title: LcrV mutants that abolish Yersinia type III injectisome function.
abstract: LcrV, the type III needle cap protein of pathogenic Yersinia, has been proposed to function as a tether between YscF, the needle protein, and YopB-YopD to constitute the injectisome, a conduit for the translocation of effector proteins into host cells. Further, insertion of LcrV-capped needles from a calcium-rich environment into host cells may trigger the low-calcium signal for effector translocation. Here, we used a genetic approach to test the hypothesis that the needle cap responds to the low-calcium signal by promoting injectisome assembly. Growth restriction of Yersinia pestis in the absence of calcium (low-calcium response [LCR(+)] phenotype) was exploited to isolate dominant negative lcrV alleles with missense mutations in its amber stop codon (lcrV(*327)). The addition of at least four amino acids or the eight-residue Strep tag to the C terminus was sufficient to generate an LCR(-) phenotype, with variant LcrV capping type III needles that cannot assemble the YopD injectisome component. The C-terminal Strep tag appears buried within the cap structure, blocking effector transport even in Y. pestis yscF variants that are otherwise calcium blind, a constitutive type III secretion phenotype. Thus, LcrV(*327) mutants arrest the needle cap in a state in which it cannot respond to the low-calcium signal with either injectisome assembly or the activation of type III secretion. Insertion of the Strep tag at other positions of LcrV produced variants with wild-type LCR(+), LCR(-), or dominant negative LCR(-) phenotypes, thereby allowing us to identify discrete sites within LcrV as essential for its attributes as a secretion substrate, needle cap, and injectisome assembly factor.
authors.affiliation: Howard Taylor Ricketts Laboratory, Argonne National Laboratory, Argonne, IL, USA.
authors.forename: Katherine Given
authors.initials: KG
authors.lastname: Ligtenberg
authors.forename: Nathan C
authors.initials: NC
authors.lastname: Miller
authors.forename: Anthony
authors.initials: A
authors.lastname: Mitchell
authors.forename: Gregory V
authors.initials: GV
authors.lastname: Plano
authors.forename: Olaf
authors.initials: O
authors.lastname: Schneewind
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism*; D001431:Bacteriological Techniques; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D052899:Pore Forming Cytotoxic Proteins / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D013997:Time Factors; D015008:Yersinia enterocolitica / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D015010:Yersinia pestis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
chemical_list: D000942:Antigens, Bacterial; C043908:LcrV protein, Yersinia; D052899:Pore Forming Cytotoxic Proteins