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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.


Immunofluorescence analysis of NDK intracellular accumulation.(A) P. gingivalis-infected primary GECs after treatment with probenecid, MβCD, ML9 or cytochalasin D. Uninfected, untreated cells were used as a control; (B) PNX1 siRNA-transfected P. gingivalis-infected primary GECs with or without MβCD, ML9 or cytochalasin D treatment. Non-target siRNA-transfected P. gingivalis-infected GECs were used as a control. P. gingivalis-NDK is labelled in green, detected by monoclonal P. gingivalis-NDK specific antibody, visualized with anti-rabbit AlexaFluor488 secondary antibody. Bar represents 1 μm. (C) NIH ImageJ analysis was performed for measuring cell fluorescent intensity. Cell boundaries were determined by actin labelling with phalloidin-TRITC. Corrected total cell fluorescence was calculated and measurements were normalized to the mean intensity of the uninfected, untreated cells. All data represent an average of at least three separate experiments. ** Denote P-values < 0.001. Select exact P-values are also shown.
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f6: Immunofluorescence analysis of NDK intracellular accumulation.(A) P. gingivalis-infected primary GECs after treatment with probenecid, MβCD, ML9 or cytochalasin D. Uninfected, untreated cells were used as a control; (B) PNX1 siRNA-transfected P. gingivalis-infected primary GECs with or without MβCD, ML9 or cytochalasin D treatment. Non-target siRNA-transfected P. gingivalis-infected GECs were used as a control. P. gingivalis-NDK is labelled in green, detected by monoclonal P. gingivalis-NDK specific antibody, visualized with anti-rabbit AlexaFluor488 secondary antibody. Bar represents 1 μm. (C) NIH ImageJ analysis was performed for measuring cell fluorescent intensity. Cell boundaries were determined by actin labelling with phalloidin-TRITC. Corrected total cell fluorescence was calculated and measurements were normalized to the mean intensity of the uninfected, untreated cells. All data represent an average of at least three separate experiments. ** Denote P-values < 0.001. Select exact P-values are also shown.

Mentions: Since our results revealed that inhibition of PNX1, along with Myosin-9 and actin cytoskeleton could prevent the extracellular secretion of P. gingivalis-NDK, we next examined whether the inhibition of the PNX1-hemichannel, as well as the blocking of putative coupling molecules that we identified in this study, Myosin-9, actin and lipid-rafts, can induce accumulation of P. gingivalis-NDK in the cells and subsequently inhibit the extracellular secretion. We employed fluorescence microscopy using P. gingivalis-NDK specific monoclonal antibody, to qualitatively observe increased intracellular fluorescence as a method to detect intracellular accumulation of P. gingivalis-NDK in infected primary GECs upon inhibition of PNX1, either by RNA silencing or by probenecid treatment, and treatment with MβCD, ML9 or cytochalasin D (Fig. 6A and B). All inhibitions, either alone or in combination, displayed increased P. gingivalis-NDK fluorescent staining in the PNX1 knock-down GECs. Most significant accumulation was present when we depleted PNX1-hemichannels via siRNA and treated with ML9 or cytochalasin D (Fig. 6B). A quantitative fluorescence intensity analysis shown in Fig. 6C substantiated the observed phenotypes in Fig. 6A and B and further demonstrated the conjoint significant involvement of ML9 and cytochalasin D in PNX1 knock-down GECs for extracellular mobilization of P. gingivalis-NDK. These findings collectively indicate that PNX1 hemichannels, along with the downstream partner molecules, such as Myosin-9 and the actin cytoskeleton, can mediate P. gingivalis-NDK transport to the cell membrane and later secretion outside of the host cells.


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 NDK intracellular accumulation.(A) P. gingivalis-infected primary GECs after treatment with probenecid, MβCD, ML9 or cytochalasin D. Uninfected, untreated cells were used as a control; (B) PNX1 siRNA-transfected P. gingivalis-infected primary GECs with or without MβCD, ML9 or cytochalasin D treatment. Non-target siRNA-transfected P. gingivalis-infected GECs were used as a control. P. gingivalis-NDK is labelled in green, detected by monoclonal P. gingivalis-NDK specific antibody, visualized with anti-rabbit AlexaFluor488 secondary antibody. Bar represents 1 μm. (C) NIH ImageJ analysis was performed for measuring cell fluorescent intensity. Cell boundaries were determined by actin labelling with phalloidin-TRITC. Corrected total cell fluorescence was calculated and measurements were normalized to the mean intensity of the uninfected, untreated cells. All data represent an average of at least three separate experiments. ** Denote P-values < 0.001. Select exact P-values are also shown.
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Related In: Results  -  Collection

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f6: Immunofluorescence analysis of NDK intracellular accumulation.(A) P. gingivalis-infected primary GECs after treatment with probenecid, MβCD, ML9 or cytochalasin D. Uninfected, untreated cells were used as a control; (B) PNX1 siRNA-transfected P. gingivalis-infected primary GECs with or without MβCD, ML9 or cytochalasin D treatment. Non-target siRNA-transfected P. gingivalis-infected GECs were used as a control. P. gingivalis-NDK is labelled in green, detected by monoclonal P. gingivalis-NDK specific antibody, visualized with anti-rabbit AlexaFluor488 secondary antibody. Bar represents 1 μm. (C) NIH ImageJ analysis was performed for measuring cell fluorescent intensity. Cell boundaries were determined by actin labelling with phalloidin-TRITC. Corrected total cell fluorescence was calculated and measurements were normalized to the mean intensity of the uninfected, untreated cells. All data represent an average of at least three separate experiments. ** Denote P-values < 0.001. Select exact P-values are also shown.
Mentions: Since our results revealed that inhibition of PNX1, along with Myosin-9 and actin cytoskeleton could prevent the extracellular secretion of P. gingivalis-NDK, we next examined whether the inhibition of the PNX1-hemichannel, as well as the blocking of putative coupling molecules that we identified in this study, Myosin-9, actin and lipid-rafts, can induce accumulation of P. gingivalis-NDK in the cells and subsequently inhibit the extracellular secretion. We employed fluorescence microscopy using P. gingivalis-NDK specific monoclonal antibody, to qualitatively observe increased intracellular fluorescence as a method to detect intracellular accumulation of P. gingivalis-NDK in infected primary GECs upon inhibition of PNX1, either by RNA silencing or by probenecid treatment, and treatment with MβCD, ML9 or cytochalasin D (Fig. 6A and B). All inhibitions, either alone or in combination, displayed increased P. gingivalis-NDK fluorescent staining in the PNX1 knock-down GECs. Most significant accumulation was present when we depleted PNX1-hemichannels via siRNA and treated with ML9 or cytochalasin D (Fig. 6B). A quantitative fluorescence intensity analysis shown in Fig. 6C substantiated the observed phenotypes in Fig. 6A and B and further demonstrated the conjoint significant involvement of ML9 and cytochalasin D in PNX1 knock-down GECs for extracellular mobilization of P. gingivalis-NDK. These findings collectively indicate that PNX1 hemichannels, along with the downstream partner molecules, such as Myosin-9 and the actin cytoskeleton, can mediate P. gingivalis-NDK transport to the cell membrane and later secretion outside of the host cells.

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.