title: Porphyromonas gingivalis resistance to polymyxin B is determined by the lipid A 4'-phosphatase, PGN_0524.
abstract: OBJECTIVE
  To elucidate the genetic basis for the pronounced resistance that the oral pathogen, Porphyromonas gingivalis (P. gingivalis), exhibits towards the cationic antimicrobial peptide, polymyxin B.
  
  
  METHODS
  A genetic screen of P. gingivalis clones generated by a Tn4400'-based random insertion mutagenesis strategy was performed to identify bacteria harboring novel genetic mutations that render P. gingivalis susceptible to killing by the cationic antimicrobial peptide, polymyxin B (PMB, 50 microg x mL(-1)).
  
  
  RESULTS
  P. gingivalis (ATCC 33277) is unusually resistant to the cationic antimicrobial peptide, PMB at relatively high concentrations (200 microg x mL(-1)). Approximately 2,700 independent Tn4400'-derived mutants of P. gingivalis were examined for increased sensitivity to PMB killing at a relatively low dose (50 microg x mL(-1)). A single PMB-sensitive mutant was obtained in this phenotypic screen. We determined that the Tn4400' transposon was integrated into the gene encoding the lipid A 4'-phosphatase, PGN_0524, demonstrating that this insertion event was responsible for its increased susceptibility of this clone to PMB-dependent killing. The resulting mutant strain, designated 0524-Tn4400', was highly sensitive to PMB killing relative to wild-type P. gingivalis, and exhibited the same sensitivity as the previously characterized strain, 0524KO, which bears a genetically engineered deletion in the PGN_0524 locus. Positive ion mass spectrometric structural (MALDI-TOF MS) analyses revealed that lipid A isolates from 0524-Tn4400' and 0524KO strains displayed strikingly similar MALDI-TOF MS spectra that were substantially different from the wildtype P. gingivalis lipid A spectrum. Finally, intact 0524-Tn4400' and 0524KO mutant bacteria, as well as their corresponding LPS isolates, were significantly more potent in stimulating Toll-like receptor 4 (TLR4)-dependent E-selectin expression in human endothelial cells relative to intact wild-type P. gingivalis or its corresponding LPS isolate.
  
  
  CONCLUSIONS
  The combined molecular evidence provided in this report suggests that PGN_0524, a lipid A 4'-phosphatase, is the sole genetic element conferring the ability of the periodontopathogen, P. gingivalis, to evade the killing activity of cationic antimicrobial peptides, such as PMB. These data strongly implicate PGN_0524 as a critical virulence factor for the ability of P. gingivalis to evade front-line host innate defenses that are dependent upon cationic antimicrobial peptide activity and TLR 4 sensing.
authors.affiliation: Department of Periodontics, School of Dentistry, University of Washington, Seattle, Washington 98195, USA. scoats@u.washington.edu
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Coats
authors.forename: Thao T
authors.initials: TT
authors.lastname: To
authors.forename: Sumita
authors.initials: S
authors.lastname: Jain
authors.forename: Pamela H
authors.initials: PH
authors.lastname: Braham
authors.forename: Richard P
authors.initials: RP
authors.lastname: Darveau
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002874:Chromosome Mapping; D004251:DNA Transposable Elements / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D019040:E-Selectin / Q000032:analysis / Q000276:immunology; D042783:Endothelial Cells / Q000276:immunology / Q000382:microbiology; D017353:Gene Deletion; D006801:Humans; D008050:Lipid A / Q000032:analysis / Q000276:immunology; D008070:Lipopolysaccharides / Q000032:analysis / Q000276:immunology; D016254:Mutagenesis, Insertional / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000502:physiology*; D011112:Polymyxin B / Q000494:pharmacology*; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D051197:Toll-Like Receptor 4 / Q000032:analysis / Q000276:immunology; D037521:Virulence Factors / Q000502:physiology
chemical_list: D000900:Anti-Bacterial Agents; D004251:DNA Transposable Elements; D019040:E-Selectin; D008050:Lipid A; D008070:Lipopolysaccharides; C546936:SELE protein, human; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D037521:Virulence Factors; C095015:lipid A 4'-phosphatase; D010744:Phosphoric Monoester Hydrolases; D011112:Polymyxin B