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 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. These studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses.
authors.affiliation: Lawrence Livermore National Laboratory, Livermore, CA, United States of America, 94550.|University of California Davis, NSF, Center for Biophotonics, Sacramento, CA, United States of America, 95817.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Coleman
authors.affiliation: Humboldt State University, Department of Chemistry, Arcata, CA, United States of America, 95521.
authors.forename: Jenny A
authors.initials: JA
authors.lastname: Cappuccio
authors.affiliation: Lawrence Livermore National Laboratory, Livermore, CA, United States of America, 94550.
authors.forename: Craig D
authors.initials: CD
authors.lastname: Blanchette
authors.affiliation: University of California Davis, NSF, Center for Biophotonics, Sacramento, CA, United States of America, 95817.
authors.forename: Tingjuan
authors.initials: T
authors.lastname: Gao
authors.affiliation: Lawrence Livermore National Laboratory, Livermore, CA, United States of America, 94550.
authors.forename: Erin S
authors.initials: ES
authors.lastname: Arroyo
authors.affiliation: Lawrence Livermore National Laboratory, Livermore, CA, United States of America, 94550.
authors.forename: Angela K
authors.initials: AK
authors.lastname: Hinz
authors.affiliation: Lawrence Livermore National Laboratory, Livermore, CA, United States of America, 94550.
authors.forename: Feliza A
authors.initials: FA
authors.lastname: Bourguet
authors.affiliation: Lawrence Livermore National Laboratory, Livermore, CA, United States of America, 94550.
authors.forename: Brent
authors.initials: B
authors.lastname: Segelke
authors.affiliation: Lawrence Livermore National Laboratory, Livermore, CA, United States of America, 94550.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Hoeprich
authors.affiliation: University of California Davis, NSF, Center for Biophotonics, Sacramento, CA, United States of America, 95817.
authors.forename: Thomas
authors.initials: T
authors.lastname: Huser
authors.affiliation: Lawrence Livermore National Laboratory, Livermore, CA, United States of America, 94550.
authors.forename: Ted A
authors.initials: TA
authors.lastname: Laurence
authors.affiliation: University of Texas Medical Branch, Galveston, TX, United States of America, 77555.
authors.forename: Vladimir L
authors.initials: VL
authors.lastname: Motin
authors.affiliation: University of California Davis, NSF, Center for Biophotonics, Sacramento, CA, United States of America, 95817.
authors.forename: Brett A
authors.initials: BA
authors.lastname: Chromy
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D055592:Biophysical Phenomena; D005786:Gene Expression Regulation; D008074:Lipoproteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D018625:Microscopy, Atomic Force; D046912:Multiprotein Complexes / Q000648:ultrastructure; D053758:Nanoparticles / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D015010:Yersinia pestis / Q000235:genetics / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D008074:Lipoproteins; D046912:Multiprotein Complexes; C078909:YopB protein, Yersinia; C078910:YopD protein, Yersinia