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Miriam Braunstein, Ph.D. |
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Microbiology and Immunology |
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Room 816 Mary Ellen Jones Building |
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919-966-5051, braunste@med.unc.edu |
Research
Summary
There
is a desperate need for new anti-tuberculosis strategies.
An increased understanding of the pathogenesis of M.
tuberculosis will facilitate this goal.
Following inhalation, M. tuberculosis bacilli reach the alveolar space and are taken up by macrophages. The ability of this bacterium to survive and grow within macrophages is central to its virulence. Exported (surface and secreted) proteins of M. tuberculosis are candidates for being involved in this process as their location positions them to be the first molecules to interact and potentially modify the host environment to enable survival. In other bacterial pathogens there are numerous examples of exported proteins and the respective protein export pathways contributing to virulence. In M. tuberculosis protein export also plays an important role in pathogenesis; yet, the responsible transport pathways and the majority of exported proteins remain unexplored. The research in our laboratory is focused on characterizing these aspects of M. tuberculosis and defining the role they play in virulence.
Protein
Export Pathways in Mycobacteria SecA2-dependent export pathway:
Our research has uncovered an unusual feature of protein export in mycobacteria – the presence of two functioning SecA homologues (SecA1 and SecA2). The Sec-dependent protein export pathway has been well characterized in other bacteria, notably Escherichia coli and Bacillus subtilis, and it is responsible for the transport across the cytoplasmic membrane of proteins containing recognizable signal sequences at their amino terminus. The SecA protein plays a central role in this export pathway. Based on studies in other bacteria it was generally assumed that a single essential SecA was universally present in bacteria. However, M. tuberculosis is part of a developing group of Gram-positive pathogenic bacteria that share the uncommon property of possessing two secA genes (secA1 and secA2). We have demonstrated in mycobacteria that SecA1 is an essential protein and appears to function as the “housekeeping” SecA. In contrast, SecA2 is a non-essential SecA homologue. This "extra" SecA is an accessory secretion factor that exports a specific subset of proteins. We deleted the secA2 gene from M. tuberculosis and evaluated the corresponding mutant in the mouse model of tuberculosis. This secA2 mutant is attenuated in mice, demonstrating a role for SecA2 in M. tuberculosis pathogenesis. Using cultured murine bone marrow derived macrophages we have also shown that the secA2 mutant is defective in intracellular growth within macrophages. We believe that SecA2 exports proteins that are involved in virulence. Genetic, biochemical, and molecular approaches are underway to define the role of SecA2 in pathogenesis, to identify proteins exported by SecA2, and to understand how SecA2 functions in protein export.
Protein
Export Pathways in Mycobacteria Tat-dependent export pathway:
Many bacteria also utilize a twin-arginine translocation (Tat) pathway to transport proteins across the cytoplasmic membrane. The Tat pathway operates independently of the Sec pathway and is distinguished by the ability to translocate proteins in a folded state. Substrates of the Tat pathway are synthesized as precursor proteins containing N-terminal signal sequences with the same overall structure as Sec signal sequences. The major distinction between Sec and Tat signal sequences is the presence of a characteristic twin-arginine ‘RR’ motif in the charged region of Tat signal sequences. The Tat pathway functions in some bacterial pathogens and had been shown to contribute to virulence.
Using the fast-growing nonpathogen M. smegmatis we demonstrated that the Tat pathway is functional in mycobacteria. We further showed that mycobacterial b-lactamases are Tat substrates, which require export by the Tat system to protect the bacterium from b-lactam antibiotics. We are now exploiting the Tat-dependence of the M. tuberculosis b-lactamase (BlaC) as a genetic reporter to identify the exported M. tuberculosis Tat substrates, including virulence factors. This is being achieved by selecting from a genomic M. tuberculosis expression library ORFs that promote export of a truncated ‘BlaC reporter.
