Involvement of ribonuclease Y in pilus production by M49 Streptococcus pyogenes strain via modulation of messenger RNA level of transcriptional regulator.

Streptococcus pyogenes displays a wide variety of pili, which is largely dependent on serotype. A distinct subset of S. pyogenes strains that possess the Nra transcriptional regulator demonstrates thermoregulated pilus production. Findings obtained in the present study of an Nra-positive serotype M49 strain revealed involvement of conserved virulence factor A (CvfA), also referred to as ribonuclease Y (RNase Y), in virulence factor expression and pilus production, while a cvfA deletion strain showed reduced pilus production and adherence to human keratinocytes as compared with wild-type and revertant strains. Furthermore, transcript levels of pilus subunits and srtC2 genes were decreased by cvfA deletion, which was remarkable at 25°C. Likewise, both messenger RNA (mRNA) and protein levels of Nra were remarkably decreased by cvfA deletion. Whether the expression of other pilus-related regulators, including fasX and CovR, was subject to thermoregulation was also examined. While the mRNA level of fasX, which inhibits cpa and fctA translation, was decreased by cvfA deletion at both 37°C and 25°C, CovR mRNA and protein levels, as well as its phosphorylation level were not significantly changed, suggesting that neither fasX nor CovR is necessarily involved in thermosensitive pilus production. Phenotypic analysis of the mutant strains revealed that culture temperature and cvfA deletion had varied effects on streptolysin S and SpeB activities. Furthermore, bactericidal assay data showed that cvfA deletion decreased the rate of survival in human blood. Together, the present findings indicate that CvfA is involved in regulation of pilus production and virulence-related phenotypes of the serotype M49 strain of S. pyogenes.

Contribution of Streptococcus pyogenes M87 protein to innate immune resistance and virulence.

Streptococcus pyogenes is a pre-eminent human pathogen, and classified by the hypervariable sequence of the emm gene encoding the cell surface M protein. Among a diversity of M/emm types, the prevalence of the M/emm87 strain has been steadily increasing in invasive S. pyogenes infections. Although M protein is the major virulence factor for globally disseminated M/emm1 strain, it is unclear if or how the corresponding M protein of M/emm87 strain (M87 protein) functions as a virulence factor. Here, we use targeted mutagenesis to show that the M87 protein contributes to bacterial resistance to neutrophil and whole blood killing and promotes the release of mature IL-1ß from macrophages. While deletion of emm87 did not influence epithelial cell adherence and nasal colonization, it significantly reduced S. pyogenes-induced mortality and bacterial loads in a murine systemic infection model. Our data suggest that emm87 is involved in pathogenesis by modulating the interaction between S. pyogenes and innate immune cells.

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.

Pneumolysin contributes to dysfunction of nasal epithelial barrier for promotion of pneumococcal dissemination into brain tissue.

Streptococcus pneumoniae is one of the major pathogens responsible for bacterial meningitis and neurological sequelae. The present study was conducted to identify a non-hematogenous route used by S. pneumoniae to gain access to brain tissue without causing bacteremia or pneumonia, as well as bacterial and host factors involved in this process. To investigate the molecular mechanisms and dissemination pathways of pneumococcal infection in brain tissue, mice were intranasally inoculated with S. pneumoniae strain EF3030, a clinical isolate from a patient with otitis media. Pneumococci were isolated from the frontal olfactory bulb, caudal cerebrum, and cerebellum, with neither bacteremia nor pneumonia observed in the present model. Immunostaining imaging revealed the presence of S. pneumoniae organisms in olfactory nerve fibers. Knockout of the ply gene encoding pneumolysin (PLY) markedly compromised the ability of the bacterial organisms to disseminate into brain tissue, whereas the dissemination efficiency of the complemented strain was restored to nearly the same level as the wild type. Notably, distinct upregulation of Gli1 and Snail1, which are involved in the transcriptional repression of junctional proteins, along with downregulation of E-cadherin, was detected in nasal lavage samples from mice infected with the wild-type or complemented strain, but not in those from mice infected with the ply mutant. Taken together, the present findings indicate that PLY induces Gli1-Snail1-dependent dysfunction of the nasal epithelial barrier, thus allowing pneumococcal dissemination to brain tissue that occurs in a non-hematogenous manner.IMPORTANCEBacterial meningitis, considered to be caused by bacteremia, can lead to blood-brain barrier disruption and bacterial dissemination into the central nervous system. Despite the availability of intravenously administered antibiotics with cerebrospinal fluid transferability, bacterial meningitis remains associated with high rates of morbidity and mortality. Here, we utilized Streptococcus pneumoniae strain EF3030, clinically isolated from otitis media, for the construction of a murine infection model to investigate the molecular mechanisms by which nasally colonized pneumococci disseminate into brain tissue. The obtained findings indicate that pneumolysin (PLY) induces Gli1-Snail1-dependent dysfunction of the nasal epithelial barrier, which facilitates pneumococcal dissemination to brain tissue in a non-hematogenous manner. Our results support the existence of an alternative route by which S. pneumoniae can reach the central nervous system and indicate the need for the development of novel therapeutic strategies, which would be an important contribution to the clinical management of bacterial meningitis.

Independent component analysis reveals 49 independently modulated gene sets within the global transcriptional regulatory architecture of multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii causes severe infections in humans, resists multiple antibiotics, and survives in stressful environmental conditions due to modulations of its complex transcriptional regulatory network (TRN). Unfortunately, our global understanding of the TRN in this emerging opportunistic pathogen is limited. Here, we apply independent component analysis, an unsupervised machine learning method, to a compendium of 139 RNA-seq data sets of three multidrug-resistant A. baumannii international clonal complex I strains (AB5075, AYE, and AB0057). This analysis allows us to define 49 independently modulated gene sets, which we call iModulons. Analysis of the identified A. baumannii iModulons reveals validating parallels to previously defined biological operons/regulons and provides a framework for defining unknown regulons. By utilizing the iModulons, we uncover potential mechanisms for a RpoS-independent general stress response, define global stress-virulence trade-offs, and identify conditions that may induce plasmid-borne multidrug resistance. The iModulons provide a model of the TRN that emphasizes the importance of transcriptional regulation of virulence phenotypes in A. baumannii. Furthermore, they suggest the possibility of future interventions to guide gene expression toward diminished pathogenic potential.IMPORTANCEThe rise in hospital outbreaks of multidrug-resistant Acinetobacter baumannii infections underscores the urgent need for alternatives to traditional broad-spectrum antibiotic therapies. The success of A. baumannii as a significant nosocomial pathogen is largely attributed to its ability to resist antibiotics and survive environmental stressors. However, there is limited literature available on the global, complex regulatory circuitry that shapes these phenotypes. Computational tools that can assist in the elucidation of A. baumannii's transcriptional regulatory network architecture can provide much-needed context for a comprehensive understanding of pathogenesis and virulence, as well as for the development of targeted therapies that modulate these pathways.

A genome-scale metabolic model of a globally disseminated hyperinvasive M1 strain of Streptococcus pyogenes.

Streptococcus pyogenes is responsible for a range of diseases in humans contributing significantly to morbidity and mortality. Among more than 200 serotypes of S. pyogenes, serotype M1 strains hold the greatest clinical relevance due to their high prevalence in severe human infections. To enhance our understanding of pathogenesis and discovery of potential therapeutic approaches, we have developed the first genome-scale metabolic model (GEM) for a serotype M1 S. pyogenes strain, which we name iYH543. The curation of iYH543 involved cross-referencing a draft GEM of S. pyogenes serotype M1 from the AGORA2 database with gene essentiality and autotrophy data obtained from transposon mutagenesis-based and growth screens. We achieved a 92.6% (503/543 genes) accuracy in predicting gene essentiality and a 95% (19/20 amino acids) accuracy in predicting amino acid auxotrophy. Additionally, Biolog Phenotype microarrays were employed to examine the growth phenotypes of S. pyogenes, which further contributed to the refinement of iYH543. Notably, iYH543 demonstrated 88% accuracy (168/190 carbon sources) in predicting growth on various sole carbon sources. Discrepancies observed between iYH543 and the actual behavior of living S. pyogenes highlighted areas of uncertainty in the current understanding of S. pyogenes metabolism. iYH543 offers novel insights and hypotheses that can guide future research efforts and ultimately inform novel therapeutic strategies.IMPORTANCEGenome-scale models (GEMs) play a crucial role in investigating bacterial metabolism, predicting the effects of inhibiting specific metabolic genes and pathways, and aiding in the identification of potential drug targets. Here, we have developed the first GEM for the S. pyogenes highly virulent serotype, M1, which we name iYH543. The iYH543 achieved high accuracy in predicting gene essentiality. We also show that the knowledge obtained by substituting actual measurement values for iYH543 helps us gain insights that connect metabolism and virulence. iYH543 will serve as a useful tool for rational drug design targeting S. pyogenes metabolism and computational screening to investigate the interplay between inhibiting virulence factor synthesis and growth.

Elucidation of independently modulated genes in Streptococcus pyogenes reveals carbon sources that control its expression of hemolytic toxins.

Streptococcus pyogenes can cause a wide variety of acute infections throughout the body of its human host. An underlying transcriptional regulatory network (TRN) is responsible for altering the physiological state of the bacterium to adapt to each unique host environment. Consequently, an in-depth understanding of the comprehensive dynamics of the S. pyogenes TRN could inform new therapeutic strategies. Here, we compiled 116 existing high-quality RNA sequencing data sets of invasive S. pyogenes serotype M1 and estimated the TRN structure in a top-down fashion by performing independent component analysis (ICA). The algorithm computed 42 independently modulated sets of genes (iModulons). Four iModulons contained the nga-ifs-slo virulence-related operon, which allowed us to identify carbon sources that control its expression. In particular, dextrin utilization upregulated the nga-ifs-slo operon by activation of two-component regulatory system CovRS-related iModulons, altering bacterial hemolytic activity compared to glucose or maltose utilization. Finally, we show that the iModulon-based TRN structure can be used to simplify the interpretation of noisy bacterial transcriptome data at the infection site. IMPORTANCE S. pyogenes is a pre-eminent human bacterial pathogen that causes a wide variety of acute infections throughout the body of its host. Understanding the comprehensive dynamics of its TRN could inform new therapeutic strategies. Since at least 43 S. pyogenes transcriptional regulators are known, it is often difficult to interpret transcriptomic data from regulon annotations. This study shows the novel ICA-based framework to elucidate the underlying regulatory structure of S. pyogenes allows us to interpret the transcriptome profile using data-driven regulons (iModulons). Additionally, the observations of the iModulon architecture lead us to identify the multiple regulatory inputs governing the expression of a virulence-related operon. The iModulons identified in this study serve as a powerful guidepost to further our understanding of S. pyogenes TRN structure and dynamics.

An M protein coiled coil unfurls and exposes its hydrophobic core to capture LL-37.

Surface-associated, coiled-coil M proteins of Streptococcus pyogenes (Strep A) disable human immunity through interaction with select proteins. However, coiled coils lack features typical of protein-protein interaction sites, and it is therefore challenging to understand how M proteins achieve specific binding, for example, with the human antimicrobial peptide LL-37, leading to its neutralization. The crystal structure of a complex of LL-37 with M87 protein, an antigenic M protein variant from a strain that is an emerging threat, revealed a novel interaction mode. The M87 coiled coil unfurled and asymmetrically exposed its hydrophobic core to capture LL-37. A single LL-37 molecule was bound by M87 in the crystal, but in solution additional LL-37 molecules were recruited, consistent with a 'protein trap' neutralization mechanism. The interaction mode visualized crystallographically was verified to contribute significantly to LL-37 resistance in an M87 Strep A strain and was identified to be conserved in a number of other M protein types that are prevalent in human populations. Our results provide specific detail for therapeutic inhibition of LL-37 neutralization by M proteins.

We share our environment with many different bacteria. Some are beneficial for our health, like gut bacteria, but others can cause severe disease if they infect and spread within the body's tissues. For example, the bacterium Streptococcus pyogenes can cause conditions ranging from skin infections to a rapidly spreading deep-tissue infection, giving it the nickname "flesh-eating bacterium". To prevent infection, our bodies have developed defence mechanisms that target disease-causing bacteria. These include antimicrobial molecules, such as LL-37, which is a small protein produced on the skin. LL-37 kills bacteria by puncturing their cell membrane (the bacterial equivalent of our skin); in other words, it acts like a tiny chemical dart that 'pops' the bacterial cell. However, some bacteria, including S. pyogenes, can disarm these defences. S. pyogenes captures LL-37 on its surface with so called M proteins, which prevent LL-37 from reaching and destroying the underlying membrane. However, it was unknown how exactly the two proteins interact, especially since LL-37 is a simple molecule that lacks the structural features that allow most proteins to bind to each other. Kolesinski et al. set out to determine how the M protein can 'grab' LL-37. A technique called X-ray crystallography allowed them to visualise the molecules atom by atom and to examine the configuration of the M protein after it had captured LL-37. The M protein selected for these experiments (M87) came from a strain associated with particularly severe disease, considered to be an emerging health threat. The results showed that M87 uncurled itself, thereby exposing specific parts that normally remain hidden. This way, it could capture LL-37, like a hand opening to grab an object. Kolesinski et al. have revealed a key molecular mechanism that enables a disease-causing bacterium to invade our immune defences. Identifying which regions of M87 are involved in capturing LL-37 may help design more effective therapies to combat S. pyogenes infections.

Pneumococcal BgaA Promotes Host Organ Bleeding and Coagulation in a Mouse Sepsis Model.

Streptococcus pneumoniae is a major cause of invasive diseases such as pneumonia, meningitis, and sepsis, with high associated mortality. Our previous molecular evolutionary analysis revealed that the S. pneumoniae gene bgaA, encoding the enzyme ß-galactosidase (BgaA), had a high proportion of codons under negative selection among the examined pneumococcal genes and that deletion of bgaA significantly reduced host mortality in a mouse intravenous infection assay. BgaA is a multifunctional protein that plays a role in cleaving terminal galactose in N-linked glycans, resistance to human neutrophil-mediated opsonophagocytic killing, and bacterial adherence to human epithelial cells. In this study, we performed in vitro and in vivo assays to evaluate the precise role of bgaA as a virulence factor in sepsis. Our in vitro assays showed that the deletion of bgaA significantly reduced the bacterial association with human lung epithelial and vascular endothelial cells. The deletion of bgaA also reduced pneumococcal survival in human blood by promoting neutrophil-mediated killing, but did not affect pneumococcal survival in mouse blood. In a mouse sepsis model, mice infected with an S. pneumoniae bgaA-deleted mutant strain exhibited upregulated host innate immunity pathways, suppressed tissue damage, and blood coagulation compared with mice infected with the wild-type strain. These results suggest that BgaA functions as a multifunctional virulence factor whereby it induces host tissue damage and blood coagulation. Taken together, our results suggest that BgaA could be an attractive target for drug design and vaccine development to control pneumococcal infection.

Biological Evaluation of the Effect of Root Canal Sealers Using a Rat Model.

Gutta-percha points and root canal sealers have been used for decades in endodontics for root canal obturation. With techniques such as single cone methods, the amount of sealer is larger, making their properties more critical. However, relatively few reports have comprehensively evaluated their biological effects. To this end, we evaluated three types of sealers, zinc oxide-fatty acid-, bio-glass- and methacrylate resin-containing sealers were considered. Their biological effects were evaluated using a rat subcutaneous implantation model. Each sealer was loaded inside a Teflon tube and implanted subcutaneously in the backs of rats. Inflammatory cells were observed around all samples 7 days after implantation and reduced after 28 days. Our results revealed that all samples were in contact with the subcutaneous tissue surrounding the sealer. Additionally, Ca and P accumulation was observed in only the bio-glass-containing sealer. Furthermore, each of the three sealers exhibited unique immune and inflammatory modulatory effects. In particular, bio-glass and methacrylate resin sealers were found to induce variable gene expression in adjacent subcutaneous tissues related to angiogenesis, wound healing, muscle tissue, and surrounding subcutaneous tissue. These results may help to understand the biological impacts of root canal sealers on surrounding biological tissues, guiding future research and comparisons with new generations of materials.

Streptococcus pyogenes upregulates arginine catabolism to exert its pathogenesis on the skin surface.

The arginine deiminase (ADI) pathway has been found in many kinds of bacteria and functions to supplement energy production and provide protection against acid stress. The Streptococcus pyogenes ADI pathway is upregulated upon exposure to various environmental stresses, including glucose starvation. However, there are several unclear points about the advantages to the organism for upregulating arginine catabolism. We show that the ADI pathway contributes to bacterial viability and pathogenesis under low-glucose conditions. S. pyogenes changes global gene expression, including upregulation of virulence genes, by catabolizing arginine. In a murine model of epicutaneous infection, S. pyogenes uses the ADI pathway to augment its pathogenicity by increasing the expression of virulence genes, including those encoding the exotoxins. We also find that arginine from stratum-corneum-derived filaggrin is a key substrate for the ADI pathway. In summary, arginine is a nutrient source that promotes the pathogenicity of S. pyogenes on the skin.

Epidemiological analysis of pneumococcal strains isolated at Yangon Children's Hospital in Myanmar via whole-genome sequencing-based methods.

Streptococcus pneumoniae causes over one million deaths from lower respiratory infections per annum worldwide. Although mortality is very high in Southeast Asian countries, molecular epidemiological information remains unavailable for some countries. In this study, we report, for the first time, the whole-genome sequences and genetic profiles of pneumococcal strains isolated in Myanmar. We isolated 60 streptococcal strains from 300 children with acute respiratory infection at Yangon Children's Hospital in Myanmar. We obtained whole-genome sequences and identified the species, serotypes, sequence types, antimicrobial resistance (AMR) profiles, virulence factor profiles and pangenome structure using sequencing-based analysis. Average nucleotide identity analysis indicated that 58 strains were S. pneumoniae and the other 2 strains were Streptococcus mitis. The major serotype was 19F (11 strains), followed by 6E (6B genetic variant; 7 strains) and 15 other serotypes; 5 untypable strains were also detected. Multilocus sequence typing analysis revealed 39 different sequence types, including 11 novel ones. In addition, genetic profiling indicated that AMR genes and mutations spread among pneumococcal strains in Myanmar. A minimum inhibitory concentration assay indicated that several pneumococcal strains had acquired azithromycin and tetracycline resistance, whereas no strains were found to be resistant against levofloxacin and high-dose penicillin G. Phylogenetic and pangenome analysis showed various pneumococcal lineages and that the pneumococcal strains contain a rich and mobile gene pool, providing them with the ability to adapt to selective pressures. This molecular epidemiological information can help in tracking global infection and supporting AMR control in addition to public health interventions in Myanmar.

Streptococcal H2O2 inhibits IgE-triggered degranulation of RBL-2H3 mast cell/basophil cell line by inducing cell death.

Mast cells and basophils are central players in allergic reactions triggered by immunoglobulin E (IgE). They have intracellular granules containing allergic mediators (e.g., histamine, serotonin, inflammatory cytokines, proteases and ß-hexosaminidase), and stimulation by IgE-allergen complex leads to the release of such allergic mediators from the granules, that is, degranulation. Mast cells are residents of mucosal surfaces, including those of nasal and oral cavities, and play an important role in the innate defense system. Members of the mitis group streptococci such as Streptococcus oralis, are primary colonizers of the human oral cavity. They produce hydrogen peroxide (H2O2) as a by-product of sugar metabolism. In this study, we investigated the effects of streptococcal infection on RBL-2H3 mast cell/basophil cell line. Infection by oral streptococci did not induce degranulation of the cells. Stimulation of the RBL-2H3 cells with anti-dinitrophenol (DNP) IgE and DNP-conjugated human serum albumin triggers degranulation with the release of ß-hexosaminidase. We found that S. oralis and other mitis group streptococci inhibited the IgE-triggered degranulation of RBL-2H3 cells. Since mitis group streptococci produce H2O2, we examined the effect of S. oralis mutant strain deficient in producing H2O2, and found that they lost the ability to suppress the degranulation. Moreover, H2O2 alone inhibited the IgE-induced degranulation. Subsequent analysis suggested that the inhibition of degranulation was related to the cytotoxicity of streptococcal H2O2. Activated RBL-2H3 cells produce interleukin-4 (IL-4); however, IL-4 production was not induced by streptococcal H2O2. Furthermore, an in vivo study using the murine pollen-induced allergic rhinitis model suggested that the streptococcal H2O2 reduces nasal allergic reaction. These findings reveal that H2O2 produced by oral mitis group streptococci inhibits IgE-stimulated degranulation by inducing cell death. Consequently, streptococcal H2O2 can be considered to modulate the allergic reaction in mucosal surfaces.

Role of BgaA as a Pneumococcal Virulence Factor Elucidated by Molecular Evolutionary Analysis.

Streptococcus pneumoniae is a major cause of pneumonia, sepsis, and meningitis. Previously, we identified a novel virulence factor by investigating evolutionary selective pressure exerted on pneumococcal choline-binding cell surface proteins. Herein, we focus on another pneumococcal cell surface protein. Cell wall-anchoring proteins containing the LPXTG motif are conserved in Gram-positive bacteria. Our evolutionary analysis showed that among the examined genes, nanA and bgaA had high proportions of codon that were under significant negative selection. Both nanA and bgaA encode a multi-functional glycosidase that aids nutrient acquisition in a glucose-poor environment, pneumococcal adherence to host cells, and evasion from host immunity. However, several studies have shown that the role of BgaA is limited in a mouse pneumonia model, and it remains unclear if BgaA affects pneumococcal pathogenesis in a mouse sepsis model. To evaluate the distribution and pathogenicity of bgaA, we performed phylogenetic analysis and intravenous infection assay. In both Bayesian and maximum likelihood phylogenetic trees, the genetic distances between pneumococcal bgaA was small, and the cluster of pneumococcal bgaA did not contain other bacterial orthologs except for a Streptococcus gwangjuense gene. Evolutionary analysis and BgaA structure indicated BgaA active site was not allowed to change. The mouse infection assay showed that the deletion of bgaA significantly reduced host mortality. These results indicated that both nanA and bgaA encode evolutionally conserved pneumococcal virulence factors and that molecular evolutionary analysis could be a useful alternative strategy for identification of virulence factors.

Performance of a Biodegradable Composite with Hydroxyapatite as a Scaffold in Pulp Tissue Repair.

Vital pulp therapy is an important endodontic treatment. Strategies using growth factors and biological molecules are effective in developing pulp capping materials based on wound healing by the dentin-pulp complex. Our group developed biodegradable viscoelastic polymer materials for tissue-engineered medical devices. The polymer contents help overcome the poor fracture toughness of hydroxyapatite (HAp)-facilitated osteogenic differentiation of pulp cells. However, the composition of this novel polymer remained unclear. This study evaluated a novel polymer composite, P(CL-co-DLLA) and HAp, as a direct pulp capping carrier for biological molecules. The biocompatibility of the novel polymer composite was evaluated by determining the cytotoxicity and proliferation of human dental stem cells in vitro. The novel polymer composite with BMP-2, which reportedly induced tertiary dentin, was tested as a direct pulp capping material in a rat model. Cytotoxicity and proliferation assays revealed that the biocompatibility of the novel polymer composite was similar to that of the control. The novel polymer composite with BMP-2-induced tertiary dentin, similar to hydraulic calcium-silicate cement, in the direct pulp capping model. The BMP-2 composite upregulated wound healing-related gene expression compared to the novel polymer composite alone. Therefore, we suggest that novel polymer composites could be effective carriers for pulp capping.

Genetic Characterization of Streptococcus pyogenes emm89 Strains Isolated in Japan From 2011 to 2019.

Invasive infection caused by Streptococcus pyogenes emm89 strains has been increasing in several countries linked to a recently emergent clade of emm89 strains, designated clade 3. In Japan, the features of emm89 S. pyogenes strains, such as clade classification, remains unknown. In this study, we collected emm89 strains isolated from both streptococcal toxic shock syndrome (STSS) (89 STSS isolates) and noninvasive infections (72 non-STSS isolates) in Japan from 2011 to 2019, and conducted whole-genome sequencing and comparative analysis, which resulted in classification of a large majority into clade 3 regardless of disease severity. In addition, invasive disease-associated factors were found among emm89 strains, including mutations of control of virulence sensor, and absence of the hylP1 gene encoding hyaluronidase. These findings provide new insights into genetic features of emm89 strains.

Identification of evolutionarily conserved virulence factor by selective pressure analysis of Streptococcus pneumoniae.

Evolutionarily conserved virulence factors can be candidate therapeutic targets or vaccine antigens. Here, we investigated the evolutionary selective pressures on 16 pneumococcal choline-binding cell-surface proteins since Streptococcus pneumoniae is one of the pathogens posing the greatest threats to human health. Phylogenetic and molecular analyses revealed that cbpJ had the highest codon rates to total numbers of codons under considerable negative selection among those examined. Our in vitro and in vivo assays indicated that CbpJ functions as a virulence factor in pneumococcal pneumonia by contributing to evasion of neutrophil killing. Deficiency of cbpL under relaxed selective pressure also caused a similar tendency but showed no significant difference in mouse intranasal infection. Thus, molecular evolutionary analysis is a powerful tool that reveals the importance of virulence factors in real-world infection and transmission, since calculations are performed based on bacterial genome diversity following transmission of infection in an uncontrolled population.

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.

Surface Pre-Reacted Glass Filler Contributes to Tertiary Dentin Formation through a Mechanism Different Than That of Hydraulic Calcium-Silicate Cement.

The induction of tissue mineralization and the mechanism by which surface pre-reacted glass-ionomer (S-PRG) cement influences pulpal healing remain unclear. We evaluated S-PRG cement-induced tertiary dentin formation in vivo, and its effect on the pulp cell healing process in vitro. Induced tertiary dentin formation was evaluated with micro-computed tomography (µCT) and scanning electron microscopy (SEM). The distribution of elements from the S-PRG cement in pulpal tissue was confirmed by micro-X-ray fluorescence (µXRF). The effects of S-PRG cement on cytotoxicity, proliferation, formation of mineralized nodules, and gene expression in human dental pulp stem cells (hDPSCs) were assessed in vitro. µCT and SEM revealed that S-PRG induced tertiary dentin formation with similar characteristics to that induced by hydraulic calcium-silicate cement (ProRoot mineral trioxide aggregate (MTA)). µXRF showed Sr and Si ion transfer into pulpal tissue from S-PRG cement. Notably, S-PRG cement and MTA showed similar biocompatibility. A co-culture of hDPSCs and S-PRG discs promoted mineralized nodule formation on surrounding cells. Additionally, S-PRG cement regulated the expression of genes related to osteo/dentinogenic differentiation. MTA and S-PRG regulated gene expression in hDPSCs, but the patterns of regulation differed. S-PRG cement upregulated CXCL-12 and TGF-ß1 gene expression. These findings showed that S-PRG and MTA exhibit similar effects on dental pulp through different mechanisms.