiGiuseppe Champion, P.A., Champion, M.M., Manzanillo, P.M. and J.S. Cox. ESX-1 Secreted Virulence Factors Are Recognized by Multiple Cytosolic AAA ATPases in Pathogenic Mycobacteria. Molecular Microbiology 2009; 73(5):950-62.
Abdallah, A.M., Gey van Pittius, N., DiGiuseppe Champion, P.A., Cox, J.S., Luirink, J., Vandenbroucke-Grauls, C.M.J.E., Appelmelk, B.J. and Bitter, W. Type VII Secretion, Mycobacterium shows the way. Review, Nature Reviews Microbiology, 5(11):883-91.
DiGiuseppe Champion, P.A. and J.S. Cox, Protein secretion in Mycobacteria. Review, Cellular Microbiology 9 (6), 1376–1384.
DiGiuseppe Champion, P.A., S.A. Stanley, M.M. Champion, E.J. Brown, and J.S. Cox, C-terminal signal sequence promotes virulence factor secretion in Mycobacterium tuberculosis. Science 2006 15 September Vol. 313. no. 5793, pp. 1583 – 1584.