Porphyromonas gingivalis Resistance to Polymyxin B Is Determined by the Lipid A 4’-Phosphatase, PGN_0524

Aim To elucidate the genetic basis for the pronounced resistance that the oral pathogen, Porphyromonas gingivalis （P. gingivalis）, exhibits towards the cationic antimicrobial peptide, polymyxin B. Methodology A genetic screen of P. gingivalis clones generated by a Tn4400-based random insertion mutagenesis strategy was performed to identify bacteria harboring novel genetic mutations that render P. gingivalis susceptible to killing by the cationic antimicrobial peptide, polymyxin B （PMB, 50μg·mL^-1）. Results P. gingivalis （ATCC 33277） is unusually resistant to the cationic antimicrobial peptide, PMB at relatively high concentrations （200μg·mL^-1）. Approximately 2,700 independent Tn4400 ＇-derived mutants ofP. gingivalis were examined for increased sensitivity to PMB killing at a relatively low dose （50 μg·mL^-1）. A single PMB-sensitive mutant was obtained in this phenotypic screen. We determined that the Tn4400＇ transposon was integrated into the gene encoding the lipid A 4＇-phosphatase, PGN 0524, demonstrating that this insertion event was responsible for its increased susceptibility of this clone to PMB-dependent killing. The resulting mutant strain, designated 0524-Tn4400＇, was highly sensitive to PMB killing relative to wild-type P. gingivalis, and exhibited the same sensitivity as the previously characterized strain, 0524KO, which bears a genetically engineered deletion in the PGN_0524 locus. Positive ion mass spectrometric structural （MALDI-TOF MS） analyses revealed that lipid A isolates from 0524-Tn4400＂ and 0524KO strains displayed strikingly similar MALDI-TOF MS spectra that were substantially different from the wildtype P gingivalis lipid A spectrum. Finally, intact 0524- Tn4400＇ and 0524KO mutant bacteria, as well as their corresponding LPS isolates, were significantly more potent in stimulating Toll-like receptor 4 （TLR4）-dependent E-selectin expression in human endothelial cells relative to intact wild-type P.. gingivalis or its corresponding LPS isolate. Conclusion The combined molecular evidence provided in this report suggests that PGN 0524, a lipid A 4＇-phosphatase, is the sole genetic element conferring the ability of the periodontopathogen, P. gingivalis, to evade the killing activity of cationic antimicrobial peptides, such as PMB. These data strongly implicate PGN_0524 as a critical virulence factor for the ability of P.. gingivalis to evade front-line host innate defenses that are dependent upon cationic antimicrobial peptide activity and TLR 4 sensing.

Role of Rac1/p38 and ERK-Dependent Cytosolic Phospholipase A2 Activation in Porphyromonas gingivalis-Evoked Induction in Matrix Metalloproteinase-9 (MMP-9) Release by Salivary Gland Cells

Matrix metalloproteinase-9 (MMP-9) is a highly glycosylated endopeptidase implicated in a wide rage of oral mucosal inflammatory and neoplastic diseases, including chronic periodontitis, a persistent mucosal inflammation attributed primarily to infection by oral anaerobe, P. gingivalis. In this study, we explored the role of Rac1 and mitogen-activated protein kinases (MAPKs) in the processes of MMP-9 release in sublingual salivary gland cells exposed to P. gingivalis key endotoxin, cell wall lipopolysaccharide (LPS). We demonstrate that the LPS-elicited induction in the acinar cell MMP-9 release is associated with MAPK, ERK and p38 activation, and occurs with the involvement of Rac1 and cytosolic phospholipase A2 (cPLA2). Further, we reveal that the LPS-induced MMP-9 release involves ERK-mediated phosphorylation of cPLA2 on Ser505 that is essential for its membrane translocation with Rac1, and that this process requires p38 activation. Moreover, we show that phosphorylation and membrane localization of p38 with Rac1-GTP play a pivotal role in cPLA2-dependent induction in MMP-9 release. Thus collectively, our findings infer that P. gingivalis LPS-induced up-regulation in the acinar cell MMP-9 release requires ERK-dependent recruitment of cPLA2 to the membrane localized Rac1/p38 complex.

Unfolded p53 in non-neuronal cells supports bacterial etiology of Alzheimer’s disease

Alzheimer’s disease has proven to be largely intractable to treatment,despite years of research,and numerous trials of therapies that target the hallmarks of the disease-amyloid plaques and neurofibrillary tangles.The etiology of Alzheimer’s disease remains elusive.There is a growing body of evidence for an infectious trigger of Alzheimer’s disease,and,in particular,the focus has been on the oral pathogen Porphyromonas gingivalis(P.gingivalis).Reports of the expression of a misfolded form of p53 in non-neuronal cells(fibroblasts,peripheral blood mononuclear cells,and B cells)and serum,which appears several years before clinical symptoms manifest,may provide further support for the role of bacteria in general,and P.gingivalis in particular,in the initiation of the disease.This review presents a model of the pathway from initial oral infection with P.gingivalis to amyloid plaque formation and neuronal degeneration,via the steps of chronic periodontitis;secretion of the inflammagens lipopolysaccharide and gingipains into the bloodstream;induction of an inflammatory response in both peripheral cells and tissues;disruption of the blood-brain barrier,and entry into the central nervous system of the inflammagens and the P.gingivalis bacteria themselves.In this model,the misfolded p53(or“unfolded p53”;up53)is induced in non-neuronal cells and upregulated in serum as a result of oxidative stress due to lipopolysaccharide from P.gingivalis.up53 is therefore a potential biomarker for early diagnosis of the presence of a causative agent of Alzheimer’s disease.Fastidious dental hygiene and aggressive antibiotic treatment may prevent the patient progressing to clinical Alzheimer’s disease if serum up53 is detected at this pre-symptomatic stage.

牙龈卟啉单胞菌感染的食管癌细胞外泌体中miRNA表达谱分析

目的研究P.gingivalis对KYSE-30细胞外泌体中miRNA表达谱及其相关信号通路的影响。方法采用高通量测序技术筛选KYSE-30细胞外泌体和P.gingivalis感染的KYSE-30细胞外泌体中差异表达的miRNA,通过生物信息学的方法预测差异miRNA的相关靶基因,并对靶基因进行GO富集分析和KEGG通路分析。结果这些差异表达的miRNA所预测的靶基因显著参与GnRH信号通路、TGF-β信号通路、Hippo信号通路、p53信号通路、Ras信号通路、mTOR信号通路、PI3K-Akt信号通路等,这些信号通路不同程度地参与食管癌的发生发展。结论这些差异表达的miRNA所涉及到的信号通路可能暗示着P.gingivalis通过影响外泌体miRNA的表达,从而调控细胞内的相关信号通路,进而参与食管癌的增殖、侵袭、转移等发展进程。

Antibacteria activities of phenolics from"Jinshiji"thinned-young apple on halitosis-related bacteria

This article aims to explain the inhibitory mechanism of thinned-young apple polyphenols(YAP)toward Fusobacterium nucleatum(F.nucleatum),Porphyromonas gingivalis(P.gingivalis)and Prevotella intermedia(P.intermedia).YAP has optimal antibacterial concentration against F.nucleatum(10.00 mg/mL),P.gingivalis(8.00 mg/mL)and P.intermedia(8.00 mg/mL),with viability of 47.97%,38.01%and 36.94%,respectively.Scanning electron microscopy(SEM)and confocal laser scanning microscopy(CLSM)observation revealed that YAP could cause the morphological changes of the halitosis-related bacterial cells and induce cell apoptosis.With the increase of YAP treatment concentrations,the permeability of the outer membranes(OM)and inner membranes(IM)of halitosis-related bacteria dramatically increased,resulting in the release of proteins and nucleic acids.In addition,bacterial cell membrane potential(MP)decreased after exposure to YAP.The results of this study provide new antibacterial agents for halitosis-related bacteria and find a new way for the development of thinned-young apples.