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Nucleoside-Diphosphate-Kinase of P. gingivalis is Secreted from Epithelial Cells In the Absence of a Leader Sequence Through a Pannexin-1 Interactome

View Article: PubMed Central - PubMed

ABSTRACT

Nucleoside-diphosphate-kinases (NDKs) are leaderless, multifunctional enzymes. The mode(s) of NDK secretion is currently undefined, while extracellular translocation of bacterial NDKs is critical for avoidance of host pathogen clearance by opportunistic pathogens such as Porphyromonas gingivalis. P. gingivalis-NDK during infection inhibits extracellular-ATP (eATP)/P2X7-receptor mediated cell death in gingival epithelial cells (GECs) via eATP hydrolysis. Furthermore, depletion of pannexin-1-hemichannel (PNX1) coupled with P2X7-receptor blocks the infection-induced eATP release in GECs, and P. gingivalis-NDK impacts this pathway. Ultrastructural and confocal microscopy of P. gingivalis-co-cultured GECs or green-fluorescent-protein (GFP)-P. gingivalis-NDK transfected GECs revealed a perinuclear/cytoplasmic localization of NDK. eATP stimulation induced NDK recruitment to the cell periphery. Depletion of PNX1 by siRNA or inhibition by probenecid resulted in significant blocking of extracellular NDK activity and secretion using ATPase and ELISA assays. Co-immunoprecipitation-coupled Mass-spectrometry method revealed association of P. gingivalis-NDK to the myosin-9 motor molecule. Interestingly, inhibition of myosin-9, actin, and lipid-rafts, shown to be involved in PNX1-hemichannel function, resulted in marked intracellular accumulation of NDK and decreased NDK secretion from infected GECs. These results elucidate for the first time PNX1-hemichannels as potentially main extracellular translocation pathway for NDKs from an intracellular pathogen, suggesting that PNX1-hemichannels may represent a therapeutic target for chronic opportunistic infections.

No MeSH data available.


Related in: MedlinePlus

Confocal Fluorescence analysis of NDK and Myosin-9 co-localization in P. gingivalis-infected primary GECs (Top) at 6 h post infection, or (Middle) at 24 h post infection with P. gingivalis. Uninfected GECs were used as staining controls (Bottom). Myosin-9 was labelled in red by rabbit anti-human Myosin-9 monoclonal antibody, visualized with anti-rabbit AlexaFluor594 secondary antibody. P. gingivalis-NDK is labelled in green, detected by monoclonal P. gingivalis-NDK specific antibody, visualized with anti-mouse AlexaFluor488 secondary antibody. Cell nuclei were visualized with DAPI. Mander’s co-localization (yellow) coefficients for the two molecules ranged from 0.465 at 6 h to 0.791 at 24 h. Bars represent 5 μm.
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f4: Confocal Fluorescence analysis of NDK and Myosin-9 co-localization in P. gingivalis-infected primary GECs (Top) at 6 h post infection, or (Middle) at 24 h post infection with P. gingivalis. Uninfected GECs were used as staining controls (Bottom). Myosin-9 was labelled in red by rabbit anti-human Myosin-9 monoclonal antibody, visualized with anti-rabbit AlexaFluor594 secondary antibody. P. gingivalis-NDK is labelled in green, detected by monoclonal P. gingivalis-NDK specific antibody, visualized with anti-mouse AlexaFluor488 secondary antibody. Cell nuclei were visualized with DAPI. Mander’s co-localization (yellow) coefficients for the two molecules ranged from 0.465 at 6 h to 0.791 at 24 h. Bars represent 5 μm.

Mentions: To obtain further insight about the interaction between NDK and Myosin-9, we performed confocal fluorescence microscopy to analyze co-localization of NDK and Myosin-9 within infected cells at early stages of infection (6 h after infection), as well as at 24 h after infection, when NDK secretion has been observed to peak in primary GECs11921. An increasing level of NDK/Myosin-9 co-localization was detected from 6 h to 24 h, with Mander’s coefficient of co-localization ranging from 0.465 at 6 h to 0.791 at 24 h (Fig. 4), further suggesting a time dependent significant increase in the interaction of NDK with Myosin-9.


Nucleoside-Diphosphate-Kinase of P. gingivalis is Secreted from Epithelial Cells In the Absence of a Leader Sequence Through a Pannexin-1 Interactome
Confocal Fluorescence analysis of NDK and Myosin-9 co-localization in P. gingivalis-infected primary GECs (Top) at 6 h post infection, or (Middle) at 24 h post infection with P. gingivalis. Uninfected GECs were used as staining controls (Bottom). Myosin-9 was labelled in red by rabbit anti-human Myosin-9 monoclonal antibody, visualized with anti-rabbit AlexaFluor594 secondary antibody. P. gingivalis-NDK is labelled in green, detected by monoclonal P. gingivalis-NDK specific antibody, visualized with anti-mouse AlexaFluor488 secondary antibody. Cell nuclei were visualized with DAPI. Mander’s co-localization (yellow) coefficients for the two molecules ranged from 0.465 at 6 h to 0.791 at 24 h. Bars represent 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121656&req=5

f4: Confocal Fluorescence analysis of NDK and Myosin-9 co-localization in P. gingivalis-infected primary GECs (Top) at 6 h post infection, or (Middle) at 24 h post infection with P. gingivalis. Uninfected GECs were used as staining controls (Bottom). Myosin-9 was labelled in red by rabbit anti-human Myosin-9 monoclonal antibody, visualized with anti-rabbit AlexaFluor594 secondary antibody. P. gingivalis-NDK is labelled in green, detected by monoclonal P. gingivalis-NDK specific antibody, visualized with anti-mouse AlexaFluor488 secondary antibody. Cell nuclei were visualized with DAPI. Mander’s co-localization (yellow) coefficients for the two molecules ranged from 0.465 at 6 h to 0.791 at 24 h. Bars represent 5 μm.
Mentions: To obtain further insight about the interaction between NDK and Myosin-9, we performed confocal fluorescence microscopy to analyze co-localization of NDK and Myosin-9 within infected cells at early stages of infection (6 h after infection), as well as at 24 h after infection, when NDK secretion has been observed to peak in primary GECs11921. An increasing level of NDK/Myosin-9 co-localization was detected from 6 h to 24 h, with Mander’s coefficient of co-localization ranging from 0.465 at 6 h to 0.791 at 24 h (Fig. 4), further suggesting a time dependent significant increase in the interaction of NDK with Myosin-9.

View Article: PubMed Central - PubMed

ABSTRACT

Nucleoside-diphosphate-kinases (NDKs) are leaderless, multifunctional enzymes. The mode(s) of NDK secretion is currently undefined, while extracellular translocation of bacterial NDKs is critical for avoidance of host pathogen clearance by opportunistic pathogens such as Porphyromonas gingivalis. P. gingivalis-NDK during infection inhibits extracellular-ATP (eATP)/P2X7-receptor mediated cell death in gingival epithelial cells (GECs) via eATP hydrolysis. Furthermore, depletion of pannexin-1-hemichannel (PNX1) coupled with P2X7-receptor blocks the infection-induced eATP release in GECs, and P. gingivalis-NDK impacts this pathway. Ultrastructural and confocal microscopy of P. gingivalis-co-cultured GECs or green-fluorescent-protein (GFP)-P. gingivalis-NDK transfected GECs revealed a perinuclear/cytoplasmic localization of NDK. eATP stimulation induced NDK recruitment to the cell periphery. Depletion of PNX1 by siRNA or inhibition by probenecid resulted in significant blocking of extracellular NDK activity and secretion using ATPase and ELISA assays. Co-immunoprecipitation-coupled Mass-spectrometry method revealed association of P. gingivalis-NDK to the myosin-9 motor molecule. Interestingly, inhibition of myosin-9, actin, and lipid-rafts, shown to be involved in PNX1-hemichannel function, resulted in marked intracellular accumulation of NDK and decreased NDK secretion from infected GECs. These results elucidate for the first time PNX1-hemichannels as potentially main extracellular translocation pathway for NDKs from an intracellular pathogen, suggesting that PNX1-hemichannels may represent a therapeutic target for chronic opportunistic infections.

No MeSH data available.


Related in: MedlinePlus