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Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles

Journal Article · · PLoS ONE
 [1];  [2];  [3];  [4];  [3];  [3];  [3];  [3];  [3];  [4];  [3];  [5];  [4]
  1. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Univ. of California, Davis, CA (United States)
  2. Humboldt State Univ., Arcata, CA (United States)
  3. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
  4. Univ. of California, Davis, CA (United States)
  5. Univ. of Texas Medical Branch, Galveston, TX (United States)
+ Show Author Affiliations

Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteins as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. Ultimately, these studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses.

Research Organization:
Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
Sponsoring Organization:
USDOE; National Science Foundation (NSF)
Grant/Contract Number:
AC52-07NA27344
OSTI ID:
1259528
Alternate ID(s):
OSTI ID: 1466148
Journal Information:
PLoS ONE, Journal Name: PLoS ONE Journal Issue: 3 Vol. 11; ISSN 1932-6203
Publisher:
Public Library of ScienceCopyright Statement
Country of Publication:
United States
Language:
English

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Cited By (2)

Cell-Free Co-Translational Approaches for Producing Mammalian Receptors: Expanding the Cell-Free Expression Toolbox Using Nanolipoproteins journal July 2019
Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development journal July 2017

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