title: Type IV Pilus Alignment Subcomplex Proteins PilN and PilO Form Homo- and Heterodimers in Vivo.
abstract: Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and is resistant to many antibiotics. Type IV pili (T4P) are among the key virulence factors used by P. aeruginosa for host cell attachment, biofilm formation, and twitching motility, making this system a promising target for novel therapeutics. Point mutations in the conserved PilMNOP alignment subcomplex were previously shown to have distinct effects on assembly and disassembly of T4P, suggesting that it may function in a dynamic manner. We introduced mutations encoding Cys substitutions into pilN and/or pilO on the chromosome to maintain normal stoichiometry and expression levels and captured covalent PilNO heterodimers, as well as PilN and PilO homodimers, in vivo Most covalent PilN or PilO homodimers had minimal functional impact in P. aeruginosa, suggesting that homodimers are a physiologically relevant state. However, certain covalent homo- or heterodimers eliminated twitching motility, suggesting that specific PilNO configurations are essential for T4P function. These data were verified using soluble N-terminal truncated fragments of PilN and PilO Cys mutants, which purified as a mixture of homo- and heterodimers at volumes consistent with a tetramer. Deletion of genes encoding alignment subcomplex components, PilM or PilP, but not other T4P components, including the motor ATPases PilB or PilT, blocked in vivo formation of disulfide-bonded PilNO heterodimers, suggesting that both PilM and PilP influence the heterodimer interface. Combined, our data suggest that T4P function depends on rearrangements at PilN and PilO interfaces.
authors.affiliation: From the Department of Biochemistry and Biomedical Sciences and the Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario L8S 4K1 and.
authors.forename: Tiffany L
authors.initials: TL
authors.lastname: Leighton
authors.affiliation: From the Department of Biochemistry and Biomedical Sciences and the Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario L8S 4K1 and.
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Yong
authors.affiliation: the Program in Molecular Structure and Function, The Hospital for Sick Children and Department of Biochemistry, University of Toronto, Toronto M5G 0A4 Ontario, Canada howell@sickkids.ca.
authors.forename: P Lynne
authors.initials: PL
authors.lastname: Howell
authors.affiliation: From the Department of Biochemistry and Biomedical Sciences and the Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario L8S 4K1 and burrowl@mcmaster.ca.
authors.forename: Lori L
authors.identifier: 0000-0003-0838-5040
authors.initials: LL
authors.lastname: Burrows
mesh_terms: D019943:Amino Acid Substitution; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D036781:Fimbriae Proteins / Q000235:genetics / Q000378:metabolism*; D010861:Fimbriae, Bacterial / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D055503:Protein Multimerization; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*
chemical_list: D036781:Fimbriae Proteins; D003545:Cysteine
keywords: Pseudomonas aeruginosa (P. aeruginosa); cysteine-mediated cross-linking; protein-protein interaction; twitching motility; type II secretion system (T2SS); type IV pili