A novel kinase function of a nucleoside-diphosphate-kinase homologue in Porphyromonas gingivalis is critical in subversion of host cell apoptosis by targeting heat-shock protein 27.
ABSTRACT: We have previously shown that a homologue of a conserved nucleoside-diphosphate-kinase (Ndk) family of multifunctional enzymes and secreted molecule in Porphyromonas gingivalis can modulate select host molecular pathways including downregulation of reactive-oxygen-species generation to promote bacterial survival in human gingival epithelial cells (GECs). In this study, we describe a novel kinase function for bacterial effector, P. gingivalis-Ndk, in abrogating epithelial cell death by phosphorylating heat-shock protein 27 (HSP27) in GECs. Infection by P. gingivalis was recently suggested to increase phosphorylation of HSP27 in cancer-epithelial cells; however, the mechanism and biological significance of antiapoptotic phospho-HSP27 during infection has never been characterised. Interestingly, using glutathione S-transferase-rNdk pull-down analysed by mass spectrometry, we identified HSP27 in GECs as a strong binder of P. gingivalis-Ndk and further verified using confocal microscopy and ELISA. Therefore, we hypothesised P. gingivalis-Ndk can phosphorylate HSP27 for inhibition of apoptosis in GECs. We further employed P. gingivalis-Ndk protein constructs and an isogenic P. gingivalis-ndk-deficient-mutant strain for functional examination. P. gingivalis-infected GECs displayed significantly increased phospho-HSP27 compared with ndk-deficient-strain during 24 hr infection. Phospho-HSP27 was significantly increased by transfection of GFP-tagged-Ndk into uninfected-GECs, and in vitro phosphorylation assays revealed direct phosphorylation of HSP27 at serines 78 and 82 by P. gingivalis-Ndk. Depletion of HSP27 via siRNA significantly reversed resistance against staurosporine-mediated-apoptosis during infection. Transfection of recombinant P. gingivalis-Ndk protein into GECs substantially decreased staurosporine-induced-apoptosis. Finally, ndk-deficient-mutant strain was unable to inhibit staurosporine-induced Cytochrome C release/Caspase-9 activation. Thus, we show for the first time the phosphorylation of HSP27 by a bacterial effector-P. gingivalis-Ndk-and a novel function of Ndks that is directly involved in inhibition of host cell apoptosis and the subsequent bacterial survival.
Project description: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)/P2X<sub>7</sub>-receptor mediated cell death in gingival epithelial cells (GECs) via eATP hydrolysis. Furthermore, depletion of pannexin-1-hemichannel (PNX1) coupled with P2X<sub>7</sub>-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.
Project description:Porphyromonas gingivalis, a periodontal pathogen, has been implicated as a causative agent of preterm delivery of low-birth-weight infants. We previously reported that P. gingivalis activated cellular DNA damage signaling pathways and ERK1/2 that lead to G1 arrest and apoptosis in extravillous trophoblast cells (HTR-8 cells) derived from the human placenta. In the present study, we further examined alternative signaling pathways mediating cellular damage caused by P. gingivalis. P. gingivalis infection of HTR-8 cells induced phosphorylation of p38 and Jun N-terminal protein kinase (JNK), while their inhibitors diminished both G1 arrest and apoptosis. In addition, heat shock protein 27 (HSP27) was phosphorylated through both p38 and JNK, and knockdown of HSP27 with small interfering RNA (siRNA) prevented both G1 arrest and apoptosis. Furthermore, regulation of G1 arrest and apoptosis was associated with p21 expression. HTR-8 cells infected with P. gingivalis exhibited upregulation of p21, which was regulated by p53 and HSP27. These results suggest that P. gingivalis induces G1 arrest and apoptosis via novel molecular pathways that involve p38 and JNK with its downstream effectors in human trophoblasts.
Project description:Porphyromonas gingivalis, an opportunistic pathogen usurps gingival epithelial cells (GECs) as primary intracellular niche for its colonization in the oral mucosa. However, the precise characterization of the intracellular trafficking and fate of P. gingivalis in GECs remains incomplete. Therefore, we employed high-resolution three-dimensional-transmission-electron-microscopy to determine the subcellular location of P. gingivalis in human primary GECs upon invasion. Serial sections of infected-GECs and their tomographic reconstruction depicted ER-rich-double-membrane autophagosomal-vacuoles harboring P. gingivalis. Western-blotting and fluorescence confocal microscopy showed that P. gingivalis significantly induces LC3-lipidation in a time-dependent-manner and co-localizes with LC3, ER-lumen-protein Bip, or ER-tracker, which are major components of the phagophore membrane. Furthermore, GECs that were infected with FMN-green-fluorescent transformant-strain (PgFbFP) and selectively permeabilized by digitonin showed rapidly increasing large numbers of double-membrane-vacuolar-P. gingivalis over 24 hours of infection with a low-ratio of cytosolically free-bacteria. Moreover, inhibition of autophagy using 3-methyladenine or ATG5 siRNA significantly reduced the viability of intracellular P. gingivalis in GECs as determined by an antibiotic-protection-assay. Lysosomal marker, LAMP-1, showed a low-degree colocalization with P. gingivalis (?20%). PgFbFP was used to investigate the fate of vacuolar- versus cytosolic-P. gingivalis by their association with ubiquitin-binding-adaptor-proteins, NDP52 and p62. Only cytosolic-P. gingivalis had a significant association with both markers, which suggests cytosolically-free bacteria are likely destined to the lysosomal-degradation pathway whereas the vacuolar-P. gingivalis survives. Therefore, the results reveal a novel mechanism for P. gingivalis survival in GECs by harnessing host autophagy machinery to establish a successful replicative niche and persistence in the oral mucosa.
Project description:Porphyromonas gingivalis, a major opportunistic pathogen in the etiology of chronic periodontitis, successfully survives in human gingival epithelial cells (GECs). P. gingivalis abrogates the effects of a host danger molecule, extracellular ATP (eATP)/P2X7 signaling, such as the generation of reactive oxygen species (ROS) via the mitochondria and NADPH oxidases (NOX) from primary GECs. However, antimicrobial functions of ROS production are thoroughly investigated in myeloid-lineage immune cells and have not been well-understood in epithelial cells. Therefore, this study characterizes antibacterial NOX2 generated ROS and host downstream effects in P. gingivalis infected human primary GECs. We examined the expression of NOX isoforms in the GECs and demonstrate eATP stimulation increased the mRNA expression of NOX2 (p < 0.05). Specific peptide inhibition of NOX2 significantly reduced eATP-mediated ROS as detected by DCFDA probe. The results also showed P. gingivalis infection can temporally modulate NOX2 pathway by reorganizing the localization and activation of cytosolic molecules (p47phox, p67phox, and Rac1) during 24 h of infection. Investigation into downstream biocidal factors of NOX2 revealed an eATP-induced increase in hypochlorous acid (HOCl) in GECs detected by R19-S fluorescent probe, which is significantly reduced by a myeloperoxidase (MPO) inhibitor. MPO activity of the host cells was assayed and found to be positively affected by eATP treatment and/or infection. However, P. gingivalis significantly reduced the MPO product, bactericidal HOCl, in early times of infection upon eATP stimulation. Analysis of the intracellular levels of a major host-antioxidant, glutathione during early infection revealed a substantial decrease (p < 0.05) in reduced glutathione indicative of scavenging of HOCl by P. gingivalis infection and eATP treatment. Examination of the mRNA expression of key enzymes in the glutathione synthesis pathway displayed a marked increase (p < 0.05) in glutamate cysteine ligase (GCL) subunits GCLc and GCLm, glutathione synthetase, and glutathione reductase during the infection. These suggest P. gingivalis modulates the danger signal eATP-induced NOX2 signaling and also induces host glutathione synthesis to likely avoid HOCl mediated clearance. Thus, we characterize for the first time in epithelial cells, an eATP/NOX2-ROS-antibacterial pathway and demonstrate P. gingivalis can circumvent this important antimicrobial defense system potentially for successful persistence in human epithelial tissues.
Project description:<h4>Background and objective</h4>The potential of probiotics on the prevention and control of periodontitis and other chronic inflammatory conditions has been suggested. <i>Lactobacillus</i> and <i>Bifidobacterium</i> species influence <i>P. gingivalis</i> interaction with gingival epithelial cells (GECs) but may not act in a unique way. In order to select the most appropriate probiotic against <i>P. gingivalis</i>, we aimed to evaluate the effect of several strains on <i>Porphyromonas gingivalis</i> biofilm formation and transcription virulence-associated factors (PgVAFs).<h4>Methods</h4>Cell-free pH neutralized supernatants (CFS) and living <i>Lactobacillus</i> spp. and <i>Bifidobacterium</i> spp. were tested against <i>P. gingivalis</i> ATCC 33277 and W83, in mono- and multi-species (with <i>Streptococcus oralis</i> and <i>S. gordonii</i>) biofilms. Relative transcription of <i>P. gingivalis</i> genes (<i>fimA, mfa1, kgp, rgp, ftsH</i> and <i>luxS</i>) was determined in biofilms and under GECs co-infection.<h4>Results</h4>Probiotics CFS reduced <i>P. gingivalis</i> ATCC 33277 levels in mono-species biofilms and living probiotics reduced <i>P. gingivalis</i> abundance in multi-species biofilms. <i>L. acidophilus</i> LA5 down-regulated transcription of most PgVAFs in biofilms and GECs.<h4>Conclusions</h4>Probiotics affect <i>P. gingivalis</i> biofilm formation by down-regulating overall PgVAFs with the most pronounced effect observed for <i>L. acidophilus</i> LA5.
Project description:<h4>Background</h4>Helicobacter pylori-mediated gastric carcinogenesis is initiated by a plethora of signaling events in the infected gastric epithelial cells (GECs). The E3 ubiquitin ligase seven in absentia homolog 2 (Siah2) is induced in GECs in response to H. pylori infection. Posttranslational modifications of Siah2 orchestrate its function as well as stability. The aim of this study was to evaluate Siah2 phosphorylation status under the influence of H. pylori infection and its impact in gastric cancer progression.<h4>Methods</h4>H. pylori-infected various GECs, gastric tissues from H. pylori-infected GC patients and H. felis-infected C57BL/6 mice were evaluated for Siah2 phosphorylation by western blotting or immunofluorescence microscopy. Coimmunoprecipitation assay followed by mass spectrometry were performed to identify the kinases interacting with Siah2. Phosphorylation sites of Siah2 were identified by using various plasmid constructs generated by site-directed mutagenesis. Proteasome inhibitor MG132 was used to investigate proteasome degradation events. The importance of Siah2 phosphorylation on tumorigenicity of infected cells were detected by using phosphorylation-null mutant and wild type Siah2 stably-transfected cells followed by clonogenicity assay, cell proliferation assay, anchorage-independent growth and transwell invasion assay.<h4>Results</h4>Siah2 was phosphorylated in H. pylori-infected GECs as well as in metastatic GC tissues at residues serine<sup>6</sup> (Ser<sup>6</sup>) and threonine<sup>279</sup> (Thr<sup>279</sup>). Phosphorylation of Siah2 was mediated by MRCK?, a Ser/Thr protein kinase. MRCK? was consistently expressed in uninfected GECs and noncancer gastric tissues but its level decreased in infected GECs as well as in metastatic tissues which had enhanced Siah2 expression. Infected murine gastric tissues showed similar results. MRCK? could phosphorylate Siah2 but itself got ubiquitinated from this interaction leading to the proteasomal degradation of MRCK? and use of proteasomal inhibitor MG132 could rescue MRCK? from Siah2-mediated degradation. Ser<sup>6</sup> and Thr<sup>279</sup> phosphorylated-Siah2 was more stable and tumorigenic than its non-phosphorylated counterpart as revealed by the proliferation, invasion, migration abilities and anchorage-independent growth of stable-transfected cells.<h4>Conclusions</h4>Increased level of Ser<sup>6</sup> and Thr<sup>279</sup>-phosphorylated-Siah2 and downregulated MRCK? were prominent histological characteristics of Helicobacter-infected gastric epithelium and metastatic human GC. MRCK?-dependent Siah2 phosphorylation stabilized Siah2 which promoted anchorage-independent survival and proliferative potential of GECs. Phospho-null mutants of Siah2 (S6A and T279A) showed abated tumorigenicity.
Project description:The recent introduction of "oxygen-independent" flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) is of major interest to both eukaryotic and prokaryotic microbial biologists. Accordingly, we demonstrate for the first time that an obligate anaerobe, the successful opportunistic pathogen of the oral cavity, Porphyromonas gingivalis, can be genetically engineered for expression of the non-toxic green FbFP. The resulting transformants are functional for studying dynamic bacterial processes in living host cells. The visualization of the transformed P. gingivalis (PgFbFP) revealed strong fluorescence that reached a maximum emission at 495 nm as determined by fluorescence microscopy and spectrofluorometry. Human primary gingival epithelial cells (GECs) were infected with PgFbFP and the bacterial invasion of host cells was analyzed by a quantitative fluorescence microscopy and antibiotic protection assays. The results showed similar levels of intracellular bacteria for both wild type and PgFbFP strains. In conjunction with organelle specific fluorescent dyes, utilization of the transformed strain provided direct and accurate determination of the live/metabolically active P. gingivalis' trafficking in the GECs over time. Furthermore, the GECs were co-infected with PgFbFP and the ATP-dependent Clp serine protease-deficient mutant (ClpP-) to study the differential fates of the two strains within the same host cells. Quantitative co-localization analyses displayed the intracellular PgFbFP significantly associated with the endoplasmic reticulum network, whereas the majority of ClpP- organisms trafficked into the lysosomes. Hence, we have developed a novel and reliable method to characterize live host cell-microbe interactions and demonstrated the adaptability of FMN-green fluorescent protein for studying persistent host infections induced by obligate anaerobic organisms.
Project description:Cell surface nucleotide-metabolizing enzyme, ectonucleotidase-CD73, has emerged as a central component of the cellular homeostatic-machinery that counterbalances the danger-molecule (extracellular-ATP)-driven proinflammatory response in immune cells. While the importance of CD73 in microbial host fitness and symbiosis is gradually being unraveled, there remains a significant gap in knowledge of CD73 and its putative role in epithelial cells. Here, we depict a novel host-pathogen adaptation mechanism where CD73 takes a center role in the intracellular persistence of <i>Porphyromonas gingivalis</i>, a major colonizer of oral mucosa, using human primary gingival epithelial cell (GEC) system. Temporal analyses revealed, upon invasion into the GECs, <i>P. gingivalis</i> can significantly elevate the host-surface CD73 activity and expression. The enhanced and active CD73 significantly increases <i>P. gingivalis</i> intracellular growth in the presence of substrate-AMP and simultaneously acts as a negative regulator of reactive oxygen species (ROS) generation upon eATP treatment. The inhibition of CD73 by siRNA or by a specific inhibitor markedly increases ROS production. Moreover, CD73 and <i>P. gingivalis</i> cross-signaling significantly modulates pro-inflammatory interleukin-6 (IL-6) in the GECs. Conversely, exogenous treatment of the infected GECs with IL-6 suppresses the intracellular bacteria via amplified ROS generation. However, the decreased bacterial levels can be restored by overexpressing functionally active CD73. Together, these findings illuminate how the local extracellular-purine-metabolism, in which CD73 serves as a core molecular switch, can alter intracellular microbial colonization resistance. Further, host-adaptive pathogens such as <i>P. gingivalis</i> can target host ectonucleotidases to disarm specific innate defenses for successful intracellular persistence in mucosal epithelia.
Project description:<h4>Objectives</h4>This study set out to analyze the difference of heat shock protein 27 (HSP27) and its phosphorylation in patients with lower extremity arteriosclerosis obliterans (LEASO) at different stages. This research also examined their clinical significance in this disease.<h4>Methods</h4>Blood samples from 60 patients with LEASO were collected and divided into two groups according to ankle-brachial index (ABI): group A (ABI ? 0.43) and group B (ABI > 0.43). The expression of HSP27 in each stage of Fontaine was measured by ELISA, and the difference of HSP27 concentration and ABI between the two groups was analyzed. Meanwhile, three normal femoral artery specimens (normal group) and three atherosclerotic femoral artery specimens (lesion group) were collected, and HSP27 and its Phospho-HSP27 (Ser15), Phospho-HSP27 (Ser78) and Phospho-HSP27 (Ser82) were detected by western blotting. The data of the protein level between the normal group and the lesion group was made a statistical analysis.<h4>Results</h4>HSP27 concentration in group A was (40.73 ± 15.99) ng/ml, and ABI was 0.26 ± 0.20. HSP27 concentration in group B was (66.30 ± 24.70) ng/ml, and ABI was 0.64 ± 0.20. The protein expression of HSP27 and its phosphorylation in the normal group was 0.82 ± 0.13, 0.66 ± 0.12, 0.91 ± 0.24 and 0.90 ± 0.16, respectively; the protein expression of the lesion group was 0.45 ± 0.08, 0.42 ± 0.09, 0.39 ± 0.12 and 0.58 ± 0.11.<h4>Conclusion</h4>Patients with higher LEASO Fontaine stage and lower ABI had a lower HSP27 concentration. Serum HSP27 concentration was negatively correlated with the severity of LEASO, while HSP27 concentration was positively correlated with ABI value. The content of HSP27 and its phosphorylation of lesion group is significantly lower than that of normal group, which may be closely related to the occurrence and development of atherosclerosis.
Project description:Porphyromonas gingivalis is a pathogen in severe periodontal disease. Able to exploit an intracellular lifestyle within primary gingival epithelial cells (GECs), a reservoir of P. gingivalis can persist within the gingival epithelia. This process is facilitated by manipulation of the host cell signal transduction cascades which can impact cell cycle, cell death, and cytokine responses. Using microarrays, we investigated the ability of P. gingivalis 33277 to regulate microRNA (miRNA) expression in GECs. One of several miRNAs differentially regulated by GECs in the presence of P. gingivalis was miRNA-203 (miR-203), which was upregulated 4-fold compared to uninfected controls. Differential regulation of miR-203 was confirmed by quantitative reverse transcription-PCR (qRT-PCR). Putative targets of miR-203, suppressor of cytokine signaling 3 (SOCS3) and SOCS6, were evaluated by qRT-PCR. SOCS3 and SOCS6 mRNA levels were reduced >5-fold and >2-fold, respectively, in P. gingivalis-infected GECs compared to controls. Silencing of miR-203 using a small interfering RNA construct reversed the inhibition of SOCS3 expression. A dual luciferase assay confirmed binding of miR-203 to the putative target binding site of the SOCS3 3' untranslated region. Western blot analysis demonstrated that activation of signal transducer and activator of transcription 3 (Stat3), a downstream target of SOCS, was diminished following miR-203 silencing. This study shows that induction of miRNAs by P. gingivalis can modulate important host signaling responses.