LptO (PG0027) Is Required for Lipid A 1-Phosphatase Activity in Porphyromonas gingivalis W50.
Porphyromonas gingivalisproduces outer membrane vesicles (OMVs) rich in virulence factors, including cysteine proteases and A-LPS, one of the two lipopolysaccharides (LPSs) produced by this organism. Previous studies had suggested that A-LPS and PG0027, an outer membrane (OM) protein, may be involved in OMV formation. Their roles in this process were examined by using W50 parent and the ΔPG0027mutant strains. Inactivation ofPG0027caused a reduction in the yield of OMVs. Lipid A from cells and OMVs ofP. gingivalisW50 and the ΔPG0027mutant strains were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Lipid A from W50 cells contained bis-P-pentaacyl, mono-P-pentaacyl, mono-P-tetraacyl, non-P-pentaacyl, and non-P-tetraacyl species, whereas lipid A from ΔPG0027mutant cells contained only phosphorylated species; nonphosphorylated species were absent. MALDI-TOF/TOF tandem MS of mono-P-pentaacyl (m/z1,688) and mono-P-tetraacyl (m/z1,448) lipid A from ΔPG0027showed that both contained lipid A 1-phosphate, suggesting that the ΔPG0027mutant strain lacked lipid A 1-phosphatase activity. The total phosphatase activities in the W50 and the ΔPG0027mutant strains were similar, whereas the phosphatase activity in the periplasm of the ΔPG0027mutant was lower than that in W50, supporting a role for PG0027 in lipid A dephosphorylation. W50 OMVs were enriched in A-LPS, and its lipid A did not contain nonphosphorylated species, whereas lipid A from the ΔPG0027mutant (OMVs and cells) contained similar species. Thus, OMVs inP. gingivalisare apparently formed in regions of the OM enriched in A-LPS devoid of nonphosphorylated lipid A. Conversely, dephosphorylation of lipid A through a PG0027-dependent process is required for optimal formation of OMVs. Hence, the relative proportions of nonphosphorylated and phosphorylated lipid A appear to be crucial for OMV formation in this organism.IMPORTANCEGram-negative bacteria produce outer membrane vesicles (OMVs) by "blebbing" of the outer membrane (OM). OMVs can be used offensively as delivery systems for virulence factors and defensively to aid in the colonization of a host and in the survival of the bacterium in hostile environments. Earlier studies using the oral anaerobePorphyromonas gingivalisas a model organism to study the mechanism of OMV formation suggested that the OM protein PG0027 and one of the two lipopolysaccharides (LPSs) synthesized by this organism, namely, A-LPS, played important roles in OMV formation. We suggest a novel mechanism of OMV formation inP. gingivalisinvolving dephosphorylation of lipid A of A-LPS controlled/regulated by PG0027, which causes destabilization of the OM, resulting in blebbing and generation of OMVs.
Institute of Dentistry, Barts and The London School of Medicine & Dentistry, Queen Mary University of London, London, United Kingdom m.rangarajan@qmul.ac.uk.
Minnie
M
Rangarajan
Institute of Dentistry, Barts and The London School of Medicine & Dentistry, Queen Mary University of London, London, United Kingdom.
Joseph
J
Aduse-Opoku
Institute of Dentistry, Barts and The London School of Medicine & Dentistry, Queen Mary University of London, London, United Kingdom.
Ahmed
A
Hashim
Cellular Pathology, Barts Health NHS Trust, London, United Kingdom.
Graham
G
McPhail
Nanovision Centre, Advanced Electron Microscopy, School of Engineering & Materials Science, Queen Mary University of London, London, United Kingdom.
Zofia
Z
Luklinska
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.
M Florencia
MF
Haurat
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.
Mario F
MF
Feldman
Institute of Dentistry, Barts and The London School of Medicine & Dentistry, Queen Mary University of London, London, United Kingdom.
Michael A
MA
Curtis
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008050:Lipid A / Q000096:biosynthesis; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D016966:Porphyromonas gingivalis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D008050:Lipid A; C546357:lipid A 1-phosphatase, Porphyromonas gingivalis; D010744:Phosphoric Monoester Hydrolases
MALDI-TOF MS; MALDI-TOF/TOF MS; PG0027; Porphyromonas gingivalis; electron microscopy; phosphatase activity