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

Abstract

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 andmore » 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.« less

Authors:
 [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)
Publication Date:
Research Org.:
Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
Sponsoring Org.:
National Science Foundation (NSF); USDOE National Nuclear Security Administration (NNSA)
OSTI Identifier:
1259528
Alternate Identifier(s):
OSTI ID: 1466148
Report Number(s):
LLNL-JRNL-737902
Journal ID: ISSN 1932-6203
Grant/Contract Number:  
AC52-07NA27344; PHY 0120999; 06-SI-003; 01-ERD- 045
Resource Type:
Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 11; Journal Issue: 3; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; Biological and medical sciences

Citation Formats

Coleman, Matthew A., Cappuccio, Jenny A., Blanchette, Craig D., Gao, Tingjuan, Arroyo, Erin S., Hinz, Angela K., Bourguet, Feliza A., Segelke, Brent, Hoeprich, Paul D., Huser, Thomas, Laurence, Ted A., Motin, Vladimir L., and Chromy, Brett A. Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles. United States: N. p., 2016. Web. doi:10.1371/journal.pone.0150166.
Coleman, Matthew A., Cappuccio, Jenny A., Blanchette, Craig D., Gao, Tingjuan, Arroyo, Erin S., Hinz, Angela K., Bourguet, Feliza A., Segelke, Brent, Hoeprich, Paul D., Huser, Thomas, Laurence, Ted A., Motin, Vladimir L., & Chromy, Brett A. Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles. United States. https://doi.org/10.1371/journal.pone.0150166
Coleman, Matthew A., Cappuccio, Jenny A., Blanchette, Craig D., Gao, Tingjuan, Arroyo, Erin S., Hinz, Angela K., Bourguet, Feliza A., Segelke, Brent, Hoeprich, Paul D., Huser, Thomas, Laurence, Ted A., Motin, Vladimir L., and Chromy, Brett A. Fri . "Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles". United States. https://doi.org/10.1371/journal.pone.0150166. https://www.osti.gov/servlets/purl/1259528.
@article{osti_1259528,
title = {Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles},
author = {Coleman, Matthew A. and Cappuccio, Jenny A. and Blanchette, Craig D. and Gao, Tingjuan and Arroyo, Erin S. and Hinz, Angela K. and Bourguet, Feliza A. and Segelke, Brent and Hoeprich, Paul D. and Huser, Thomas and Laurence, Ted A. and Motin, Vladimir L. and Chromy, Brett A.},
abstractNote = {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.},
doi = {10.1371/journal.pone.0150166},
journal = {PLoS ONE},
number = 3,
volume = 11,
place = {United States},
year = {2016},
month = {3}
}

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Works referenced in this record:

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Atomic force microscopy differentiates discrete size distributions between membrane protein containing and empty nanolipoprotein particles
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Quantifying Interactions of a Membrane Protein Embedded in a Lipid Nanodisc using Fluorescence Correlation Spectroscopy
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Amino acid and structural variability of Yersinia pestis LcrV protein
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Minimal YopB and YopD translocator secretion by Yersinia is sufficient for Yop-effector delivery into target cells
journal, February 2007


Yersinia pestis YopD 150–287 fragment is partially unfolded in the native state
journal, March 2008


Type III secretion gets an LcrV tip
journal, May 2006


Different Apolipoproteins Impact Nanolipoprotein Particle Formation
journal, November 2007

  • Chromy, Brett A.; Arroyo, Erin; Blanchette, Craig D.
  • Journal of the American Chemical Society, Vol. 129, Issue 46, p. 14348-14354
  • DOI: 10.1021/ja074753y

Hydrogen Production by a Hyperthermophilic Membrane-Bound Hydrogenase in Water-Soluble Nanolipoprotein Particles
journal, June 2009

  • Baker, Sarah E.; Hopkins, Robert C.; Blanchette, Craig D.
  • Journal of the American Chemical Society, Vol. 131, Issue 22
  • DOI: 10.1021/ja809251f

Insertion of Membrane Proteins into Discoidal Membranes Using a Cell-Free Protein Expression Approach
journal, August 2008

  • Katzen, Federico; Fletcher, Julia E.; Yang, Jian-Ping
  • Journal of Proteome Research, Vol. 7, Issue 8
  • DOI: 10.1021/pr800265f

The type III secretion injectisome
journal, November 2006


The Salmonella type III secretion translocon protein SspC is inserted into the epithelial cell plasma membrane upon infection
journal, September 2000


LcrV is a channel size-determining component of the Yop effector translocon of Yersinia
journal, February 2001


Type III export: new uses for an old pathway
journal, April 2001


Protective Anti‐V Antibodies Inhibit Pseudomonas and Yersinia Translocon Assembly within Host Membranes
journal, July 2005

  • Goure, Julien; Broz, Petr; Attree, Olivier
  • The Journal of Infectious Diseases, Vol. 192, Issue 2
  • DOI: 10.1086/430932

The V-Antigen of Yersinia Forms a Distinct Structure at the Tip of Injectisome Needles
journal, October 2005


Vaccination of Mice with a Yop Translocon Complex Elicits Antibodies That Are Protective against Infection with F1- Yersinia pestis
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