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.

A novel kinase function of a nucleoside-diphosphate-kinase homologue inPorphyromonas gingivalisis critical in subversion of host cell apoptosis by targeting heat-shock protein 27

“…Nucleoside diphosphate kinase can work both directly or indirectly with molecules in the host cell to facilitate membrane trafficking, manipulate metabolic systems, and affect host survival signalling pathways (Atanasova et al, ; Atanasova & Yilmaz, ; Atanasova & Yilmaz, ; Chakrabarty, ; Choi et al, ; Spooner & Yilmaz, ). As a secreted virulence factor from P .…”

“…Nucleoside diphosphate kinases are highly conserved multifunctional enzymes expressed in both eukaryotes and prokaryotes that balance the pools of nucleoside triphosphates, such as ATP or GTP, by catalysing the transfer of orthophosphates (Chakrabarty, ; Ray & Mathews, ; Spooner & Yilmaz, ). Ndks form either homo‐tetramers or ‐hexamers with phosphotransferase activity that function in a variety of cellular housekeeping functions such as DNA cleavage/repair, transcriptional regulation, cell proliferation, and apoptosis (Atanasova et al, ; Kumar et al, ; Yu, Rao, & Zhang, ). In addition to homeostatic functions, bacterial and human Ndk homologues have been implicated in the progression of a variety of different chronic diseases (e.g., cancer) and opportunistic infections (Atanasova & Yilmaz, ; Chakrabarty, ; Yu et al, ).…”

“…The extracellular release of P . gingivalis ‐Ndk from host epithelial cell does not occur as a result of compromised host membranes or cell death (Choi et al, ; Yilmaz et al, ) but rather occurs in a regulated mode using host trafficking machinery via Myosin‐9 and leaving host cells through pannexin‐1 hemi‐channels (Atanasova et al, ). However, P .…”

“…However, P . gingivalis ‐Ndk can also be found in the cytoplasm of the infected host cells (Atanasova et al, ; Choi et al, ).…”

Activation of vitamin D in the gingival epithelium and its role in gingival inflammation and alveolar bone loss

“…Mean fluorescence, cell area, and the integrated density for each cell were measured by the software. The corrected total cell fluorescence (CTCF) was calculated as follows: CTCF = Integrated density − (Area of selected cell × Mean fluorescence of background readings) . A minimum of 20 cells, originating from at least three separate experiments, were evaluated for each experimental condition .…”