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Ramsdell, Talia Lynn

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Ramsdell

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Talia Lynn

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Ramsdell, Talia Lynn
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    Molecular Motors of ESX-Type Secretion Systems
    (2012-12-17) Ramsdell, Talia Lynn; Fortune, Sarah Merritt; Husson, Robert; Rubin, Eric; Sassetti, Christopher; Marti, Matthias
    Tuberculosis is an enormous global health problem. Despite decades of research, the mechanism(s) by which Mycobacterium tuberculosis (Mtb) mediates virulence remains incompletely understood. The ESX-1 secretion system is critical for Mtb to survive and cause disease in vivo, but its primary function and mechanism of action are unclear. The many inherent challenges of working with this slow-growing pathogen often limit the experimental approaches that can be used to address these questions. Thus, we have developed a model system in the nonpathogenic bacterium Bacillus subtilis to study ESX-type secretion systems. Here, we demonstrate that the B. subtilis yuk operon encodes an ESX-type secretion system responsible for the secretion of YukE. Additionally, we demonstrate that the yuk system is active in B. subtilis during conditions of nutrient deprivation and is required for normal biofilm formation. Interestingly, this is similar to our findings that the Mtb ESX-1 system plays dual roles in protein secretion and modulating cell wall integrity. One defining feature of all ESX loci is the presence of an FtsK/SpoIIIE family ATPase. Interestingly, these ATPases have a domain structure unique to ESX-associated ATPases, where each protein contains multiple (2-3) enzymatic domains. We used our B. subtilis system to dissect the mechanism of action of this unique class of motor proteins. We find that the yuk-encoded ATPase YukBA dimerizes to form a hexamer of enzymatic subunits that are differentially required for secretion. Strikingly, we find a unique requirement for rotational symmetry in the nucleotide binding activity of the subunits. Finally, we compared the energy requirements of the Mtb ESX-1 system and the B. subtilis yuk system. We find that these systems have some overlapping ATPase requirements for protein secretion and cell wall integrity/biofilm formation, suggesting that there is a conservation of function among ESX-type systems. We also find that some ATPase domains are differentially required for function between these two systems, which we postulate is due to the split protein architecture of the ESX-1-encoded ATPases. Together, these findings highlight the power of using a B. subtilis model system to understand the function and mechanism of action of ESX-type secretion systems.
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    EspA Acts as a Critical Mediator of ESX1-Dependent Virulence in Mycobacterium tuberculosis by Affecting Bacterial Cell Wall Integrity
    (Public Library of Science, 2010) Garces, Alejandra; Woodworth, Joshua S.; Krastins, Bryan; Atmakuri, Krishnamohan; Chase, Michael; Rothchild, Alissa C.; Ramsdell, Talia Lynn; Lopez, Mary; Behar, Samuel M.; Sarracino, David A.; Fortune, Sarah
    Mycobacterium tuberculosis (Mtb) requires the ESX1 specialized protein secretion system for virulence, for triggering cytosolic immune surveillance pathways, and for priming an optimal CD8+ T cell response. This suggests that ESX1 might act primarily by destabilizing the phagosomal membrane that surrounds the bacterium. However, identifying the primary function of the ESX1 system has been difficult because deletion of any substrate inhibits the secretion of all known substrates, thereby abolishing all ESX1 activity. Here we demonstrate that the ESX1 substrate EspA forms a disulfide bonded homodimer after secretion. By disrupting EspA disulfide bond formation, we have dissociated virulence from other known ESX1-mediated activities. Inhibition of EspA disulfide bond formation does not inhibit ESX1 secretion, ESX1-dependent stimulation of the cytosolic pattern receptors in the infected macrophage or the ability of Mtb to prime an adaptive immune response to ESX1 substrates. However, blocking EspA disulfide bond formation severely attenuates the ability of Mtb to survive and cause disease in mice. Strikingly, we show that inhibition of EspA disulfide bond formation also significantly compromises the stability of the mycobacterial cell wall, as does deletion of the ESX1 locus or individual components of the ESX1 system. Thus, we demonstrate that EspA is a major determinant of ESX1-mediated virulence independent of its function in ESX1 secretion. We propose that ESX1 and EspA play central roles in the virulence of Mtb in vivo because they alter the integrity of the mycobacterial cell wall.