title: Mutation of the conserved calcium-binding motif in Neisseria gonorrhoeae PilC1 impacts adhesion but not piliation.
abstract: Neisseria gonorrhoeae PilC1 is a member of the PilC family of type IV pilus-associated adhesins found in Neisseria species and other type IV pilus-producing genera. Previously, a calcium-binding domain was described in the C-terminal domains of PilY1 of Pseudomonas aeruginosa and in PilC1 and PilC2 of Kingella kingae. Genetic analysis of N. gonorrhoeae revealed a similar calcium-binding motif in PilC1. To evaluate the potential significance of this calcium-binding region in N. gonorrhoeae, we produced recombinant full-length PilC1 and a PilC1 C-terminal domain fragment. We show that, while alterations of the calcium-binding motif disrupted the ability of PilC1 to bind calcium, they did not grossly affect the secondary structure of the protein. Furthermore, we demonstrate that both full-length wild-type PilC1 and full-length calcium-binding-deficient PilC1 inhibited gonococcal adherence to cultured human cervical epithelial cells, unlike the truncated PilC1 C-terminal domain. Similar to PilC1 in K. kingae, but in contrast to the calcium-binding mutant of P. aeruginosa PilY1, an equivalent mutation in N. gonorrhoeae PilC1 produced normal amounts of pili. However, the N. gonorrhoeae PilC1 calcium-binding mutant still had partial defects in gonococcal adhesion to ME180 cells and genetic transformation, which are both essential virulence factors in this human pathogen. Thus, we conclude that calcium binding to PilC1 plays a critical role in pilus function in N. gonorrhoeae.
authors.affiliation: Department of Medicine, Division of Infectious Diseases.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Cheng
authors.forename: Michael D L
authors.initials: MD
authors.lastname: Johnson
authors.forename: Christine
authors.initials: C
authors.lastname: Burillo-Kirch
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Mocny
authors.forename: James E
authors.initials: JE
authors.lastname: Anderson
authors.forename: Christopher K
authors.initials: CK
authors.lastname: Garrett
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Redinbo
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Thomas
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D001422:Bacterial Adhesion; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004252:DNA Mutational Analysis; D004847:Epithelial Cells / Q000382:microbiology; D036781:Fimbriae Proteins / Q000235:genetics / Q000378:metabolism*; D010861:Fimbriae, Bacterial / Q000235:genetics / Q000502:physiology*; D006801:Humans; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009344:Neisseria gonorrhoeae / Q000235:genetics / Q000502:physiology*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
chemical_list: D018829:Adhesins, Bacterial; D050505:Mutant Proteins; C079208:pilC protein, Neisseria gonorrhoeae; D036781:Fimbriae Proteins; D002118:Calcium