TY - JOUR
T1 - 3D correlative electron microscopy reveals continuity of Brucellacontaining vacuoles with the endoplasmic reticulum
AU - Sedzicki, Jaroslaw
AU - Tschon, Therese
AU - Low, Shyan Huey
AU - Willemart, Kevin
AU - Goldie, Kenneth N
AU - Letesson, Jean-Jacques
AU - Stahlberg, Henning
AU - Dehio, Christoph
N1 - Funding Information:
This work was supported by the Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation; SNSF) (grant 31003A_173119 to C.D.); a European Research Council (ERC) advanced grant (FicModFun) (340330 to C.D.); the Research and Technology Development (RTD) project TargetInfectX in the frame of SystemsX.ch (www.systemX.ch) to C.D. (grant 51RTP0_151029); the Swiss Initiative for Systems Biology; and by grant NCCR TransCure from the SNSF to H.S. The research at UNamur was supported by the Interuniversity Attraction Poles Program initiated by the Federaal Wetenschapsbeleid (Belgian Science Policy Office) (PAI no. P7/28).
Funding Information:
We are grateful to the Jean Celli (Washington State University, Pullman, WA) for providing the JC44 plasmid. We thank Dr Jennifer Lippincott-Schwartz for providing the Emerald-Sec61β construct. We thank Dr Gisou van der Goot for providing the calnexin-GFP construct. The authors acknowledge the technical assistance of Marcel Düggelin (ZMB, University of Basel, Switzerland) and Daniel Mathys (SNI, University of Basel, Switzerland) for the assistance with FIB/SEM imaging.We thank the Imaging Core Facility (IMCF, University of Basel, Switzerland) and in particular Alexia Ferrand for the technical assistance provided on the OMX microscope. We thank Laurent Gelman (Friedrich Miescher Institute, Basel, Switzerland) for the technical assistance provided with the Elyra microscope. We would like to thank Maxime Québatte for critical reading of the manuscript. This work was supported by the Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation; SNSF) (grant 31003A_173119 to C.D.); a European Research Council (ERC) advanced grant (FicModFun) (340330 to C.D.); the Research and Technology Development (RTD) project TargetInfectX in the frame of SystemsX.ch (www.systemX.ch) to C.D. (grant 51RTP0_151029); the Swiss Initiative for Systems Biology; and by grant NCCR TransCure from the SNSF to H.S. The research at UNamur was supported by the Interuniversity Attraction Poles Program initiated by the Federaal Wetenschapsbeleid (Belgian Science Policy Office) (PAI no. P7/28).
Publisher Copyright:
© 2018. Published by The Company of Biologists Ltd.
Copyright:
Copyright 2019 Elsevier B.V., All rights reserved.
PY - 2018/2/1
Y1 - 2018/2/1
N2 - Entry of the facultative intracellular pathogenBrucellainto host cells results in the formation of endosomalBrucella-containing vacuoles (eBCVs) that initially traffic along the endocytic pathway. eBCV acidification triggers the expression of a type IV secretion system that translocates bacterial effector proteins into host cells. This interferes with lysosomal fusion of eBCVs and supports their maturation to replicativeBrucella-containing vacuoles (rBCVs). Bacteria replicate in rBCVs to large numbers, eventually occupying most of the cytoplasmic volume. As rBCV membranes tightly wrap each individual bacterium, they are constantly being expanded and remodeled during exponential bacterial growth. rBCVs are known to carry endoplasmic reticulum (ER) markers; however, the relationship of the vacuole to the genuine ER has remained elusive. Here, we have reconstructed the 3-dimensional ultrastructure of rBCVs and associated ER by correlative structured illumination microscopy (SIM) and focused ion beam/scanning electron microscopic tomography (FIB/SEM). StudyingB. abortus-infected HeLa cells and trophoblasts derived fromB. melitensis-infected mice, we demonstrate that rBCVs are complex and interconnected compartments that are continuous with neighboring ER cisternae, thus supporting a model that rBCVs are extensions of genuine ER.
AB - Entry of the facultative intracellular pathogenBrucellainto host cells results in the formation of endosomalBrucella-containing vacuoles (eBCVs) that initially traffic along the endocytic pathway. eBCV acidification triggers the expression of a type IV secretion system that translocates bacterial effector proteins into host cells. This interferes with lysosomal fusion of eBCVs and supports their maturation to replicativeBrucella-containing vacuoles (rBCVs). Bacteria replicate in rBCVs to large numbers, eventually occupying most of the cytoplasmic volume. As rBCV membranes tightly wrap each individual bacterium, they are constantly being expanded and remodeled during exponential bacterial growth. rBCVs are known to carry endoplasmic reticulum (ER) markers; however, the relationship of the vacuole to the genuine ER has remained elusive. Here, we have reconstructed the 3-dimensional ultrastructure of rBCVs and associated ER by correlative structured illumination microscopy (SIM) and focused ion beam/scanning electron microscopic tomography (FIB/SEM). StudyingB. abortus-infected HeLa cells and trophoblasts derived fromB. melitensis-infected mice, we demonstrate that rBCVs are complex and interconnected compartments that are continuous with neighboring ER cisternae, thus supporting a model that rBCVs are extensions of genuine ER.
KW - CLEM
KW - Correlative light and electron microscopy
KW - Endoplasmic reticulum
KW - FIB/SEM tomography
KW - RBCV
KW - Replicative Brucellacontaining vacuole
KW - Sec61
KW - Brucella melitensis/pathogenicity
KW - Microscopy, Electron, Scanning
KW - Type IV Secretion Systems/ultrastructure
KW - Humans
KW - Vacuoles/microbiology
KW - Brucella abortus/pathogenicity
KW - Animals
KW - Trophoblasts/microbiology
KW - Cytoplasm/microbiology
KW - Host-Pathogen Interactions/genetics
KW - Endoplasmic Reticulum/microbiology
KW - Mice
KW - HeLa Cells
UR - http://www.scopus.com/inward/record.url?scp=85042395629&partnerID=8YFLogxK
U2 - 10.1242/jcs.210799
DO - 10.1242/jcs.210799
M3 - Article
C2 - 29361547
SN - 0021-9533
VL - 131
JO - Journal of Cell Science
JF - Journal of Cell Science
IS - 4
M1 - jcs210799
ER -