title: Diffusion of antibiotics through the PilQ secretin in Neisseria gonorrhoeae occurs through the immature, sodium dodecyl sulfate-labile form.
abstract: In strains of Neisseria gonorrhoeae harboring the mtr and penB determinants that decrease permeation of antibiotics into the periplasm, mutation or deletion of the PilQ secretin of type IV pili increases resistance to penicillin by ∼3-fold, indicating a role for PilQ in antibiotic permeation. In this study, we examined spontaneously arising mutants with decreased susceptibility to penicillin. One class of mutants had a phenotype indistinguishable from that of a previously characterized pilQ2 mutation that interfered with the formation of SDS-resistant PilQ multimers. A second class of mutants contained frameshift mutations in genes upstream of pilQ in the pilMNOPQ operon that increased resistance to levels similar to those of the pilQ2 mutation. In-frame deletions of these genes were constructed, but only the frameshift mutations increased antibiotic resistance, suggesting that the mutations had polar effects on PilQ. Consistent with this result, titration of wild-type PilQ levels revealed a direct correlation between resistance and expression levels of PilQ. To determine which form of PilQ, the monomer or the multimer, was responsible for antibiotic permeation, we manipulated and quantified these forms in different mutants. Deletion of PilW, which is responsible for the maturation of PilQ into SDS-resistant multimers, had no effect on resistance. Moreover, Western blot analysis revealed that while SDS-resistant multimer levels were decreased by 26% in frameshift mutants, the levels of PilQ monomers were decreased by 48%. These data suggest that immature, SDS-labile complexes, not mature, SDS-resistant PilQ complexes, serve as the route of entry of antibiotics into the periplasm.
  
  
  OBJECTIVE
  The capacity of antibiotics to reach their target is crucial for their activity. In Neisseria gonorrhoeae, the PilQ secretin of type IV pili plays an important role in antibiotic influx when diffusion of antibiotics through porins is limited (e.g., in most resistant strains). On Western blots, PilQ exists both as a mature higher-order multimer and an immature, SDS-labile monomer. In this study, we examined spontaneously arising mutations in PilQ and in the genes upstream of PilQ in the pilMNOPQ operon that increase resistance to penicillin. We provide evidence that PilQ monomers associate by mass action to form immature multimers and that these complexes likely mediate the diffusion of antibiotics across the outer membrane.
authors.affiliation: Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Sobhan
authors.initials: S
authors.lastname: Nandi
authors.affiliation: Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Shauna
authors.initials: S
authors.lastname: Swanson
authors.affiliation: Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Joshua
authors.initials: J
authors.lastname: Tomberg
authors.affiliation: Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA nicholas@med.unc.edu.
authors.forename: Robert A
authors.identifier: http://orcid.org/0000-0003-2507-8974
authors.initials: RA
authors.lastname: Nicholas
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D036781:Fimbriae Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009344:Neisseria gonorrhoeae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D010403:Penicillin Resistance; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*
chemical_list: D000900:Anti-Bacterial Agents; C084295:pilQ protein, bacteria; D036781:Fimbriae Proteins; D012967:Sodium Dodecyl Sulfate