Opportunistic Pathogen Porphyromonas gingivalis Modulates Danger Signal ATP-Mediated Antibacterial NOX2 Pathways in Primary Epithelial Cells

Abstract

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.

Cellular metabolic rates and oxidative stress profiles in primary fibroblast cells isolated from virgin females, reproductively experienced females, and male Sprague-Dawley rats

“…Excitation and emission were measured in the violet spectrum at 404/526 nm, respectively (Mandavilli and Janes ; Roberts et al. ). We used a concentration of 1–50 μmol/L Glutathione (Sigma cat no.…”

“…Cells were thenwashed another three times with sterile PBS, and imaged in phenol red-free FluoroBrite DMEM. Excitation and emission were measured in the violet spectrum at 404/526 nm, respectively (Mandavilli and Janes 2010;Roberts et al 2017). We used a concentration of 1-50 lmol/L Glutathione (Sigma cat no.…”

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

“…The ATP hydrolytic activity of P . gingivalis‐ Ndk (~0.1 μM s −1 ; Choi et al, ; Yilmaz et al, ) is especially important in diminishing the danger signal extracellular ATP (eATP)/P2X7‐receptor signalling in primary GECs that generates robust cellular ROS upstream of hypochlorous acid production, a potent eliminator of invading pathogens (Choi et al, ; Roberts et al, ; Roberts & Yilmaz, ; Yilmaz et al, ). Moreover, P .…”

“…Furthermore, P. gingivalis-Ndk is biologically active with similar GTP and ATP hydrolysis activity as other Ndk homologues in both higher organisms and bacterial species (Choi et al, 2013;Sun et al, 2013). The ATP hydrolytic activity of P. gingivalis-Ndk (~0.1 μM s −1 ; Choi et al, 2013;Yilmaz et al, 2008) is especially important in diminishing the danger signal extracellular ATP (eATP)/P2X7-receptor signalling in primary GECs that generates robust cellular ROS upstream of hypochlorous acid production, a potent eliminator of invading pathogens (Choi et al, 2013;Roberts et al, 2017;Roberts & Yilmaz, 2015;Yilmaz et al, 2008). Moreover, P. gingivalis-Ndk is also of importance in the modulation of P2X7-dependent inflammasome activation and the attenuation of secretion of pro-inflammatory cytokine IL-1β as well as pro-inflammatory danger signal HMGB1 in primary GECs (Johnson et al, 2015;Olsen & Yilmaz, 2016).…”

Myd88 plays a major role in the keratinocyte response to infection with Porphyromonas gingivalis

“…Although the oral epithelium was previously thought to play a mainly passive barrier role, the active role of keratinocytes in the host antimicrobial response is now well appreciated . In fact, a special interaction exists between P gingivalis and keratinocytes, involving intracellular trafficking of the bacterium and cellular changes following the infection . Keratinocytes express antimicrobial peptides in response to pathogenic and non‐pathogenic microbial species and coordinate the innate and adaptive responses to oral bacteria .…”