The Putative Cytochrome b5 Domain-Containing Protein CaDap1 Homologue Is Involved in Antifungal Drug Tolerance, Cell Wall Chitin Maintenance, and Virulence in Candida albicans.

Candida albicans (Ca), a prominent opportunistic fungal pathogen in humans, has garnered considerable attention due to its infectious properties. Herein, we have identified and characterized CaCDAP1 (Ca orf19.1034), a homolog of ScDAP1 found in Saccharomyces cerevisiae. CaCDAP1 encodes a 183-amino acid protein with a conserved cytochrome b5-like heme-binding domain. The deletion of CaDAP1 renders Ca cells susceptible to caspofungin and terbinafine. CaDAP1 deletion confers resistance to Congo Red and Calcofluor White, and sensitivity to sodium dodecyl sulfate. The deletion of CaDAP1 results in a 50% reduction in chitin content within the cell wall, the downregulation of phosphorylation levels in CaMkc1, and the upregulation of phosphorylation levels in CaCek1. Notably, CaDAP1 deletion results in the abnormal hyphal development of Ca cells and diminishes virulence in a mouse systemic infection model. Thus, CaDAP1 emerges as a critical regulator governing cellular responses to antifungal drugs, the synthesis of cell wall chitin, and virulence in Ca.

Bacterial growth stage determines the yields, protein composition, and periodontal pathogenicity of Porphyromonas gingivalis outer membrane vesicles.

Introduction: P. gingivalis (W83), as the keystone pathogen in chronic periodontitis, has been found to be tightly bound to systemic diseases. Outer membrane vesicles (OMVs) produced by P. gingivalis (W83) are thought to serve key functions in bacterial virulence and pathogenicity. This study aims to comprehend the biological functions of P. gingivalis OMVs isolated from different growth stages by comparing their physicochemical properties and pathogenicity. Methods: Protein composition was analyzed via isotope-labeled relative and absolute quantification (iTRAQ). Macrophage polarization and the expression of IL-6 and IL-1ß were detected. The proliferation, migration, osteogenic differentiation, and IL-1b/NLRP3 expression of periodontal ligament stem cells (PDLSCs) were evaluated. P. gingivalis/P. gingivalis OMVs-induced periodontal models were also constructed in Sprague Dawley rats. Results: The protein composition of P. gingivalis OMVs isolated from different growth stages demonstrated obvious differences ranging from 25 KDa to 75 KDa. In the results of flow cytometry, we found that in vitro experiments the M1 subtype of macrophages was more abundant in the late-log OMVs and stationary OMVs groups which boosted the production of inflammatory cytokines more than pre-log OMVs. Compared to pre-log OMVs, late-log OMVs and stationary OMVs had more pronounced inhibitory effects on proliferation, migration, and early osteogenesis of PDLSCs. The NLRP3 inflammasome was activated to a larger extent in the stationary OMVs group. Micro-computed tomography (Micro CT), hematoxylin-eosin staining (HE), and tartrate acid phosphatase (TRAP) results showed that the periodontal damage in the stationary OMVs group was worse than that in the pre-log OMVs and late-log OMVs group, but almost equal to that in the positive control group (P. gingivalis). Discussion: In general, both in vivo and in vitro experiments showed that late-log OMVs and stationary OMVs have more significant pathogenicity in periodontal disease.

Let-7a promotes periodontal bone regeneration of bone marrow mesenchymal stem cell aggregates via the Fas/FasL-autophagy pathway.

Periodontal bone regeneration using bone marrow mesenchymal stem cell (BMMSC) transplantation is a promising method; however, the method for osteogenic differentiation of BMMSCs needs to be improved. In this research, we sought to identify the roles of let-7a in the osteogenesis of BMMSCs and to provide a potential method for periodontal bone regeneration. Our previous study revealed that Fas/FasL is a target of let-7a. In this study, we demonstrated that let-7a overexpression significantly enhanced BMMSC-CAs osteogenesis both in vitro and in vivo. Mechanistically, upregulation of Fas/FasL using the rfas/rfaslg plasmid obstructed the osteogenesis of BMMSCs by inhibiting autophagy. Furthermore, we confirmed that overexpression of let-7a activated autophagy and alleviated the inhibited osteogenesis by the autophagy inhibitor 3-MA and the rfas/rfaslg plasmid of BMMSCs. In general, our findings showed that let-7a promoted the osteogenesis of BMMSCs through the Fas/FasL-autophagy pathway, suggesting that the application of let-7a in BMMSC-CAs based periodontal bone regeneration could be a promising strategy.

Dental Pulp Stem Cell-Derived Exosomes Regulate Anti-Inflammatory and Osteogenesis in Periodontal Ligament Stem Cells and Promote the Repair of Experimental Periodontitis in Rats.

Purpose: Dental pulp stem cell-derived exosomes (DPSC-EXO), which have biological characteristics similar to those of metrocytes, have been found to be closely associated with tissue regeneration. Periodontitis is an immune inflammation and tissue destructive disease caused by plaque, resulting in alveolar bone loss and periodontal epithelial destruction. It is not clear whether DPSC-EXO can be used as an effective therapy for periodontal regeneration. The purpose of this study was not only to verify the effect of DPSC-EXO on reducing periodontitis and promoting periodontal tissue regeneration, but also to reveal the possible mechanism. Methods: DPSC-EXO was isolated by ultracentrifugation. Then it characterized by transmission electron microscope (TEM), nanoparticle tracking analysis (NTA) and Western Blot. In vitro, periodontal ligament stem cells (PDLSCs) were treated with DPSC-EXO, the abilities of cell proliferation, migration and osteogenic potential were evaluated. Furthermore, we detected the expression of IL-1ß, TNF-αand key proteins in the IL-6/JAK2/STAT3 signaling pathway after simulating the inflammatory environment by LPS. In addition, the effect of DPSC-EXO on the polarization phenotype of macrophages was detected. In vivo, the experimental periodontitis in rats was established and treated with DPSC-EXO or PBS. After 4 weeks, the maxillae were collected and detected by micro-CT and histological staining. Results: DPSC-EXO promoted the proliferation, migration and osteogenesis of PDLSCs in vitro. DPSC-EXO also regulated inflammation by inhibiting the IL-6/JAK2/STAT3 signaling pathway during acute inflammatory stress. In addition, the results showed that DPSC-EXO could polarize macrophages from the M1 phenotype to the M2 phenotype. In vivo, we found that DPSC-EXO could effectively reduce alveolar bone loss and promote the healing of the periodontal epithelium in rats with experimental periodontitis. Conclusion: DPSC-EXO plays an important role in inhibiting periodontitis and promoting tissue regeneration. This study provides a promising acellular therapy for periodontitis.

Exopolysaccharides metabolism and cariogenesis of Streptococcus mutans biofilm regulated by antisense vicK RNA.

Background: Streptococcus mutans (S. mutans) is a pivotal cariogenic pathogen contributing to its multiple virulence factors, one of which is synthesizing exopolysaccharides (EPS). VicK, a sensor histidine kinase, plays a major role in regulating genes associated with EPS synthesis and adhesion. Here we first identified an antisense vicK RNA (ASvicK) bound with vicK into double-stranded RNA (dsRNA). Objective: This study aims to investigate the effect and mechanism of ASvicK in the EPS metabolism and cariogenesis of S. mutans. Methods: The phenotypes of biofilm were detected by scanning electron microscopy (SEM), gas chromatography-mass spectrometery (GC-MS) , gel permeation chromatography (GPC) , transcriptome analysis and Western blot. Co-immunoprecipitation (Co-ip) assay and enzyme activity experiment were adopted to investigate the mechanism of ASvicK regulation. Caries animal models were developed to study the relationship between ASvicK and cariogenicity of S. mutans. Results: Overexpression of ASvicK can inhibit the growth of biofilm, reduce the production of EPS and alter genes and protein related to EPS metabolism. ASvicK can adsorb RNase III to regulate vicK and affect the cariogenicity of S. mutans. Conclusions: ASvicK regulates vicK at the transcriptional and post-transcriptional levels, effectively inhibits EPS synthesis and biofilm formation and reduces its cariogenicity in vivo.

Outer membrane vesicles of Porphyromonas gingivalis trigger NLRP3 inflammasome and induce neuroinflammation, tau phosphorylation, and memory dysfunction in mice.

Background: Porphyromonas gingivalis (Pg), the keystone pathogen in chronic periodontitis, is reported to initiate Alzheimer's disease pathologies in preclinical studies. However, the specific mechanisms and signaling pathways acting on the brain still need to be further explored. Outer membrane vesicles are derived from Gram-negative bacteria and contain many virulence factors of bacteria. We hypothesized that outer membrane vesicles are an important weapon of Porphyromonas gingivalis to initiate Alzheimer's disease pathologies. Methods: The outer membrane vesicles of Porphyromonas gingivalis (Pg OMVs, 4 mg/kg) or saline were delivered to 14-month-old mice by oral gavage every other day for eight weeks. Behavioral alterations were assessed by the open field test, Morris water maze, and Y-maze test. Blood-brain barrier permeability, neuroinflammation, tau phosphorylation, and NLRP3 inflammasome-related protein were analyzed. Results: Pg OMVs impaired memory and learning ability of mice and decreased tight junction-related gene expression ZO-1, occludin, claudin-5, and occludin protein expression in the hippocampus. Pg OMVs could be detected in the hippocampus and cortex three days after oral gavage. Furthermore, Pg OMVs activated both astrocytes and microglia and elevated IL-1ß, tau phosphorylation on the Thr231 site, and NLRP3 inflammasome-related protein expression in the hippocampus. In in vitro studies, Pg OMV (5 µg/ml) stimulation increased the mRNA and immunofluorescence of NLRP3 in BV2 microglia, which were significantly inhibited by the NLRP3 inhibitor MCC950. In contrast, the tau phosphorylation in N2a neurons was enhanced after treatment with conditioned media from Pg OMV-stimulated microglia, which was attenuated after pretreatment with MCC950. Conclusions: These results indicate that Pg OMVs prompt memory dysfunction, neuroinflammation, and tau phosphorylation and trigger NLRP3 inflammasome in the brain of middle-aged mice. We propose that Pg OMVs play an important role in activating neuroinflammation in the AD-like pathology triggered by Porphyromonas gingivalis, and NLRP3 inflammasome activation is a possible mechanism.

The lipid flippase subunit Cdc50 is required for antifungal drug resistance, endocytosis, hyphal development and virulence in Candida albicans.

Cdc50 is the non-catalytic subunit of the flippase that establishes phospholipid asymmetry in membranes and functions in vesicle-mediated trafficking in Saccharomyces cerevisiae. Here, we have identified the homologous gene CaCDC50 that encodes a protein of 396 amino acids with two conserved transmembrane domains in Candidaalbicans. Deletion of CaCDC50 results in C. albicans cells becoming sensitive to the antifungal drugs azoles, terbinafine and caspofungin, as well as to the membrane-perturbing agent sodium dodecyl sulfate. We also show that CaCDC50 is involved in both endocytosis and vacuolar function. CaCDC50 confers tolerance to high concentrations of cations, although it is not required for osmolar response. Moreover, deletion of CaCDC50 leads to severe defects in hyphal development of C. albicans cells and highly attenuated virulence in the mouse model of systemic infection. Therefore, CaCDC50 regulates cellular responses to antifungal drugs, cell membrane stress, endocytosis, filamentation and virulence in the human fungal pathogen C. albicans.