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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.

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Immunofluorescence analysis of P. gingivalis-NDK cellular localization in GECs. (A) 12 h of P. gingivalis infection; (B) transfection with GFP-linked P. gingivalis-NDK. (C) 3 mM ATP stimulation of 12 h P. gingivalis-infected GECs; P. gingivalis-NDK in infected cells (A,C) was detected using rabbit anti-P. gingivalis NDK antibody and visualized with anti-rabbit AlexaFluor488 secondary antibody; (D) 3 mM ATP of GECs transfected with GFP-linked P. gingivalis-NDK (E) No-infection, no-transfection control cells showed no unspecific staining of NDK. (F) Transfection with GFP-containing plasmid, without P. gingivalis-NDK insertion (empty vector), showed an unspecific uniform GFP expression throughout the transfected cells as expected. These are representative images of at least three separate experiments performed in duplicates. All images represent maximum image projections of z-sections performed on laser confocal microscope. Bars represent 15 μm.
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f2: Immunofluorescence analysis of P. gingivalis-NDK cellular localization in GECs. (A) 12 h of P. gingivalis infection; (B) transfection with GFP-linked P. gingivalis-NDK. (C) 3 mM ATP stimulation of 12 h P. gingivalis-infected GECs; P. gingivalis-NDK in infected cells (A,C) was detected using rabbit anti-P. gingivalis NDK antibody and visualized with anti-rabbit AlexaFluor488 secondary antibody; (D) 3 mM ATP of GECs transfected with GFP-linked P. gingivalis-NDK (E) No-infection, no-transfection control cells showed no unspecific staining of NDK. (F) Transfection with GFP-containing plasmid, without P. gingivalis-NDK insertion (empty vector), showed an unspecific uniform GFP expression throughout the transfected cells as expected. These are representative images of at least three separate experiments performed in duplicates. All images represent maximum image projections of z-sections performed on laser confocal microscope. Bars represent 15 μm.

Mentions: The NDK species have been suggested to be cytoplasmic proteins13. Accordingly, we aimed to further examine the subcellular localization of P. gingivalis-NDK within host cells using confocal fluorescence microscopy, which revealed that P. gingivalis-NDK was primarily found in the perinuclear area of the infected GECs, and some in the cytoplasm (Fig. 2A). We also utilized a green-fluorescent-protein (GFP) construct of P. gingivalis-NDK, and introduced it into primary GECs by transfection (Fig. 2B and D). This approach was taken to verify whether the cytoplasmic localization is dependent on the presence of whole bacteria3. Similarly, the GFP-NDK transfected GECs also displayed localization of NDK largely in the perinuclear and some in the cytoplasmic area (Fig. 2B). Since we previously showed that P. gingivalis infection induces the release of ATP from infected GECs, and that P. gingivalis secretes NDK to modulate host cell death mediated by eATP during the infection, we investigated the potential effect of eATP treatment on NDK trafficking in GECs. Interestingly, eATP treatment of GECs induced NDK translocation towards the cell periphery in both the infected and the GFP-NDK-transfected (infection-free) cells (Fig. 2C and D), suggesting that NDK enzyme translocation is likely activated by eATP-stimulation. A quantitative analysis of NDK’s subcellular localizations through fluorescence intensity measurements using NIH image analysis further supported the mobilizing effect of eATP on the cytoplasmic NDK (Supplementary Fig. 1).


Nucleoside-Diphosphate-Kinase of P. gingivalis is Secreted from Epithelial Cells In the Absence of a Leader Sequence Through a Pannexin-1 Interactome
Immunofluorescence analysis of P. gingivalis-NDK cellular localization in GECs. (A) 12 h of P. gingivalis infection; (B) transfection with GFP-linked P. gingivalis-NDK. (C) 3 mM ATP stimulation of 12 h P. gingivalis-infected GECs; P. gingivalis-NDK in infected cells (A,C) was detected using rabbit anti-P. gingivalis NDK antibody and visualized with anti-rabbit AlexaFluor488 secondary antibody; (D) 3 mM ATP of GECs transfected with GFP-linked P. gingivalis-NDK (E) No-infection, no-transfection control cells showed no unspecific staining of NDK. (F) Transfection with GFP-containing plasmid, without P. gingivalis-NDK insertion (empty vector), showed an unspecific uniform GFP expression throughout the transfected cells as expected. These are representative images of at least three separate experiments performed in duplicates. All images represent maximum image projections of z-sections performed on laser confocal microscope. Bars represent 15 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5121656&req=5

f2: Immunofluorescence analysis of P. gingivalis-NDK cellular localization in GECs. (A) 12 h of P. gingivalis infection; (B) transfection with GFP-linked P. gingivalis-NDK. (C) 3 mM ATP stimulation of 12 h P. gingivalis-infected GECs; P. gingivalis-NDK in infected cells (A,C) was detected using rabbit anti-P. gingivalis NDK antibody and visualized with anti-rabbit AlexaFluor488 secondary antibody; (D) 3 mM ATP of GECs transfected with GFP-linked P. gingivalis-NDK (E) No-infection, no-transfection control cells showed no unspecific staining of NDK. (F) Transfection with GFP-containing plasmid, without P. gingivalis-NDK insertion (empty vector), showed an unspecific uniform GFP expression throughout the transfected cells as expected. These are representative images of at least three separate experiments performed in duplicates. All images represent maximum image projections of z-sections performed on laser confocal microscope. Bars represent 15 μm.
Mentions: The NDK species have been suggested to be cytoplasmic proteins13. Accordingly, we aimed to further examine the subcellular localization of P. gingivalis-NDK within host cells using confocal fluorescence microscopy, which revealed that P. gingivalis-NDK was primarily found in the perinuclear area of the infected GECs, and some in the cytoplasm (Fig. 2A). We also utilized a green-fluorescent-protein (GFP) construct of P. gingivalis-NDK, and introduced it into primary GECs by transfection (Fig. 2B and D). This approach was taken to verify whether the cytoplasmic localization is dependent on the presence of whole bacteria3. Similarly, the GFP-NDK transfected GECs also displayed localization of NDK largely in the perinuclear and some in the cytoplasmic area (Fig. 2B). Since we previously showed that P. gingivalis infection induces the release of ATP from infected GECs, and that P. gingivalis secretes NDK to modulate host cell death mediated by eATP during the infection, we investigated the potential effect of eATP treatment on NDK trafficking in GECs. Interestingly, eATP treatment of GECs induced NDK translocation towards the cell periphery in both the infected and the GFP-NDK-transfected (infection-free) cells (Fig. 2C and D), suggesting that NDK enzyme translocation is likely activated by eATP-stimulation. A quantitative analysis of NDK’s subcellular localizations through fluorescence intensity measurements using NIH image analysis further supported the mobilizing effect of eATP on the cytoplasmic NDK (Supplementary Fig. 1).

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