| BackgroundAggregatibacter actinomycetemcomitans(A.actinomycetemcomitans) is one of the bacteria attracting high attention in the bacterial etiology research of periodontitis in recent years, which has been recognized one of the periodontal pathogen, especially is closely related with aggressive periodontitis. A.actinomycetemcomitans resides in the human mouth in biofilm forms. It discharges vesicles during the growth period which containing virulence factors such as endotoxin, leukotoxin (LT) and bone resorption factors, et al. And the endotoxin, cytolethal distending toxin (CDT) such as leukotoxin and other soluble virulent factors mediate the resistance to host defense, periodontal tissue destruction by A.actinomycetemcomitans in the occurrence and development of periodontitis, which plays an important role in the pathogenic process.Phosphorylcholine (PC) is an integral component of plant and animal cells, rich in nerve tissue, especially in myelin and egg yolk. It has been reported that PC exists in a variety of pathogens, including gram-positive bacteria and Gram-negative bacteria. Recent studies showed that PC may be an important virulence factor of the pathogens. Currently, the mechnism of phosphorylcholine in the pathogenesis of most pathogens is still unclear. Studies reported that the choline of the bacterial cell wall combines with the endothelial cells depending on the platelet-activating factor receptor (PAFR)-mediated invasion by S.pn and H.influenzae.PAFR is located in a variety of endothelial and the epithelial cells, the platelet activating factor (PAF), which is the natural ligand for PAFR, contains phosphorylcholine. So the phosphorylcholine in the bacterial cell wall, binds to the platelet activating factorreceptor via analog platelet activating factor and helps the bacteria invade into host cells.Previous studies have found that PC can be expressed in most of the oral supragingival and subgingival bacteria, such as oral streptococci, Fusobacterium, A.actinomycetemcomitans, various Actinomyces and Neisseria, et al. Schenkein et al found a significant virulence difference between PC positive and negative A.actinomycetemcomitans strains. It suggested that PC may act as an important virulence factor of A.actinomycetemcomitans strains during the pathogenic process of periodontal disease. However, the signaling pathway mediating A.actinomycetemcomitans adhesion and invasion into host cells has not been reported. This study intends to isolate clinical A.actinomycetemcomitans strains and to identify the PC positive A.actinomycetemcomitans strains and then to explore the mechanisms for A.actinomycetemcomitans adhesion to and invasion into host cells after combination of PC with PAFR. It is expected that these studies will provide evidences for further elucidating pathogenic mechanisms of A.actinomycetemcomitans.Isolation and Identification of PC-positive A. actinomycetemcomitansNon-attached plaque in periodontal pockets of seven patients with aggressive periodontitis were collected. After cultured on selective medium TSBV plates, seven strains of bacteria were found that the surfaces were rough, catalase test were positive and Gram stain was negative and final identification were confirmed by specific16SrDNA hybridization.SDS-PAGE electrophoresis was done with the whole cell of the isolated clinical A.actinomycetemcomitans strains. Coomassie blue staining was used to observe the bacterial protein expression. Western blot was taken to identify the phosphorylcholine positive A.actinomycetemcomitans strains using the TEPC-15monoclonal antibody to response with phosphorylcholine. At last one phosphorylcholine positive A.actinomycetemcomitans strain was found through Western blotting.Role of PAFR in the pathogenesis of PC-positive A. actinomycetemcomitans1. Role of PC in the adhesion and invasion of HUVEC by A.actinomycetemcomitans For adhesion and invasion assays, cultured HUVEC confluent monolayers in24-well plates(approximately1×10cells/well), were pretreated with phosphorylcholine monoclonal antibody TEPC-15(2.5μg/ml) for30min; and infected with PC positive or negative A.actinomycetemcomitans strains (1×108cfu/mL) respectively for4h at37℃. Then, the effect of adherence and invasion of A.actinomycetemcomitans is assessed as the percentage of total non-treated A.actinomycetemcomitans strains adhered to or internalized by HUVEC. Data are representative of three independent experiments. It was found that after pretreated with anti-PC monoclonal antibody TEPC-15, the adhesion to and invasion of HUVEC by PC-positive A.actinomycetemcomitans were obviously declined to31.87±4.22%and24.63±3.55%of the control, respectively. However, PC negative A.actinomycetemcomitans showed no significant difference in adhesion and invasion of HUVEC after pretreated with TEPC-15antibody. This shows that PC helps PC positive A.actinomycetemcomitans adhesion and invasion of HUVEC.2. Role of PAFR in adhesion and invasion of HUVEC by A.actinomycetemcomitans For adhesion and invasion assays, cultured HUVEC confluent monolayers in24-well plates(approximately1×105cells/well), were pretreated with PAFR antagonist CV3988(50nm,100nm、200nm、500nm)and anti-human PAFR monoclonal antibody(2.5μg/ml) for30min; and infected with PC positive or negative A.actinomycetemcomitans strains (1×108cfu/mL) respectively for4h at37℃. The effect of adherence and invasion of A.actinomycetemcomitans is assessed as above. It was found100,200and500nm of PAFR antagonists significantly reduced adhesion and invasion of PC positive A.actinomycetemcomitans on HUVEC, adhesion were36.29±3.52%,19.04±3.35%and7.69±3.19%of the control;invasion were12.12±1.58%,7.08±0.29%and2.60±2.26%of the control.Similarly, the A.actinomycetemcomitans adhesion and invasion of HUVEC was significantly declined after the HUVEC was pretreated with anti-PAFR antibody, the adhesion and invasion were50.05±5.28%and39.09±6.50%of the control.3. Role of PAFR in bacteria influence cell viability HUVEC confluent monolayers in96-well plates were pretreated were pretreated with PAFR antagonists and anti-PAFR antibody for30min; and infected with PC positive or negative A.actinomycetemcomitans strains (1×108cfu/mL) respectively for24h at37℃. A.actinomycetemcomitans cytotoxicity was assessed quantitatively by monitoring mitochondrial reduction activity using the MTT assay. It was found that HUVEC incubating with PC positive A.actinomycetemcomitans, the cell viability was significantly increased, from25.39±9.33%to91.12±3.14%,94.12±2.15%and65.5±1.87%, respectively after pretreated with PAFR antagonist (200nm and500nm) and anti-PAFR antibody (25μg/ml).However, incubating with PC negative A.actinomycetemcomitans, the HUVEC cell viability was not significantly increased as compared the pretreated group with the non-pretreated group.Role of phosphoinositide signaling pathway mediated by PAFR in the pathogenesis of PC-positive A.actinomycetemcomitans1.Adhesion and invasion assay of A.actinomycetemcomitans after PLC being inhibitedTo explore whether the G-protein coupled receptor pathway is involved in the process of A.actinomycetemcomitans adhesion to and invasion into HUVEC, PLC inhibitor U73122(5μM) and U73343(5μM) which was the inactive analog of U73122were used to pretreat HUVEC. It was found that the PC positive A.actinomycetemcomitans internalized into HUVEC was dramatically decreased to12.62±2.1%of the control in the U73122group. However, there was no significantly difference of bacterial adhesion between the pretreated and non-pretreated groups with U73122. At the same time, PC positive A.actinomycetemcomitans invasion and adhesion to HUVEC did not show a significant difference after HUVEC was pretreated with U73343(P>0.05), indicating that U73343did not block the combination of PC and PAFR to initiate the signal pathway.1. A.actinomycetemcomitans effect on cell viability after PLC being inhibited HUVECs were pretreated with U73343, U73122for30min and infected with PC positive or negative A.actinomycetemcomitans strains(1×108cfu/mL). And the cytotoxicity was assessed quantitatively as above.For PC positive A.actinomycetemcomitans-induced cell death, HUVEC pretreated with U73122showed a significant effect of protecting HUVEC from being induced death, cell viability increased from39.31±6.43%to93.09±5.46%. For PC negative A.actinomycetemcomitans infection of HUVEC, the pretreatment of HUVEC with U73122and U73343showed no significant differences between pretreated and non-pretreated groups with U73122and U73343on cell viability.3. Measurement of intracelluar Ca2+To further confirm that the inhibition of PC positive A.actinomycetemcomitans internalizing into HUVEC by U73122was mediated through blocking the binding of the PLC and the G protein coupled receptor PAFR, the intracellular Ca2+mobilization during the process of PC positive A.actinomycetemcomitans internalizing into HUVEC after pretreated with U73122was measured by fura-2/AM. It was found that U73122almost completely inhibited the increase of intracellular Ca+concentration.Role of clathrin and β-arrestins in the pathogenic process of A. actinomycetemcomitans1. Adhesion and invasion assay of A.actinomycetemcomitans after clathrin being inhibitedTo investigate whether the clathrin was involved in the A.actinomycetemcomitans endocytosis process during A.actinomycetemcomitans invading HUVEC, the impact of clathrin inhibitors, monodansylcadaverine (MDC) and chlorpromazine(CPZ)were studied in A. actinomycetemcomitans adhesion and invasion of HUVEC process. It was found that MDC and CPZ effectively inhibited the PC positive A.actinomycetemcomitans invasion of HUVEC to41.33±2.59%and43.22±2.67%of the control, respectively.However, there was no significant difference between the MDC or CPZ pretreated and nonpretreated groups on adherence, which showed that the MDC and CPZ inhibition of clathrin could not stop A.actinomycetemcomitans adhesion to HUVEC. 2. Role of β-arrestin-land β-arrestin-2during A.actinomycetemcomitans adhesion and invasion of HUVECTo study the role of β-arrestins in the process of A.actinomycetemcomitans adhesion and invasion of HUVEC, double-stranded siRNAs for β-arrestin-1, β-arrestin-2and control were chemically synthesized and transfected in HUVEC, and then the effects on A. actinomycetemcomitans adhesion and invasion of HUVEC were investigated. First, it was detected that intracellular level of β-arrestins was targetedly reduced after transfection compared with untransfected group and negative control group. The invasion of PC positive A.actinomycetemcomitans into HUVEC was effectively blocked by β-arrestins siRNA, the invasion was53.12±2.81%and56.00±2.37%of the control, respectively.However, there was no significant difference on the number of bacteria adhered to the cells, which indicated that β-arrestins siRNA could not inhibit the PC binding to PAFR on the cell surface. Also, there was no significant difference in the negative control groups on the adhesion and invasion of HUVEC.Statistical analysisAll values are expressed as mean±standard deviation. Statistical analysis was performed with Student’s t-test for comparison of two groups, Bonferroni was used to correcte test level; one-way ANOVA was used for multiple comparisons, ie, homogeneity of variance using LSD, heterogeneity of variance using Dunnett’s T3; Spearman was used for correlation analysis. Differences with P<0.05were considered to be statistically significant. The SPSS (version13.0) statistical package was used (SPSS Inc., Chicago, IL).Conclusions1. Phosphorylcholine positive A.actinomycetemcomitans strain was successfully identified from the clinical isolates which provided the foundation for the following studies.2. It showed that PC helped PC positive A.actinomycetemcomitans adhesion and invasion of host cells, and PAFR played an important part in PC positive Aa adhesion, invasion, and inducing cell death. 3. PAFR mediated phosphoinositide signaling pathway is involved in the PC-positive A. actinomycetemcomitans adhesion, invasion of cells and inducing cell death.4. Clathrin and β-arrestins participated in the PAFR-mediated PC positive A.actinomycetemcomitans invasion of HUVEC. |
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