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Tuberculosis is a severe global health problem. Currently, nearly two million people die each year from tuberculosis and one third of the world's population is believed infected with Mycobacterium tuberculosis, the bacterium responsible for this disease. The situation is even more alarming when drug resistant (MDR and XDR) M. tuberculosis and the HIV co-epidemic are considered. In order to control tuberculosis, new drugs and vaccines are needed. A better understanding of the basic biology and pathogenesis of M. tuberculosis will aid such development efforts.
Protein Export Pathways of M. tuberculosis
Research from my laboratory demonstrated the existence of conserved protein export pathways in mycobacteria: the general secretion (Sec) pathway and the twin-arginine translocation (Tat) pathway. Together, these two systems probably account for the majority of protein transport across the cytoplasmic membrane of mycobacteria. My research also identified a specialized system that is dedicated to a select subset of secreted and surface proteins. This SecA2-dependent export system is important to virulence as shown by the attenuated phenotype of a M. tuberculosis secA2 mutant in macrophages and mice. Macrophages infected with the secA2 mutant produce higher levels of pro-inflammatory cytokines and effectors. We hypothesize that the role of SecA2 in virulence is to export proteins so they are positioned to interact with the host and limit protective immune responses. We are working to better understand this role of SecA2 in virulence by identifying host pathways influenced by SecA2. Our most recent data suggest that MyD88-dependent pathways are involved. We are also applying proteomic approaches to identify the proteins exported by the SecA2 pathway. So far, we have identified a small subset of secreted proteins and surface lipoproteins that are SecA2-dependent. Finally, we are working to understand how SecA2 functions in protein export. Specifically, we want to know how SecA2 recognizes the appropriate substrates and exports them.
Exported Proteins of M. tuberculosis
The M. tuberculosis proteins exported to the bacterial surface or secreted are ideally positioned to modify the host and promote virulence. Although the subject of investigation for some time, many exported M. tuberculosis proteins remain to be identified and characterized.
My laboratory developed genetic reporter systems that can be used directly in M. tuberculosis for large-scale identification of exported proteins. These systems use b-lactamase to report on export of a protein to which the enzyme is fused. Because b-lactam antibiotics target the cell wall, b-lactamases must be exported to protect bacterial from drug. We demonstrated that the TEM-1 b-lactamase, derived from a clinical isolate of Escherichia coli, can report on protein export by Sec or Tat pathways in a b-lactam sensitive mutant of M. tuberculosis. We engineered this reporter onto the end of a transposon and are currently using it in M. tuberculosis for genome-wide identification of Open Reading Frams (ORFs) that encode for exported proteins. In most cases, the transposon insertion that identifies an exported protein also eliminates function. We are systematically screeing our b-lactam resistant transposon mutant library (current count 98 unique ORFs identified with active transposon insertions) in macrophages to identify new M. tuberculosis exported proteins that function in intracellular growth. These studies are significant in serving to assign biological properties to unknown ORFs of M. tuberculosis. My laboratory also showed that the same b-lactamase reporter works during growth of M. tuberculosis in b-lactam treated THP1 macrophage-like cells. This is a significant finding, since a limitation of current methods to identify exported proteins of intracellular pathogens is the reliance on in vitro growth conditions. This discovery will allow us to use the same genetic reporter to identify new M. tuberculosis proteins only exported or expressed in the context of the host.
We are also working to specifically identify the Tat exported proteins of M. tuberculosis with the goal of determining the role this pathway plays in virulence. Our studies of the Tat export pathway identified the native b-lactamase (BlaC) of M. tuberculosis as a Tat exported protein. BlaC can also function as a reporter of proteinn export in M. tuberculosis. Interestingly, it differs from TEM-1 b-lactamase in that BlaC only works with Tat exported proteins. We are taking advantage of the Tat-specific nature of the BlaC reporter to identify the Tat exported proteins of M. tuberculosis.
These b-lactamase reporters represent long-awaited genetic tools for large scale identification and study of exported proteins directly in M. tuberculosis. We expect that this research will identify new proteins important to pathogenesis. Our long-term plans include characterizing the function of the new exported virulence factors we identify.
In summary, my research is focused on understanding the protein export systems and exported proteins of M. tuberculosis and the roles they play in pathogenesis. This research will lead to an increased understanding of the pathogenic mechanisms employed by M. tuberculosis and it has potential to reveal new targets and strategies for disease intervention. It may also help in construction of recombinant mycobacterial strains, for use as novel vaccines, with enhanced ability to export protective antigens.
McCann JR, McDonough JA, Sullivan JT, Feltcher ME, Braunstein M. (2011): Genome-wide identification of Mycobacterium tuberculosis exported proteins with roles in intracellular growth. J Bacteriol. 193(4):854-61.
Feltcher ME, Sullivan JT, Braunstein M. (2010): Protein export systems of Mycobacterium tuberculosis: novel targets for drug development? Future Microbiol. 5(10):1581-97. Review.
McElvania Tekippe E, Allen IC, Hulseberg PD, Sullivan JT, McCann JR, Sandor M, Braunstein M, Ting JP. (2010): Granuloma formation and host defense in chronic Mycobacterium tuberculosis infection requires PYCARD/ASC but not NLRP3 or caspase-1. PLoS One. 5(8):e12320
Rigel NW, Gibbons HS, McCann JR, McDonough JA, Kurtz S, Braunstein M. (2009) The Accessory SecA2 System of Mycobacteria Requires ATP Binding and the Canonical SecA1. J Biol Chem. 284(15):9927-36.
*McDonough, J.A., *McCann, J.R., McElvania Tekippe, E. Silverman, J.S., Rigel, N.W. and Braunstein, M. (2008): Identification of functional Tat signal sequences in Mycobacterium tuberculosis proteins. *co-first authors.
Hou, J.M., D’Lima, N.G., Rigel, N.W., Gibbons, H.S., McCann, J.R., *Braunstein, M. and Teschke, C.M. (2008). ATPase activity of Mycobacterium tuberculosis SecA1 and SecA2 proteins and its importance to SecA2 function in macrophages. Journal of Bacteriology. 2008 190:4880-4887. *co-corresponding author
Rigel, N.W. and Braunstein, M. (2008) A new twist on an old pathway – accessory Sec systems. Molecular Microbiology 69:291-302
McDonough, J. A. and Braunstein, M. (2008). Protein transport pathways in Mycobacterium tuberculosis. In Handbook of Tuberculosis:Molecular Biology and Biochemistry. S.H.E. Kaufmann and Rubin, E. eds. (Wiley-VCH Publisher). p. 111-130
Mohamedmohaideen, N.N., Palaninathan, S.K., Morin, P.M., Williams, B.J., Braunstein, M., Tichy, S.E., Locker, J., Russell, D.H., Jacobs, W.R. Jr., and Sacchettini. J.C. (2008) The structure and function of the virulence-associated high temperature requirement A of M. tuberculosis. Biochemistry 47:6092-6102.
McCann, J.R., McDonough, J.M., Pavelka, M.S. and Braunstein, M. (2007). ß-lactamase can function as a reporter of bacterial protein export during Mycobacterium tuberculosis infection of host cells. Microbiology, 153:3350-3359.
Lu, D., Garcia-Contreras, L., Xu, D., Kurtz, S.L., Liu, J., Braunstein, M., McMurray, D.N. and Hickey, A.J. (2007). Poly (Lactide-co-Glycolide) microspheres in respirable sizes can enhance an in vitro T cell response to Recombinant Mycobacterium tuberculosis Antigen 85B. Pharmaceutical Research, 24:1834-1843
Hinchey, J., Lee, S., Manjunatha, V., Chen, B., Basaraba, R.J., Jeon, B.Y., Derrick, S.C., Chan, J., Braunstein, M., Orme, I.M., Morris, S.L., Jacobs, W.R. and Porcelli, S.A. (2007). Enhanced priming of adaptive immunity by a mutant of Mycobacterium tuberculosis that is defective in inhibition of host cell apoptosis. Journal of Clinical Investigation, 171: 2279-2288.
Gibbons, H.S., Wolschendorf, F., Abshire, M., Niederwies, M. and Braunstein, M. (2007) Identification of two Mycobacterium smegmatis lipoproteins exported by the SecA2-dependent export pathway. Journal of Bacteriology, 189: 5090-5100
Guo, X.V., Monteleone, M., Klotzsche, M., Kamionka, A., Hillen, W., Braunstein, M., Ehrt, S. and Schnappinger, D. (2007). Silencing essential protein secretion in Mycobacterium smegmatis by using tetracycline repressors. Journal of Bacteriology, 189: 4614-4623.
Kurtz, S., McKinnon ,K.P., Runge, M.S., Ting, J.P. and Braunstein, M. (2006). The SecA2 secretion factor of Mycobacterium tuberculosis promotes growth in macrophages and limits host cell activation. Infection and Immunity, 74: 6855-6864.
McDonough, J.A., Hacker, K.E., Flores, A.R., Pavelka, M.S. and Braunstein, M. (2005). The twin-arginine translocation (Tat) pathway of Mycobacterium smegmatis is functional and required for the export of mycobacterial beta-lactamases. Journal of Bacteriology, 187:7667-7679.
Williams, K.L., Lich, J.D., Rallabhandi, P., Reed, W., Kurtz, S., Coffield, N., Su, L., Vogel, S.N., Braunstein, M. and Ting, J.P.-Y. (2005). Monarch-1 is an antagonist of TLR-induced inflammatory signal transduction. Journal of Biological Chemistry, 280:39914-39924.
Kurtz, S. and Braunstein, M. (2005) Protein secretion and export in Mycobacterium tuberculosis. In Mycobacterium: Molecular Microbiology. Parish, T. ed. (Horizon Press). p. 71-138.