Molecular Genetics of Mycobacterial Protein Secretion and Pathogenesis
Patricia A. Champion
One in three people globally are infected with Mycobacterium tuberculosis, the causative agent of Tuberculosis (TB). Although only 5-10% of infected people develop active disease, there are still approximately 1.4 million deaths annually. This burden of infection and disease is in part due to a lack of a viable vaccine that protects adults from pulmonary disease, the most common form of TB. To develop the vaccines and therapeutics needed to control the TB epidemic, we first need a better understanding of M. tuberculosis biology and the mechanisms it uses to circumvent the host immune response.
An key mechanism that bacteria use to promote their survival is by the targeted transport of bacterial proteins, small molecules or nucleic acids directly into the host (secretion). In the host these molecules directly interact with and disrupt the function of cellular processes which are required for identifying and eliminating the bacteria. Targeted protein secretion is used by several successful bacterial pathogens, including M. tuberculosis. Esx-1(ESAT-6 system-1) is a specific protein export system that is conserved across diverse bacterial species. Through a better understanding of protein transport systems including Esx-1 we will gain important insight into how mycobacteria interact with the host and cause disease. The long-term goal of my research program is to define the molecular mechanisms underlying mycobacterial pathogenesis. To address this goal, our current research is focused on identifying novel genes and mechanisms required for mycobacterial Esx-1 export. We have devised several new assays to measure Esx-1 export. We are using these assays to identify novel genes in M. marinum required for Esx-1 export and virulence. We are currently defining how each gene promotes Esx-1 export and virulence. TO do so we use a multidisciplinary approach including bacterial genetics, molecular biology, proteomics and biochemistry. We expect that this course of research will not only expand our understanding of the Esx-1 system, but may also lead to the identification of novel targets for anti-virulence based therapeutics against TB.
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.
Mba Medie, F., Champion, M.M., Williams, E.A., and DiGiuseppe Champion, P.A. (2014). Homeostasis of N-a terminal acetylation of EsxA correlates with virulence in M. marinum. Infect. Immun. doi: 10.1128/IAI.02153-14. Published ahead of print 18 August 2014.
Champion, M.M, Williams, E.A., Pinapati, R.S. and DiGiuseppe Champion, P.A. (2014). Correlation of Phenotypic Profiles Using Targeted Proteomics Identifies Mycobacterial Esx-1 Substrates. J. Proteome Res. doi: 10.1021/pr500484w. Published 8 August 2014.
Kennedy, G.M, Hooley, G.H., Champion M.M., Mba Medie, F.M. and DiGiuseppe Champion, P.A. (2014). A novel ESX-1 locus reveals that surface associated ESX-1 substrates mediate virulence in Mycobacterium marinum J. Bacteriol. May;196(10):1877-88. doi: 10.1128/JB.01502-14. Published ahead of print 7 March 2014.
DiGiuseppe Champion, P.A. (2013). Disconnecting in vitro ESX-1 secretion from mycobacterial virulence. J Bacteriol. 2013 Dec; 195(24):5418-20. doi: 10.1128/JB.01145-13.
Champion, M.M., Williams, E.A., Kennedy, G.M and DiGiuseppe Champion, P.A. (2012). Direct Detection of Bacterial Protein Secretion Using Whole Colony Proteomics. Molecular and Cellular Proteomics, Sept ;12 (9): 596-604
Kennedy, G. K., Morisaki, J.K. and DiGiuseppe Champion, P.A. (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 Dovichi, N.J. (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. (2010). 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 (2009). 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. (2007). Type VII Secretion, Mycobacterium shows the way. Review, Nature Reviews Microbiology. 5(11):883-91.
DiGiuseppe Champion, P.A. and J.S. Cox (2007). 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 (2006). C-terminal signal sequence promotes virulence factor secretion in Mycobacterium tuberculosis. Science. 2006 15 September Vol. 313. no. 5793, pp. 1583 – 1584.