@article{MietrachSchlosserGeibel2019,
  author    = {Mietrach, Nicole and Schlosser, Andreas and Geibel, Sebastian},
  title     = {An extracellular domain of the EsaA membrane component of the type VIIb secretion system: expression, purification and crystallization},
  series = {Acta Crystallographica Section F},
  volume    = {75},
  journal   = {Acta Crystallographica Section F},
  number    = {12},
  doi       = {10.1107/S2053230X1901495X},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-213681},
  pages     = {725-730},
  year      = {2019},
  abstract  = {The membrane protein EsaA is a conserved component of the type VIIb secretion system. Limited proteolysis of purified EsaA from Staphylococcus aureus USA300 identified a stable 48 kDa fragment, which was mapped by fingerprint mass spectrometry to an uncharacterized extracellular segment of EsaA. Analysis by circular dichroism spectroscopy showed that this fragment folds into a single stable domain made of mostly α-helices with a melting point of 34.5°C. Size-exclusion chromatography combined with multi-angle light scattering indicated the formation of a dimer of the purified extracellular domain. Octahedral crystals were grown in 0.2 M ammonium citrate tribasic pH 7.0, 16\% PEG 3350 using the hanging-drop vapor-diffusion method. Diffraction data were analyzed to 4.0 {\AA} resolution, showing that the crystals belonged to the enantiomorphic tetragonal space groups P41212 or P43212, with unit-cell parameters a = 197.5, b = 197.5, c = 368.3 {\AA}, α = β = γ = 90°.},
  language  = {en}
}
@phdthesis{Mietrach2020,
  author    = {Mietrach, Nicole Aline},
  title     = {Structural and functional elucidation of the Type VIIb secretion system from Staphylococcus aureus},
  doi       = {10.25972/OPUS-21482},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-214824},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {The Type VII secretion system (T7SS) is linked to virulence and long-term pathogenesis in a broad range of Gram-positive bacteria, including the human commensal and pathogen Staphylococcus aureus. The Type VIIb secretion system (T7SSb) is responsible for the export of small toxic proteins, which induce antibacterial immune responses and mediate bacterial persistence in the host. In addition, it is also involved in bacterial competition. The T7SSb requires several proteins to build up the secretion machinery. This work focuses on the structural and functional investigation of the motor ATPase EssC and the putative pore forming, multi-pass membrane component EsaA. Both proteins are indispensable for substrate secretion. EssC belongs to the FtsK/SpoIIIE ATPase family and is conserved among the T7SSs. It contains three C-terminal, cytosolic ATPase domains, designated as EssC- D1, -D2 and -D3, whereby EssC-D3 is the most distal one. In this thesis, I am presenting the crystal structure of the EssC-D3 at 1.7 {\AA} resolution. As the deletion of EssC-D3 abrogates substrate export, I have demonstrated that this domain comprises a hydrophobic, surface-exposed pocket, which is required for substrate secretion. More specifically, I have identified two amino acids involved in the secretion process. In addition, my results indicate that not only EssC-D3 is important for substrate interaction but also EssC-D2 and/or EssC-D1. Unlike in the related Yuk T7SSb of Bacillus subtilis, the ATPase activity of D3 domain contributes to substrate secretion. Mutation of the modified Walker B motif in EssC-D3 diminishes substrate secretion completely. The membrane protein EsaA encompasses an extracellular segment spanning through the cell wall of S. aureus. I was able to reveal that this part folds into a stable domain, which was crystallized and diffracted up to 4 {\AA}. The first attempts to dissolve the structure failed due to a lack of homologues structures. Therefore, crystals for single-wavelength anomalous dispersion, containing selenomethionyl-substitutes, were produced and the structure solution is still in progress. Preliminary experiments addressing the function of the extracellular domain indicate an important role in substrate secretion and bacterial competition.},
  subject      = {Secretion},
  language  = {en}
}
@article{MielichSuessWagnerMietrachetal.2017,
  author    = {Mielich-S{\"u}ss, Benjamin and Wagner, Rabea M. and Mietrach, Nicole and Hertlein, Tobias and Marincola, Gabriella and Ohlsen, Knut and Geibel, Sebastian and Lopez, Daniel},
  title     = {Flotillin scaffold activity contributes to type VII secretion system assembly in Staphylococcus aureus},
  series = {PLoS Pathogens},
  volume    = {13},
  journal   = {PLoS Pathogens},
  number    = {11},
  doi       = {10.1371/journal.ppat.1006728},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170035},
  pages     = {e1006728},
  year      = {2017},
  abstract  = {Scaffold proteins are ubiquitous chaperones that promote efficient interactions between partners of multi-enzymatic protein complexes; although they are well studied in eukaryotes, their role in prokaryotic systems is poorly understood. Bacterial membranes have functional membrane microdomains (FMM), a structure homologous to eukaryotic lipid rafts. Similar to their eukaryotic counterparts, bacterial FMM harbor a scaffold protein termed flotillin that is thought to promote interactions between proteins spatially confined to the FMM. Here we used biochemical approaches to define the scaffold activity of the flotillin homolog FloA of the human pathogen Staphylococcus aureus, using assembly of interacting protein partners of the type VII secretion system (T7SS) as a case study. Staphylococcus aureus cells that lacked FloA showed reduced T7SS function, and thus reduced secretion of T7SS-related effectors, probably due to the supporting scaffold activity of flotillin. We found that the presence of flotillin mediates intermolecular interactions of T7SS proteins. We tested several small molecules that interfere with flotillin scaffold activity, which perturbed T7SS activity in vitro and in vivo. Our results suggest that flotillin assists in the assembly of S. aureus membrane components that participate in infection and influences the infective potential of this pathogen.},
  language  = {en}
}