ABSTRACT
BACKGROUND: Arg-gingipain A (RgpA) is the primary virulence factor of Porphyromonas gingivalis and contains hemagglutinin adhesin (HA), which helps bacteria adhere to cells and proteins. Hemagglutinin's functional domains include cleaved adhesin (CA), which acts as a hemagglutination and hemoglobin-binding actor. Here, we confirmed that the HA and CA genes are immunogenic, and using adjuvant chemokine to target dendritic cells (DCs) enhanced protective autoimmunity against P. gingivalis-induced periodontal disease. METHODS: C57 mice were immunized prophylactically with pVAX1-CA, pVAX1-HA, pVAX1, and phosphate-buffered saline (PBS) through intramuscular injection every 2 weeks for a total of three administrations before P. gingivalis-induced periodontitis. The DCs were analyzed using flow cytometry and ribonucleic acid sequencing (RNA-seq) transcriptomic assays following transfection with CA lentivirus. The efficacy of the co-delivered molecular adjuvant CA DNA vaccine was evaluated in vivo using flow cytometry, immunofluorescence techniques, and micro-computed tomography. RESULTS: After the immunization, both the pVAX1-CA and pVAX1-HA groups exhibited significantly elevated P. gingivalis-specific IgG and IgG1, as well as a reduction in bone loss around periodontitis-affected teeth, compared to the pVAX1 and PBS groups (p < 0.05). The expression of CA promoted the secretion of HLA, CD86, CD83, and DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) in DCs. Furthermore, the RNA-seq analysis revealed a significant increase in the chemokine (C-C motif) ligand 19 (p < 0.05). A notable elevation in the quantities of DCs co-labeled with CD11c and major histocompatibility complex class II, along with an increase in interferon-gamma (IFN-γ) cells, was observed in the inguinal lymph nodes of mice subjected to CCL19-CA immunization. This outcome effectively illustrated the preservation of peri-implant bone mass in rats afflicted with P. gingivalis-induced peri-implantitis (p < 0.05). CONCLUSIONS: The co-administration of a CCL19-conjugated CA DNA vaccine holds promise as an innovative and targeted immunization strategy against P. gingivalis-induced periodontitis and peri-implantitis.
ABSTRACT
Protein S-acyl transferases (PATs) catalyze S-acylation, a reversible post-translational modification critical for membrane association, trafficking, and stability of substrate proteins. Many plant proteins are potentially S-acylated but few have corresponding PATs identified. By using genomic editing, confocal imaging, pharmacological, genetic, and biochemical assays, we demonstrate that three Arabidopsis class C PATs positively regulate BR signaling through S-acylation of BRASSINOSTEROID-SIGNALING KINASE1 (BSK1). PAT19, PAT20, and PAT22 associate with the plasma membrane (PM) and the trans-Golgi network/early endosome (TGN/EE). Functional loss of all three genes results in a plethora of defects, indicative of reduced BR signaling and rescued by enhanced BR signaling. PAT19, PAT20, and PAT22 interact with BSK1 and are critical for the S-acylation of BSK1, and for BR signaling. The PM abundance of BSK1 was reduced by functional loss of PAT19, PAT20, and PAT22 whereas abolished by its S-acylation-deficient point mutations, suggesting a key role of S-acylation in its PM targeting. Finally, an active BR analog induces vacuolar trafficking and degradation of PAT19, PAT20, or PAT22, suggesting that the S-acylation of BSK1 by the three PATs serves as a negative feedback module in BR signaling.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Protein Serine-Threonine Kinases , Acylation , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Brassinosteroids/metabolism , Gene Expression Regulation, Plant , Signal Transduction , Transferases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolismABSTRACT
In this study, we explored the direct effect of vascular endothelial growth factor (VEGF) on temporomandibular joint osteoarthritis (TMJ-OA) by analyzing the transformation of mouse condylar cartilage treated in vitro with various concentrations of VEGF. Tissue samples from 126 condyles of four-week-old male C57 mice were randomly divided into 21 groups and treated with VEGF (0 ng/mL, 100 ng/mL, 500 ng/mL, 1 µg/mL, or 2 µg/mL). Furthermore, the samples were treated at different time points (1 d, 2 d, 4 d, and 7 d) and stained with hematoxylin and eosin (HE) and Safranin O and Fast Green stains to observe their morphology. The Mankin score was used to evaluate changes to the condylar cartilage tissues, and immunohistochemistry was performed to observe the expressions of VEGF receptor 2 (VEGFR2), matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 13 (MMP13), and tumor necrosis factor-related activation-induced cytokine (TRANCE). An HE staining analysis revealed that the experimental groups treated with VEGF exhibited the destruction of their condylar cartilage and a proliferation of their hypertrophic cells, in comparison to the control group. Safranin O and Fast Green staining showed that the experimental groups had decreased levels of proteoglycan and degenerative changes in their condylar cartilage. The Mankin score of the samples increased with increasing concentration and treatment time of VEGF, and the differences between the groups were statistically significant (P < 0.05). Immunohistochemistry demonstrated that the expression levels of VEGFR2, MMP9, MMP13, and TRANCE significantly increased in the experimental groups, in comparison to those in the control group, suggesting that VEGF promoted TMJ-OA in mice in vitro.