Streptococcus pyogenes Transcriptome Changes in the Inflammatory Environment of Necrotizing Fasciitis.

Streptococcus pyogenes is a major cause of necrotizing fasciitis, a life-threatening subcutaneous soft-tissue infection. At the host infection site, the local environment and interactions between the host and bacteria have effects on bacterial gene expression profiles, while the gene expression pattern of S. pyogenes related to this disease remains unknown. In this study, we used a mouse model of necrotizing fasciitis and performed RNA-sequencing (RNA-seq) analysis of S. pyogenes M1T1 strain 5448 by isolating total RNA from infected hind limbs obtained at 24, 48, and 96 h postinfection. RNA-seq analysis results identified 483 bacterial genes whose expression was consistently altered in the infected hindlimbs compared to their expression under in vitro conditions. Genes showing consistent enrichment during infection included 306 encoding molecules involved in virulence, carbohydrate utilization, amino acid metabolism, trace-metal transport, and the vacuolar ATPase transport system. Surprisingly, drastic upregulation of 3 genes, encoding streptolysin S precursor (sagA), cysteine protease (speB), and secreted DNase (spd), was noted in the present mouse model (log2 fold change, >6.0, >9.4, and >7.1, respectively). Conversely, the number of consistently downregulated genes was 177, including those associated with the oxidative stress response and cell division. These results suggest that in necrotizing fasciitis, S. pyogenes shows an altered metabolism, decreased cell proliferation, and upregulation of expression of major toxins. Our findings are considered to provide critical information for developing novel treatment strategies and vaccines for necrotizing fasciitis.IMPORTANCE Necrotizing fasciitis, a life-threatening subcutaneous soft-tissue infection, is principally caused by S. pyogenes The inflammatory environment at the site of infection causes global gene expression changes for survival of the bacterium and pathogenesis. However, no known study regarding transcriptomic profiling of S. pyogenes in cases of necrotizing fasciitis has been presented. We identified 483 bacterial genes whose expression was consistently altered during infection. Our results showed that S. pyogenes infection induces drastic upregulation of the expression of virulence-associated genes and shifts metabolic pathway usage. In particular, high-level expression of toxins, such as cytolysins, proteases, and nucleases, was observed at infection sites. In addition, genes identified as consistently enriched included those related to metabolism of arginine and histidine as well as carbohydrate uptake and utilization. Conversely, genes associated with the oxidative stress response and cell division were consistently downregulated during infection. The present findings provide useful information for establishing novel treatment strategies.

Streptococcus pneumoniae Evades Host Cell Phagocytosis and Limits Host Mortality Through Its Cell Wall Anchoring Protein PfbA.

Streptococcus pneumoniae is a Gram-positive bacterium belonging to the oral streptococcus species, mitis group. This pathogen is a leading cause of community-acquired pneumonia, which often evades host immunity and causes systemic diseases, such as sepsis and meningitis. Previously, we reported that PfbA is a ß-helical cell surface protein contributing to pneumococcal adhesion to and invasion of human epithelial cells in addition to its survival in blood. In the present study, we investigated the role of PfbA in pneumococcal pathogenesis. Phylogenetic analysis indicated that the pfbA gene is highly conserved in S. pneumoniae and Streptococcus pseudopneumoniae within the mitis group. Our in vitro assays showed that PfbA inhibits neutrophil phagocytosis, leading to pneumococcal survival. We found that PfbA activates NF-κB through TLR2, but not TLR4. In addition, TLR2/4 inhibitor peptide treatment of neutrophils enhanced the survival of the S. pneumoniae ΔpfbA strain as compared to a control peptide treatment, whereas the treatment did not affect survival of a wild-type strain. In a mouse pneumonia model, the host mortality and level of TNF-α in bronchoalveolar lavage fluid were comparable between wild-type and ΔpfbA-infected mice, while deletion of pfbA decreased the bacterial burden in bronchoalveolar lavage fluid. In a mouse sepsis model, the ΔpfbA strain demonstrated significantly increased host mortality and TNF-α levels in plasma, but showed reduced bacterial burden in lung and liver. These results indicate that PfbA may contribute to the success of S. pneumoniae species by inhibiting host cell phagocytosis, excess inflammation, and mortality by interacting with TLR2.

Injection of Dental Pulp Stem Cells Promotes Healing of Damaged Bladder Tissue in a Rat Model of Chemically Induced Cystitis.

Dental pulp stem cells (DPSCs) are reported as sources of mesenchymal stem cells (MSCs). MSCs are used as cell therapy options for various diseases. The present study examined the healing effects of DPSC injection on damaged bladder tissue in a chemically induced cystitis rat model. Cystitis was induced by hydrochloride injection into the bladder of female F344/NSlc rats. On the following day, DPSCs suspended in phosphate-buffered saline (PBS) were injected into the bladder and maintained for 1 h (DPSC injection group), while PBS alone was injected as the standard for comparison (PBS injection group). After 2 days following injection, considerable submucosal edema, vascular structure destruction, hemorrhage, and inflammatory cell invasion were observed both in the DPSC and PBS injection groups, with no difference in their degree of submucosal edema and hemorrhage. Six days after injection, vascular structure regeneration was observed in both groups; however, unlike the DPSC injection group, the PBS injection group showed traces of submucosal edema and hemorrhage. These results correlated with tissue concentrations of myeloperoxidase (MPO) and the inflammatory cytokines IL-1ß, IL-6, and TNF-α. Furthermore, the intercontraction interval was prolonged, and the frequency of nociceptive behaviors was reduced in the DPSC injection group compared with the PBS injection group. DPSCs were found on the bladder epithelium until day 3 after injection. In the DPSC-conditioned media (CM), the trophic factors FGF-2, VEGF, and the C-C and C-X-C families of chemokines were detected. The results of DPSC injection into the cystitis rat model suggested that the injected cells promote the healing of the damaged bladder tissue by exerting various trophic effects while localizing on the bladder epithelium and that MSC injection is a potential novel therapy for interstitial cystitis/painful bladder syndrome.

Trophic Effects and Regenerative Potential of Mobilized Mesenchymal Stem Cells From Bone Marrow and Adipose Tissue as Alternative Cell Sources for Pulp/Dentin Regeneration.

Dental pulp stem cell (DPSC) subsets mobilized by granulocyte-colony-stimulating factor (G-CSF) are safe and efficacious for complete pulp regeneration. The supply of autologous pulp tissue, however, is very limited in the aged. Therefore, alternative sources of mesenchymal stem/progenitor cells (MSCs) are needed for the cell therapy. In this study, DPSCs, bone marrow (BM), and adipose tissue (AD)-derived stem cells of the same individual dog were isolated using G-CSF-induced mobilization (MDPSCs, MBMSCs, and MADSCs). The positive rates of CXCR4 and G-CSFR in MDPSCs were similar to MADSCs and were significantly higher than those in MBMSCs. Trophic effects of MDPSCs on angiogenesis, neurite extension, migration, and antiapoptosis were higher than those of MBMSCs and MADSCs. Pulp-like loose connective tissues were regenerated in all three MSC transplantations. Significantly higher volume of regenerated pulp and higher density of vascularization and innervation were observed in response to MDPSCs compared to MBMSC and MADSC transplantation. Collagenous matrix containing dentin sialophosphoprotein (DSPP)-positive odontoblast-like cells was the highest in MBMSCs and significantly higher in MADSCs compared to MDPSCs. MBMSCs and MADSCs, therefore, have potential for pulp regeneration, although the volume of regenerated pulp tissue, angiogenesis, and reinnervation, were less. Thus, in conclusion, an alternative cell source for dental pulp/dentin regeneration are stem cells from BM and AD tissue.