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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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Related in: MedlinePlus

A proposed pathway of NDK secretion from infected host cells.Upon infection with P. gingivalis, an initial activation of ATP release from the cell arises, and the P2X7-receptor/Pannexin-1 (PNX1)-hemichannel is activated12125. P. gingivalis-NDK is accumulated in the cytoplasm and mostly in the perinuclear area. The accumulated P. gingivalis-NDK is activated upon ATP release, which can act as an autocrine danger signal to the host by stimulating the P2X7-receptor/PNX1-hemichannel. Following activation, P. gingivalis-NDK interacts Myosin-9 motor molecule and is trafficked along the Myosin-9 and actin filaments to the cell periphery. NDK translocates to the extracellular space through the forming P2X7-receptor/PNX1 channel. Secreted NDK hydrolyzes the danger-signal eATP, thus attenuating the stimulation of the P2X7-receptor/PNX1-hemichannel complex, and the downregulating the associated downstream events, such as reactive oxygen species production and intracellular bacterial killing118192125.
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f7: A proposed pathway of NDK secretion from infected host cells.Upon infection with P. gingivalis, an initial activation of ATP release from the cell arises, and the P2X7-receptor/Pannexin-1 (PNX1)-hemichannel is activated12125. P. gingivalis-NDK is accumulated in the cytoplasm and mostly in the perinuclear area. The accumulated P. gingivalis-NDK is activated upon ATP release, which can act as an autocrine danger signal to the host by stimulating the P2X7-receptor/PNX1-hemichannel. Following activation, P. gingivalis-NDK interacts Myosin-9 motor molecule and is trafficked along the Myosin-9 and actin filaments to the cell periphery. NDK translocates to the extracellular space through the forming P2X7-receptor/PNX1 channel. Secreted NDK hydrolyzes the danger-signal eATP, thus attenuating the stimulation of the P2X7-receptor/PNX1-hemichannel complex, and the downregulating the associated downstream events, such as reactive oxygen species production and intracellular bacterial killing118192125.

Mentions: In light of our results and the currently available literature, we propose a novel molecular pathway for P. gingivalis-NDK extracellular translocation (Fig. 7). Our study elucidates for the first time a potential shared mechanism of unconventional translocation of intracellular NDKs outside of host cells, involving PNX1/P2X7-complex-mediated large pore formation. We also realize that NDKs might have multiple redundant pathways for exhibiting their function and biology varying from one organism to another. Our findings may have implications in various fields of research, from basic bacterial-host interaction and possible control of persistent intracellular bacterial infections, to management of some NDK secretion and PNX1- related chronic conditions. The elucidated molecular mode of action can also be a valuable contribution to future studies designed to dissect the importance of PNX1-hemichannel and pnx1-allelic variations in other chronic diseases.


Nucleoside-Diphosphate-Kinase of P. gingivalis is Secreted from Epithelial Cells In the Absence of a Leader Sequence Through a Pannexin-1 Interactome
A proposed pathway of NDK secretion from infected host cells.Upon infection with P. gingivalis, an initial activation of ATP release from the cell arises, and the P2X7-receptor/Pannexin-1 (PNX1)-hemichannel is activated12125. P. gingivalis-NDK is accumulated in the cytoplasm and mostly in the perinuclear area. The accumulated P. gingivalis-NDK is activated upon ATP release, which can act as an autocrine danger signal to the host by stimulating the P2X7-receptor/PNX1-hemichannel. Following activation, P. gingivalis-NDK interacts Myosin-9 motor molecule and is trafficked along the Myosin-9 and actin filaments to the cell periphery. NDK translocates to the extracellular space through the forming P2X7-receptor/PNX1 channel. Secreted NDK hydrolyzes the danger-signal eATP, thus attenuating the stimulation of the P2X7-receptor/PNX1-hemichannel complex, and the downregulating the associated downstream events, such as reactive oxygen species production and intracellular bacterial killing118192125.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: A proposed pathway of NDK secretion from infected host cells.Upon infection with P. gingivalis, an initial activation of ATP release from the cell arises, and the P2X7-receptor/Pannexin-1 (PNX1)-hemichannel is activated12125. P. gingivalis-NDK is accumulated in the cytoplasm and mostly in the perinuclear area. The accumulated P. gingivalis-NDK is activated upon ATP release, which can act as an autocrine danger signal to the host by stimulating the P2X7-receptor/PNX1-hemichannel. Following activation, P. gingivalis-NDK interacts Myosin-9 motor molecule and is trafficked along the Myosin-9 and actin filaments to the cell periphery. NDK translocates to the extracellular space through the forming P2X7-receptor/PNX1 channel. Secreted NDK hydrolyzes the danger-signal eATP, thus attenuating the stimulation of the P2X7-receptor/PNX1-hemichannel complex, and the downregulating the associated downstream events, such as reactive oxygen species production and intracellular bacterial killing118192125.
Mentions: In light of our results and the currently available literature, we propose a novel molecular pathway for P. gingivalis-NDK extracellular translocation (Fig. 7). Our study elucidates for the first time a potential shared mechanism of unconventional translocation of intracellular NDKs outside of host cells, involving PNX1/P2X7-complex-mediated large pore formation. We also realize that NDKs might have multiple redundant pathways for exhibiting their function and biology varying from one organism to another. Our findings may have implications in various fields of research, from basic bacterial-host interaction and possible control of persistent intracellular bacterial infections, to management of some NDK secretion and PNX1- related chronic conditions. The elucidated molecular mode of action can also be a valuable contribution to future studies designed to dissect the importance of PNX1-hemichannel and pnx1-allelic variations in other chronic diseases.

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