title: CRISPR/Cas9 Screens Reveal Requirements for Host Cell Sulfation and Fucosylation in Bacterial Type III Secretion System-Mediated Cytotoxicity.
abstract: Type III secretion systems (T3SSs) inject bacterial effector proteins into host cells and underlie the virulence of many gram-negative pathogens. Studies have illuminated bacterial factors required for T3SS function, but the required host processes remain largely undefined. We coupled CRISPR/Cas9 genome editing technology with the cytotoxicity of two Vibrio parahaemolyticus T3SSs (T3SS1 and T3SS2) to identify human genome disruptions conferring resistance to T3SS-dependent cytotoxicity. We identity non-overlapping genes required for T3SS1- and T3SS2-mediated cytotoxicity. Genetic ablation of cell surface sulfation reduces bacterial adhesion and thereby alters the kinetics of T3SS1-mediated cytotoxicity. Cell surface fucosylation is required for T3SS2-dependent killing, and genetic inhibition of fucosylation prevents membrane insertion of the T3SS2 translocon complex. These findings reveal the importance of ubiquitous surface modifications for T3SS function, potentially explaining the broad tropism of V. parahaemolyticus, and highlight the utility of genome-wide CRISPR/Cas9 screens to discover processes underlying host-pathogen interactions.
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Boston, MA 02115, USA; Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Carlos J
authors.initials: CJ
authors.lastname: Blondel
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Boston, MA 02115, USA; Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute, Boston, MA 02115, USA; Boston University School of Medicine, Boston, MA 02118, USA.
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Park
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Boston, MA 02115, USA; Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Troy P
authors.initials: TP
authors.lastname: Hubbard
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Boston, MA 02115, USA; Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Alline R
authors.initials: AR
authors.lastname: Pacheco
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Boston, MA 02115, USA; Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Carole J
authors.initials: CJ
authors.lastname: Kuehl
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Boston, MA 02115, USA; Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Walsh
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Boston, MA 02115, USA; Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Brigid M
authors.initials: BM
authors.lastname: Davis
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Boston, MA 02115, USA; Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Gewurz
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
authors.forename: John G
authors.initials: JG
authors.lastname: Doench
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Boston, MA 02115, USA; Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute, Boston, MA 02115, USA. Electronic address: mwaldor@research.bwh.harvard.edu.
authors.forename: Matthew K
authors.initials: MK
authors.lastname: Waldor
mesh_terms: D001422:Bacterial Adhesion; D002470:Cell Survival; D005643:Fucose / Q000378:metabolism; D055786:Gene Knockout Techniques / Q000379:methods; D018390:Gene Targeting / Q000379:methods; D054884:Host-Pathogen Interactions; D006801:Humans; D011499:Protein Processing, Post-Translational; D013431:Sulfates / Q000378:metabolism; D013499:Surface Properties; D000069296:Type III Secretion Systems / Q000378:metabolism*; D014736:Vibrio parahaemolyticus / Q000235:genetics* / Q000378:metabolism*; D037521:Virulence Factors / Q000378:metabolism*
chemical_list: D013431:Sulfates; D000069296:Type III Secretion Systems; D037521:Virulence Factors; D005643:Fucose