Porphyromonas gingivalis Resistance to Polymyxin B Is Determined by the Lipid A 4＇-Phosphatase, PGN_0524

• Published in: International journal of oral science 2009-09, Vol.1 (3), p.126-135
• Main Authors: Coats, Stephen R, To, Thao T, Jain, Sumita, Braham, Pamela H, Darveau, Richard P
• Format: Article
• Language: English
• Subjects: Anti-Bacterial Agents - pharmacology; Chromosome Mapping; Dentistry; 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; Medicine; Mutagenesis, Insertional - genetics; Open Reading Frames - genetics; Oral and Maxillofacial Surgery; Original Scientific; original-scientific-article; Orthopedics; Phosphoric Monoester Hydrolases - genetics; Phosphoric Monoester Hydrolases - physiology; Polymyxin B - pharmacology; Porphyromonas gingivalis - enzymology; Porphyromonas gingivalis - genetics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Surgical Orthopedics; Toll-Like Receptor 4 - analysis; Toll-Like Receptor 4 - immunology; Virulence Factors - physiology; 抗菌剂; 病原体; 磷酸酶; 脂多糖
• ISSN: 1674-2818; 2049-3169
• 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, 50μg·mL^-1）. Results P. gingivalis （ATCC 33277） is unusually resistant to the cationic antimicrobial peptide, PMB at relatively high concentrations （200μg·mL^-1）. Approximately 2,700 independent Tn4400 ＇-derived mutants ofP. gingivalis were examined for increased sensitivity to PMB killing at a relatively low dose （50 μg·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 an