Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About IAI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Infection and Immunity
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About IAI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Cellular Microbiology: Pathogen-Host Cell Molecular Interactions

The Translocated Salmonella Effector Proteins SseF and SseG Interact and Are Required To Establish an Intracellular Replication Niche

Jörg Deiwick, Suzana P. Salcedo, Emmanuel Boucrot, Sarah M. Gilliland, Thomas Henry, Nele Petermann, Scott R. Waterman, Jean-Pierre Gorvel, David W. Holden, Stéphane Méresse
Jörg Deiwick
1Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Méditerranée, Parc Scientifique de Luminy, Case 906-13288, Marseille Cedex 9, France
3Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: deiwick@biochem.uni-luebeck.de
Suzana P. Salcedo
2Department of Infectious Diseases, Centre for Molecular Microbiology and Infection, Imperial College London, Armstrong Road, London SW7 2AZ, United Kingdom
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Emmanuel Boucrot
1Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Méditerranée, Parc Scientifique de Luminy, Case 906-13288, Marseille Cedex 9, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Sarah M. Gilliland
2Department of Infectious Diseases, Centre for Molecular Microbiology and Infection, Imperial College London, Armstrong Road, London SW7 2AZ, United Kingdom
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Thomas Henry
1Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Méditerranée, Parc Scientifique de Luminy, Case 906-13288, Marseille Cedex 9, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nele Petermann
3Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Scott R. Waterman
2Department of Infectious Diseases, Centre for Molecular Microbiology and Infection, Imperial College London, Armstrong Road, London SW7 2AZ, United Kingdom
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jean-Pierre Gorvel
1Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Méditerranée, Parc Scientifique de Luminy, Case 906-13288, Marseille Cedex 9, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
David W. Holden
2Department of Infectious Diseases, Centre for Molecular Microbiology and Infection, Imperial College London, Armstrong Road, London SW7 2AZ, United Kingdom
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Stéphane Méresse
1Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Méditerranée, Parc Scientifique de Luminy, Case 906-13288, Marseille Cedex 9, France
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/IAI.00648-06
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • Tables
  • FIG. 1.
    • Open in new tab
    • Download powerpoint
    FIG. 1.

    Identification of new effectors required for Golgi localization of SCVs in HeLa cells. (A and B) HeLa cells were infected with GFP-expressing wild-type (wt) Salmonella or mutants lacking different effectors and fixed at 8 h postinfection. (A) Cells were immunostained for Lamp1 and the Golgi marker Giantin and scored by immunofluorescence for the intracellular positioning of bacteria. Only bacteria enclosed in a Lamp1-positive compartment that were either completely or partially surrounded by the Golgi marker were counted as being Golgi associated. Bacterial clusters that were found adjacent to the Golgi but did not fulfill the above criteria were counted as nonassociated. At least 50 host cells, corresponding to more than 100 bacteria, were scored blind in each experiment. Statistical analysis for comparison of wild-type Salmonella and mutant strains indicated a significant difference for the ssaV, sseF, and sifA strains (P < 0.001), whereas no significant difference was observed in comparison of the wt to other mutants or the complemented sseF and sifA strains (P > 0.05). (B) Confocal immunofluorescence images of the dispersed distribution of the sseF mutant in contrast with the Golgi localization of the wt strain. The cell shape is marked. Bars, 10 μm. (C) Intracellular replication of Salmonella strains. Values indicate the fold increase, calculated as the ratio of intracellular bacteria between 2 and 16 h after invasion. Statistical analysis indicated a significant difference between mutant strains and wild-type Salmonella (P < 0.001), whereas no significant difference was observed between mutant strains. (D) Complementation of the intracellular positioning of the sseF and sifA mutants to the Golgi region by ectopic expression of myc-SseF and myc-SifA, respectively. (A, C, and D) Standard deviations of the means are shown and correspond to three independent experiments.

  • FIG. 2.
    • Open in new tab
    • Download powerpoint
    FIG. 2.

    Role of membrane-targeting domains and cellular localization of ectopically expressed effectors. (A) Protein lysates of myc-tagged proteins expressed in HeLa cells were partitioned using Triton X-114. Aliquots of each fraction were analyzed by Western blotting after SDS-PAGE. S, soluble phase; TX114, membrane-enriched phase. For controls, the partitioning of GFP, Lamp1, and Rab7 was analyzed. (B) Confocal immunofluorescence analysis of HeLa cells transfected with plasmids expressing myc-tagged effectors (two upper panels) and cotransfection of myc- and HA-tagged effectors (lower panel). The cells were fixed 24 h posttransfection and labeled using appropriate antibodies. Tagged SseF and SseG colocalized with each other as well as with the Golgi marker TGN46. Bars, 10 μm. (C) Schematic diagram showing that the region of SseF homologous to the Golgi-targeting domain of SseG is not sufficient for Golgi localization of the ectopically expressed myc-tagged protein. HR, hydrophobic region.

  • FIG. 3.
    • Open in new tab
    • Download powerpoint
    FIG. 3.

    Interaction of SseF and SseG. (A) Competitive index analysis of Salmonella mutant strains. The competitive indices of wild type (wt) versus sseG (n = 3) and of wt versus sseF (n = 6) are not significantly different (P = 0.71) but are significantly (P = 0.0012) or very significantly (P = 0.0006) lower than 1, respectively. The competitive indices of sseG versus sseF sseG (n = 3; P = 0.30) and sseF versus sseF sseG (n = 4; P = 0.12) are not significantly different from 1. These data indicate that the two genes are functionally linked. (B) GST-SseF bound to beads was incubated with extracts of HeLa cells expressing myc-tagged Salmonella effectors. Total lysates and proteins bound to washed beads were analyzed by Western blotting using an anti-myc antibody. (C) HeLa cells were cotransfected with plasmids coding for myc- and HA-tagged effector proteins as indicated, lysed, and immunoprecipitated (IP) using an anti-myc antibody coupled to Sepharose beads. HA-tagged proteins present in lysates (upper panels) and coimmunoprecipitated with myc-tagged effectors (lower panel) were analyzed by Western blotting (WB). The lysate membrane was reanalyzed for myc-tagged proteins (encircled bands, middle panel). Controls indicated the effective binding of myc-tagged proteins to the beads (data not shown). The top and bottom bands seen after anti-myc immunoprecipitation (lower panel) correspond to mouse IgG heavy and light chains.

  • FIG. 4.
    • Open in new tab
    • Download powerpoint
    FIG. 4.

    Interaction of translocated SseF and SseG. HeLa cells were infected with an S. enterica serovar Typhimurium sseF sseG double mutant harboring plasmid p2888 (sseF-HA and sseG-M45) (C). As controls, double mutants harboring plasmids p2643 (sseF-HA) (A) or p2788 (sseG-M45) (B) were used. The tagged proteins were detected in extracts of bacteria grown under phosphate starvation, inducing ssrAB-regulated proteins in vitro (left panel). SseG-M45 and SseF-HA were detected in the postnuclear supernatant (PNS) of HeLa cells infected for 12 h (middle panel). The signal for SseF-HA was intensified by successive incubations of the blotting membrane with a rabbit anti-rat and a goat anti-rabbit antibody, both coupled to peroxidase. Tagged proteins were subsequently immunoprecipitated (IP) from the PNS using anti-HA or anti-M45 antibodies coupled to the Sepharose beads. Immunoprecipitated proteins were analyzed by Western blotting (WB, right panel). The fractions of translocated SseF detected in the coimmunoprecipitation or PNS differ in electrophoretic mobility (apparent molecular masses, 33.7, 32.5, and 30.5 kDa) compared to SseF-HA secreted in vitro (apparent molecular mass, 31.6 kDa). Expression and secretion of SseJ were controlled using Salmonella wild type, the double mutant strain expressing sseF-HA and sseG-M45, and an sseJ mutant. The absence of translocated SseJ with sseF-HA and sseG-M45 was used to check the specificity of the immunoprecipitations.

Tables

  • Figures
  • TABLE 1.

    Primers used in this study

    DesignationNucleotide sequence
    SseF1for5′-GCGAGATCTAAAATTCATATTCCGTCAGCGGC-3′
    SseF1rev5′-GCGGTCGACAGGTTTCATGGTTCTCCCC-3′
    SseGfor5′-GCGAGATCTAAACCTGTTAGCCCAAATGCTC-3′
    SseGrev5′-GCGGTCGACGATTACTCCGGCGCACG-3′
    SifBfor5′-GCGGGATCCAATTACTATCGGGAGAGG-3′
    SifBrev5′-GCGCTGCAGGGGATTGTAAATCCATAC-3′
    SseIfor5′-GCGGGATCCTTTCATATTGGAAGCGGATG-3′
    SseIrev5′-GCGGTCGACGGTGCGCTTACATTTTACCT-3′
    SseJfor5′-GCGGGATCCCCATTGAGTGTTGGACAGGG-3′
    SseJrev5′-GCGGTCGACTTTATTCAGTGGAATAATGATG-3′
    SseF2for5′-GGATCCAAAATTCATATTCCGTCAGCG-3′
    SseF2rev5′-GAATTCTCATGGTTCTCCCCGAGATG-3′
    Myc-rev5′-ATGGATCCCAGGTCCTCCTCGGAGATCAGC-3′
    SseF(Δ1-63)for5′-GCTGGATCCTTTATGCAATACACTATTCGTGC-3′
    SseF (Δ1-82)for5′-GCTGGATCCGCGGTAATTTCTGGCGGGGCAGG-3′
    SseF (Δ208-260)rev5′-TATCTAGAATAATCCAGTACCGCACCTATCGC-3′
    SseF (Δ208-260)for5′-GCTGGATCCTTTATGCAATACACTATTCGTGC-3′
    SseF (Δ83-145)rev5′-GCTCTAGACGCTGCAGCAACCGATAACCC-3′
    SseF (Δ83-145)for5′-GCTCTAGACTTAACTGCGCTAACACCCTTGC-3′
    SseF (83-145)for5′-ATGGATCCGTAATTTCTGGCGGGGCAGGATTACC-3′
    SseF (83-145)rev5′-GCTCTAGAACTTGCCCCACATTTTAAGGC-3′
PreviousNext
Back to top
Download PDF
Citation Tools
The Translocated Salmonella Effector Proteins SseF and SseG Interact and Are Required To Establish an Intracellular Replication Niche
Jörg Deiwick, Suzana P. Salcedo, Emmanuel Boucrot, Sarah M. Gilliland, Thomas Henry, Nele Petermann, Scott R. Waterman, Jean-Pierre Gorvel, David W. Holden, Stéphane Méresse
Infection and Immunity Nov 2006, 74 (12) 6965-6972; DOI: 10.1128/IAI.00648-06

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Infection and Immunity article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
The Translocated Salmonella Effector Proteins SseF and SseG Interact and Are Required To Establish an Intracellular Replication Niche
(Your Name) has forwarded a page to you from Infection and Immunity
(Your Name) thought you would be interested in this article in Infection and Immunity.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
The Translocated Salmonella Effector Proteins SseF and SseG Interact and Are Required To Establish an Intracellular Replication Niche
Jörg Deiwick, Suzana P. Salcedo, Emmanuel Boucrot, Sarah M. Gilliland, Thomas Henry, Nele Petermann, Scott R. Waterman, Jean-Pierre Gorvel, David W. Holden, Stéphane Méresse
Infection and Immunity Nov 2006, 74 (12) 6965-6972; DOI: 10.1128/IAI.00648-06
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

Bacterial Proteins
Salmonella enterica
Vacuoles

Related Articles

Cited By...

About

  • About IAI
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #IAIjournal

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0019-9567; Online ISSN: 1098-5522