Identification of β‐haemolysin‐encoding genes in Streptococcus anginosus

Streptococcus anginosus is an emerging pathogen, but little is known about its virulence factors. To detect the genes responsible for β‐haemolysis we performed genomic mutagenesis of the β‐haemolytic S. anginosus type strain ATCC 12395 using the vector pGhost9:ISS1. Integration site analysis of 15 non‐haemolytic mutants identified a gene cluster with high homology to the genes of the streptolysin S (SLS) encoding sag gene cluster of S. pyogenes. The gene cluster harbours 10 open reading frames displaying significant similarities to the S. pyogenes genes sagA‐sagI, with the identities on protein level ranging from 38 to 87%. Complementation assays of S. anginosus sagB and sagD integration mutants with the respective genes confirmed their importance for β‐haemolysin production and suggest the presence of post‐translational modifications in S. anginosus SLS similar to SLS of S. pyogenes. Characterization of the S. anginosus haemolysin in comparison to the S. pyogenes SLS showed that the haemolysin is surface bound, but in contrast to S. pyogenes neither fetal calf serum nor RNA was able to stabilize the haemolysin of S. anginosus in culture supernatants. Inhibition of β‐haemolysis by polyethylene glycol of different sizes was carried out, giving no evidence of a pore‐forming haemolytic mechanism. Analysis of a whole genome shotgun sequence of Streptococcus constellatus, a closely related streptococcal species that belongs to the S. anginosus group, revealed a similar sag gene cluster. Employing a genomic mutagenesis strategy we were able to determine an SLS encoding gene cluster in S. anginosus and demonstrate its importance for β‐haemolysin production in S. anginosus.     

Efficacy of stabilisation splint treatment on facial pain – 1‐year follow‐up

The aim of this randomised controlled trial was to assess the efficacy of stabilisation splint treatment on TMD‐related facial pain during a 1‐year follow‐up. Eighty patients were randomly assigned to two groups: splint group (n = 39) and control group (n = 41). The patients in the splint group were treated with a stabilisation splint and received counselling and instructions for masticatory muscle exercises. The controls received only counselling and instructions for masticatory muscles exercises. The outcome variables were the change in the intensity of facial pain (as measured with visual analogue scale, VAS) as well as the patients' subjective estimate of treatment outcome. The differences in VAS changes between the groups were analysed using variance analysis and linear regression models. The VAS decreased in both groups, the difference between the groups being not statistically significant. The group status did not significantly associate with the decrease in VAS after adjustment for baseline VAS, gender, age, length of treatment and general health status. The only statistically significant predicting factor was the baseline VAS, which was also confirmed by the mixed‐effect linear model. After 1‐year follow‐up, 27·6% of the patients in the splint group and 37·5% of the patients in the control group reported ‘very good' treatment effects. The findings of this study did not show stabilisation splint treatment to be more effective in decreasing facial pain than masticatory muscle exercises and counselling alone in the treatment of TMD‐related facial pain over a 1‐year follow‐up.     

Animal models for bisphosphonate‐related osteonecrosis of the jaws ‐ an appraisal

The prolonged use of bisphosphonates has been shown to cause a condition termed ‘bisphosphonate related osteonecrosis of the jaws’ (BRONJ). BRONJ is a disease entity which has only been described relatively recently, and its multi‐factorial aetiology is yet to be fully elucidated. Therefore, the treatment of BRONJ lesions remains a challenge, and animal models are necessary to assist researchers in better understanding the disease. This has led to the recent publication of a number of studies utilising a variety of animal models of BRONJ. This review outlines the factors to be considered when selecting an animal model for BRONJ and discusses the current literature in this rapidly progressing field of research. It is important to consider the applicability of a given model to the clinical condition presenting in humans, and to this end, thorough characterisation of the clinical, histological, radiographic and systemic features is necessary. The development of a clinical lesion is an important consideration in terms of choosing a relevant model, and it appears clear that surgical manipulation, generally involving tooth extraction, is necessary for successful induction of the classic ‘clinical’ lesion of BRONJ.     

Role of MyD88‐dependent and MyD88‐independent signaling in Porphyromonas gingivalis‐elicited macrophage foam cell formation

Clinical studies and experimental modeling identify a potential link between periodontal disease and periodontal pathogens such as Porphyromonas gingivalis and atherosclerosis and formation of macrophage foam cells. Toll‐like receptors and molecules governing their intracellular signaling pathways such as MyD88 play roles in atherosclerosis, as well as host response to P. gingivalis. The aim of this study was to define roles of MyD88 and TRIF during macrophage foam cell formation in response to P. gingivalis. In the presence of human low‐density lipoprotein (LDL) mouse bone‐marrow‐derived macrophages (BMφ) cultured with P. gingivalis responded with significant reduction in tumor necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6). The BMφ stained strongly with oil red O, regardless of whether bacterial challenge occurred concurrent with or before LDL treatment. Heat‐killed P. gingivalis stimulated foam cell formation in a similar way to live bacteria. The BMφ from MyD88‐knockout and Lps2 mice revealed a significant role for MyD88, and a minor role for TRIF in P. gingivalis‐elicited foam cell formation. Porphyromonas gingivalis‐elicited TNF‐α and IL‐6 were affected by MyD88 ablation and to a lesser extent by TRIF status. These data indicate that LDL affects the TNF‐α and IL‐6 response of macrophages to P. gingivalis challenge and that MyD88 and TRIF play important roles in P. gingivalis‐elicited foam cell formation.     

Fermentative 2‐carbon metabolism produces carcinogenic levels of acetaldehyde in Candida albicans

Acetaldehyde is a carcinogenic product of alcohol fermentation and metabolism in microbes associated with cancers of the upper digestive tract. In yeast acetaldehyde is a by‐product of the pyruvate bypass that converts pyruvate into acetyl‐Coenzyme A (CoA) during fermentation. The aims of our study were: (i) to determine the levels of acetaldehyde produced by Candida albicans in the presence of glucose in low oxygen tension in vitro; (ii) to analyse the expression levels of genes involved in the pyruvate‐bypass and acetaldehyde production; and (iii) to analyse whether any correlations exist between acetaldehyde levels, alcohol dehydrogenase enzyme activity or expression of the genes involved in the pyruvate‐bypass. Candida albicans strains were isolated from patients with oral squamous cell carcinoma (n = 5), autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) patients with chronic oral candidosis (n = 5), and control patients (n = 5). The acetaldehyde and ethanol production by these isolates grown under low oxygen tension in the presence of glucose was determined, and the expression of alcohol dehydrogenase (ADH1 and ADH2), pyruvate decarboxylase (PDC11), aldehyde dehydrogenase (ALD6) and acetyl‐CoA synthetase (ACS1 and ACS2) and Adh enzyme activity were analysed. The C. albicans isolates produced high levels of acetaldehyde from glucose under low oxygen tension. The acetaldehyde levels did not correlate with the expression of ADH1, ADH2 or PDC11 but correlated with the expression of down‐stream genes ALD6 and ACS1. Significant differences in the gene expressions were measured between strains isolated from different patient groups. Under low oxygen tension ALD6 and ACS1, instead of ADH1 or ADH2, appear the most reliable indicators of candidal acetaldehyde production from glucose.     

Heterogeneity of Streptococcus anginosus ß‐hemolysis in relation to CRISPR/Cas

Streptococcus anginosus is a commensal of the oral mucosa that can cause severe invasive infections. A considerable proportion of Streptococcus anginosus strains are ß‐hemolytic due to the presence of an SLS‐like gene cluster. However, the majority of strains do not display ß‐hemolysis. To investigate ß‐hemolysin heterogeneity in S. anginosus, we determined the presence of sag genes and correlated it with the presence of CRISPR/Cas genes in a collection of ß‐hemolytic and non‐ß‐hemolytic strains. All of the ß‐hemolytic strains carried the sag gene cluster. In contrast to other streptococci, clinical S. anginosus strains that do not display ß‐hemolysis do not harbor sag genes. Phylogenetic analysis of the ß‐hemolytic strains revealed that they belong to two previously defined clusters within S. anginosus. Correlation with CRISPR/Cas genes showed a significant difference for the presence of CRISPR/Cas in ß‐hemolytic versus non‐ß‐hemolytic isolates. The presence of the CRISPR/Cas type IIA or type IIC locus is associated with the absence of sag genes; in 65% of the non‐ß‐hemolytic strains a CRISPR/Cas locus was found, while only 24% of ß‐hemolytic strains carry CRISPR/Cas genes. Further analysis of the spacer content of the CRISPR systems revealed the presence of multiple self‐targeting sequences directed against S. anginosus genes. These results support the hypothesis that horizontal gene transfer is involved in the acquisition of ß‐hemolysin genes and that CRISPR/Cas may limit DNA uptake in S. anginosus.     

Homeobox protein MSX‐1 inhibits expression of bone morphogenetic protein 2, bone morphogenetic protein 4, and lymphoid enhancer‐binding factor 1 via Wnt/β‐catenin signaling to prevent differentiation of dental mesenchymal cells during the late bell stage

Homeobox protein MSX‐1 (hereafter referred to as MSX‐1) is essential for early tooth‐germ development. Tooth‐germ development is arrested at bud stage in Msx1 knockout mice, which prompted us to study the functions of MSX‐1 beyond this stage. Here, we investigated the roles of MSX‐1 during late bell stage. Mesenchymal cells of the mandibular first molar were isolated from mice at embryonic day (E)17.5 and cultured in vitro. We determined the expression levels of β‐catenin, bone morphogenetic protein 2 (Bmp2), Bmp4, and lymphoid enhancer‐binding factor 1 (Lef1) after knockdown or overexpression of Msx1. Our findings suggest that knockdown of Msx1 promoted expression of Bmp2, Bmp4, and Lef1, resulting in elevated differentiation of odontoblasts, which was rescued by blocking the expression of these genes. In contrast, overexpression of Msx1 decreased the expression of Bmp2, Bmp4, and Lef1, leading to a reduction in odontoblast differentiation. The regulation of Bmp2, Bmp4, and Lef1 by Msx1 was mediated by the Wnt/β‐catenin signaling pathway. Additionally, knockdown of Msx1 impaired cell proliferation and slowed S‐phase progression, while overexpression of Msx1 also impaired cell proliferation and prolonged G1‐phase progression. We therefore conclude that MSX‐1 maintains cell proliferation by regulating transition of cells from G1‐phase to S‐phase and prevents odontoblast differentiation by inhibiting expression of Bmp2, Bmp4, and Lef1 at the late bell stage via the Wnt/β‐catenin signaling pathway.     

Porphyromonas gingivalis inhibits M2 activation of macrophages by suppressing α‐ketoglutarate production in mice

Reprograming of metabolic pathways is critical in governing the polarization of macrophages into classical proinflammatory M1 or alternative anti‐inflammatory M2 phenotypes in metabolic diseases, such as diabetes. Porphyromonas gingivalis, a keystone pathogen of periodontitis, causes an imbalance in M1/M2 activation, resulting in a hyperinflammatory environment that promotes the pathogenesis of periodontitis. However, whether P. gingivalis infection modulates metabolic pathways to alter macrophage polarization remains unclear. Bone‐marrow‐derived macrophages (BMDMs) were collected from 6‐week‐old female C57BL/6 mice and stimulated with P. gingivalis, P. gingivalis‐derived LPS or IL‐4. Relative gene expression and protein production were measured by quantitative real‐time PCR, RNA sequencing and western blotting. Colorimetric assays were also performed to assess the amounts of α‐ketoglutarate (α‐KG) and succinate. P. gingivalis or P. gingivalis‐derived LPS‐induced inflammatory responses enhanced M1 macrophages and suppressed M2 macrophages, even in the presence of IL‐4. P. gingivalis inhibited Idh1/2 and Gpt1/2 mRNA expression, and increased Akgdh mRNA expression, thus decreasing the ratio of α‐KG/succinate. Supplementation of cell‐permeable dimethyl‐α‐KG dramatically restored M2 activation during P. gingivalis infection. Our study suggests that P. gingivalis maintains a hyperinflammatory state by suppressing the production of α‐KG by M2 macrophages.