title: Nucleoside-Diphosphate-Kinase of P. gingivalis is Secreted from Epithelial Cells In the Absence of a Leader Sequence Through a Pannexin-1 Interactome.
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.
authors.affiliation: Department of Periodontology, University of Florida, Gainesville, Florida, USA.
authors.forename: Kalina
authors.initials: K
authors.lastname: Atanasova
authors.affiliation: Department of Periodontology, University of Florida, Gainesville, Florida, USA.
authors.forename: Jungnam
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Oral Health Sciences, College of Dental Medicine, Medical University of South Carolina, Charleston, South Carolina, USA.
authors.forename: JoAnn
authors.initials: J
authors.lastname: Roberts
authors.affiliation: Department of Periodontology, University of Florida, Gainesville, Florida, USA.
authors.forename: Kyulim
authors.initials: K
authors.lastname: Lee
authors.affiliation: Department of Biomedical Sciences, University of the Pacific, Arthur Dugoni School of Dentistry, San Francisco, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Ojcius
authors.affiliation: Department of Oral Health Sciences, College of Dental Medicine, Medical University of South Carolina, Charleston, South Carolina, USA.|Department of Microbiology and Immunology, College of Medicine, Medical University of South Carolina, USA.
authors.forename: Özlem
authors.initials: Ö
authors.lastname: Yilmaz
mesh_terms: D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000328:Adult; D016866:Bacteroidaceae Infections / Q000382:microbiology / Q000473:pathology; D002478:Cells, Cultured; D017630:Connexins / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000201:enzymology* / Q000382:microbiology* / Q000648:ultrastructure; D005881:Gingiva / Q000473:pathology; D006801:Humans; D013058:Mass Spectrometry; D021962:Membrane Microdomains / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009218:Myosins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism*; D016966:Porphyromonas gingivalis / Q000201:enzymology*; D025941:Protein Interaction Mapping; D021382:Protein Sorting Signals; D021381:Protein Transport; D014162:Transfection
chemical_list: D000199:Actins; D017630:Connexins; D009419:Nerve Tissue Proteins; C492764:PANX1 protein, human; D021382:Protein Sorting Signals; D000255:Adenosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase; D009218:Myosins