title: A dominant-negative needle mutant blocks type III secretion of early but not late substrates in Yersinia.
abstract: Yersinia pseudotuberculosis uses a type III secretion system (T3SS) to deliver effectors into host cells. A key component of the T3SS is the needle, which is a hollow tube on the bacterial surface through which effectors are secreted, composed of the YscF protein. To study needle assembly, we performed a screen for dominant-negative yscF alleles that prevented effector secretion in the presence of wild-type (WT) YscF. One allele, yscF-L54V, prevents WT YscF secretion and needle assembly, although purified YscF-L54V polymerizes in vitro. YscF-L54V binds to its chaperones YscE and YscG, and the YscF-L54V-EG complex targets to the T3SS ATPase, YscN. We propose that YscF-L54V stalls at a binding site in the needle assembly pathway following its release from the chaperones, which blocks the secretion of WT YscF and other early substrates required for building a needle. Interestingly, YscF-L54V does not affect the activity of pre-assembled actively secreting machines, indicating that a factor and/or binding site required for YscF secretion is absent from T3SS machines already engaged in effector secretion. Thus, substrate switching may involve the removal of an early substrate-specific binding site as a mechanism to exclude early substrates from Yop-secreting machines.
authors.affiliation: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111, USA. Alison_J.Davis@tufts.edu
authors.forename: Alison J
authors.initials: AJ
authors.lastname: Davis
authors.forename: Dennise A De Jesús
authors.initials: DA
authors.lastname: Díaz
authors.forename: Joan
authors.initials: J
authors.lastname: Mecsas
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000378:metabolism*; D046911:Macromolecular Substances / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism; D020125:Mutation, Missense; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D037521:Virulence Factors / Q000378:metabolism*; D015011:Yersinia pseudotuberculosis / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D018832:Molecular Chaperones; D037521:Virulence Factors; C094426:YscG protein, Yersinia