Porphyromonas gingivalis resistance to polymyxin B is determined by the lipid A 4'-phosphatase, PGN_0524

Int J Oral Sci. 2009 Sep;1(3):126-35. doi: 10.4248/IJOS.09062.


Aim: 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.

Methodology: 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.

Conclusion: 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.

Keywords: P. gingivalis; antimicrobial peptide; lipid A phosphatase; lipopolysaccharide; polymyxin B; transposon.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Anti-Bacterial Agents / pharmacology*
  • Chromosome Mapping
  • DNA Transposable Elements / genetics
  • Drug Resistance, Bacterial / genetics*
  • E-Selectin / analysis
  • E-Selectin / immunology
  • Endothelial Cells / immunology
  • Endothelial Cells / microbiology
  • Gene Deletion
  • Humans
  • Lipid A / analysis
  • Lipid A / immunology
  • Lipopolysaccharides / analysis
  • Lipopolysaccharides / immunology
  • Mutagenesis, Insertional / genetics
  • Open Reading Frames / genetics
  • Phosphoric Monoester Hydrolases / genetics
  • Phosphoric Monoester Hydrolases / physiology*
  • Polymyxin B / pharmacology*
  • Porphyromonas gingivalis / enzymology*
  • Porphyromonas gingivalis / genetics
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Toll-Like Receptor 4 / analysis
  • Toll-Like Receptor 4 / immunology
  • Virulence Factors / physiology


  • Anti-Bacterial Agents
  • DNA Transposable Elements
  • E-Selectin
  • Lipid A
  • Lipopolysaccharides
  • SELE protein, human
  • TLR4 protein, human
  • Toll-Like Receptor 4
  • Virulence Factors
  • lipid A 4'-phosphatase
  • Phosphoric Monoester Hydrolases
  • Polymyxin B