LptO (PG0027) Is Required for Lipid A 1-Phosphatase Activity in Porphyromonas gingivalis W50.

Porphyromonas gingivalis produces 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 ΔPG0027 mutant strains. Inactivation of PG0027 caused a reduction in the yield of OMVs. Lipid A from cells and OMVs of P. gingivalis W50 and the ΔPG0027 mutant 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 ΔPG0027 mutant cells contained only phosphorylated species; nonphosphorylated species were absent. MALDI-TOF/TOF tandem MS of mono-P-pentaacyl (m/z 1,688) and mono-P-tetraacyl (m/z 1,448) lipid A from ΔPG0027 showed that both contained lipid A 1-phosphate, suggesting that the ΔPG0027 mutant strain lacked lipid A 1-phosphatase activity. The total phosphatase activities in the W50 and the ΔPG0027 mutant strains were similar, whereas the phosphatase activity in the periplasm of the ΔPG0027 mutant 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 ΔPG0027 mutant (OMVs and cells) contained similar species. Thus, OMVs in P. gingivalis are 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.IMPORTANCE Gram-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 anaerobe Porphyromonas gingivalis as 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 in P. gingivalis involving dephosphorylation of lipid A of A-LPS controlled/regulated by PG0027, which causes destabilization of the OM, resulting in blebbing and generation of OMVs.

Biodegradation process of α-tricalcium phosphate and α-tricalcium phosphate solid solution bioceramics in vivo: a comparative study.

This article reports the structure and morphology of the in vivo interface between implants composed of either a tricalcium phosphate (αTCP) or αTCP doped with 3.0 wt% dicalcium silicate (αTCP(ss)) ceramic, and natural bone of rabbit tibias. Both interfaces developed a new bone layer in direct contact with the implants after 4 and 8 weeks of implantation. The specimens were examined using analytical scanning and transmission electron microscopy, up to the lattice plane resolution level. Degradation processes of the implants developed at the interfaces encouraged osseous tissue ingrowth into the periphery of the material, changing the microstructure of the implants. The ionic exchange initiated at the implant interface with the environment was essential in the integration process of the implant, through a dissolution­precipitation­transformation mechanism. The interfaces developed normal biological and chemical activities and remained reactive over the 8-week period. Organized collagen fibrils were found at the αTCP(ss)/bone interface after 4 weeks, whereas a collagen-free layer was present around the Si-free αTCP implants. These findings suggest that the incorporation of silicate ions into αTCP ceramic promotes processes of the bone remodeling at the bone/αTCP(ss) interface, hence the solubility rate of the aTCP(ss) material decreased.

Bioactivity of diopside ceramic in human parotid saliva.

Diopside ceramic pellets with a nominal composition of 55.5 wt % SiO(2)-25.9 wt % CaO-18.0 wt % MgO were soaked in human parotid saliva (HPS) over different time intervals, to investigate the behavior of the material in a natural medium of high protein content. The results showed the formation of a hydroxyapatite (HA)-like layer on the surface of the ceramic, and suggested that the mechanism of HA-like layer formation in saliva was similar to that showed in vitro test by other silica-based materials. The HA-like layer formed at the interface was found to be compact, continuous, and composed of many small crystallites with ultrastructure similar to that of natural cortical bone and dentine. The study concluded that the high pH conditions (9.8) existing right at the ceramic/human parotid saliva interface promoted HA-like phase precipitation. At this stage of the study, it is possible to suggest that the diopside ceramic could be of interest in specific periodontal applications for bone restorative purposes. Morphology, structure, and composition of the interfacial reaction product were examined by Scanning and Transmission Electron Microscopy techniques (SEM and TEM), combined with Energy Dispersive X-say Spectroscopy (EDS). Changes in ionic concentrations were measured using Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES), while the pH right at the interface of diopside/PHS were determined with an Ion Sensitive Field Effect Transistor (ISFET-Meter) instruments.

In vitro behavior of osteoblastic cells cultured in the presence of pseudowollastonite ceramic.

Pseudowollastonite ceramic (psW) is a bioactive ceramic that binds to bone when implanted in vivo and may be useful for the treatment of skeletal defects. However, there have been no studies that examined the interaction between psW and osteoblastic cells in vitro. This study investigated the suitability of psW as a substratum for cell attachment and the ability of the material to effect osteoblasts at a distance from the material surface. Fetal rat calvarial cells were plated onto the ceramic and examined by scanning electron microscopy. The findings reported show that cells attached and proliferated on the surface to the ceramic. Attachment by cells to the material can be enhanced by preincubation of psW in serum or media containing fibronectin. The adhesion of cells can be inhibited by addition of GRGDS peptides suggesting that adhesion to psW is mediated by integrin binding to adsorbed proteins. To study the effects of psW at a distance, cells were cultured in the presence but not in direct contact with the material. Subsequent changes in proliferation, alkaline phosphatase expression, and bone nodule formation were assessed. Cells grown in wells containing psW demonstrated an increase in both the rate and total numbers of bone nodules formed, although there were no differences in proliferation or alkaline phosphatase expression. Overall, these results suggest that psW is biocompatible and osteoconductive.

αTCP ceramic doped with dicalcium silicate for bone regeneration applications prepared by powder metallurgy method: in vitro and in vivo studies.

This study reports on the in vitro and in vivo behavior of α-tricalcium phosphate (αTCP) and also αTCP doped with either 1.5 or 3.0 wt % of dicalcium silicate (C2 S). The ceramics were successfully prepared by powder metallurgy method combined with homogenization and heat treatment procedures. All materials were composed of a single-phase, αTCP in the case of a pure material, or solid solution of C2 S in αTCP for the doped αTCP, which were stable at room temperature. The ceramics were tested for bioactivity in simulated body fluid, cell culture medium containing adult mesenchymal stem cells of human origin, and in animals. Analytical scanning electron microscopy combined with chemical elemental analysis was used and Fourier transform infrared and conventional histology methods. The in vivo behavior of the ceramics matched the in vitro results, independently of the C2 S content in αTCP. Carbonated hydroxyapatite (CHA) layer was formed on the surface and within the inner parts of the specimens in all cases. A fully mineralized new bone growing in direct contact with the implants was found under the in vivo conditions. The bioactivity and biocompatibility of the implants increased with the C2 S content in αTCP. The C2 S doped ceramics also favoured a phase transformation of αTCP into CHA, important for full implant integration during the natural bone healing processes. αTCP ceramic doped with 3.0 wt % C2 S showed the best bioactive in vitro and in vivo properties of all the compositions and hence could be of interest in specific applications for bone restorative purposes.