The group A Streptococcus interleukin-8 protease SpyCEP promotes bacterial intracellular survival by evasion of autophagy

Autophagy serves an innate immune function in defending the host against invading bacteria, including group A Streptococcus (GAS). Autophagy is regulated by numerous host proteins, including the endogenous negative regulator calpain, a cytosolic protease. Globally disseminated serotype M1T1 GAS strains associated with high invasive disease potential express numerous virulence factors and resist autophagic clearance. Upon in vitro infection of human epithelial cell lines with representative wild-type GAS M1T1 strain 5448 (M1.5448), we observed increased calpain activation linked to a specific GAS virulence factor, the IL-8 protease SpyCEP. Calpain activation inhibited autophagy and decreased capture of cytosolic GAS in autophagosomes. In contrast, the serotype M6 GAS strain JRS4 (M6.JRS4), which is highly susceptible to host autophagy-mediated killing, expresses low levels of SpyCEP and does not activate calpain. Overexpression of SpyCEP in M6.JRS4 stimulated calpain activation, inhibited autophagy and significantly decreased bacterial capture in autophagosomes. These paired loss- and gain-of-function studies reveal a novel role for the bacterial protease SpyCEP in enabling GAS M1 evasion of autophagy and host innate immune clearance

Evolutionary inactivation of a sialidase in group B Streptococcus

Group B Streptococcus (GBS) is a leading cause of bacterial sepsis and meningitis in newborns. GBS possesses a protein with homology to the pneumococcal virulence factor, NanA, which has neuraminidase (sialidase) activity and promotes blood-brain barrier penetration. However, phylogenetic sequence and enzymatic analyses indicate the GBS NanA ortholog has lost sialidase function – and for this distinction we designate the gene and encoded protein nonA/NonA. Here we analyze NonA function in GBS pathogenesis, and through heterologous expression of active pneumococcal NanA in GBS, potential costs of maintaining sialidase function. GBS wild-type and ΔnonA strains lack sialidase activity, but forced expression of pneumococcal NanA in GBS induced degradation of the terminal sialic acid on its exopolysaccharide capsule. Deletion of nonA did not change GBS-whole blood survival or brain microvascular cell invasion. However, forced expression of pneumococcal NanA in GBS removed terminal sialic acid residues from the bacterial capsule, restricting bacterial proliferation in human blood and in vivo upon mouse infection. GBS expressing pneumococcal NanA had increased invasion of human brain microvascular endothelial cells. Thus, we hypothesize that nonA lost enzyme activity allowing the preservation of an effective survival factor, the sialylated exopolysaccharide capsule

糖尿病患者の在宅ケア向上をめざしたTCDS育成の試み

The Tokushima City Medical Association has cultivated the talented persons of nursing care profession by both the education of diabetes and the instruction of medical treatments to secure the quality of home care for increasing diabetic patients. They are certified to be the Tokushima City Certified Diabetes Supporter（TCDS）. In Tokushima Prefecture, the rate of aging and the certification rate of care need are ranked high in Japan, and the medical measures should be provided for the aged diabetic patients utilizing team nursing care as well as team medical care, because many of these patients are obliged to receive home medical care owing to the introduction of community-based integrated care systems. Tokushima Prefecture kept the worst of age-adjusted diabetes mortality and also the worst of crude diabetes mortality in recent years. Therefore, the program for the TCDS was arranged by the staffs composed of board certified fellows of the Japan Diabetes Society, certified diabetes physicians of Tokushima, and Tokushima local certified diabetes educators （Tokushima LCDEs）. The program includes the lectures of diabetes and medical treatments, the practical training, and the group work by the World Café system collaborated with the medical staffs across many different fields, using the dramatic skit presented by the medical doctors and the LCDE staffs after narrating the scenario for the blood glucose control of diabetic patients to be treated. The persons who have completed the training course are certified as the TCDS by The Tokushima City Medical Association. The workshop is held twice a year and the certification is renewed every three years without examination. In conclusion, it is suggested that the development of TCDS leads to the improvement of the ability of nursing care staffs to support diabetic treatments and the advancement of the quality of home medical care for the aged diabetic patients

Data set of manuscript by Takahara et al.

We ensure to provide the raw data in an openly available repository to comply with the journal's policies.</p

Hydrogen peroxide produced by oral Streptococci induces macrophage cell death.

Hydrogen peroxide (H2O2) produced by members of the mitis group of oral streptococci plays important roles in microbial communities such as oral biofilms. Although the cytotoxicity of H2O2 has been widely recognized, the effects of H2O2 produced by oral streptococci on host defense systems remain unknown. In the present study, we investigated the effect of H2O2 produced by Streptococcus oralis on human macrophage cell death. Infection by S. oralis was found to stimulate cell death of a THP-1 human macrophage cell line at multiplicities of infection greater than 100. Catalase, an enzyme that catalyzes the decomposition of H2O2, inhibited the cytotoxic effect of S. oralis. S. oralis deletion mutants lacking the spxB gene, which encodes pyruvate oxidase, and are therefore deficient in H2O2 production, showed reduced cytotoxicity toward THP-1 macrophages. Furthermore, H2O2 alone was capable of inducing cell death. The cytotoxic effect seemed to be independent of inflammatory responses, because H2O2 was not a potent stimulator of tumor necrosis factor-α production in macrophages. These results indicate that streptococcal H2O2 plays a role as a cytotoxin, and is implicated in the cell death of infected human macrophages

Hydrogen peroxide contributes to the epithelial cell death induced by the oral mitis group of streptococci.

Members of the mitis group of streptococci are normal inhabitants of the commensal flora of the oral cavity and upper respiratory tract of humans. Some mitis group species, such as Streptococcus oralis and Streptococcus sanguinis, are primary colonizers of the human oral cavity. Recently, we found that hydrogen peroxide (H2O2) produced by S. oralis is cytotoxic to human macrophages, suggesting that streptococcus-derived H2O2 may act as a cytotoxin. Since epithelial cells provide a physical barrier against pathogenic microbes, we investigated their susceptibility to infection by H2O2-producing streptococci in this study. Infection by S. oralis and S. sanguinis was found to stimulate cell death of Detroit 562, Calu-3 and HeLa epithelial cell lines at a multiplicity of infection greater than 100. Catalase, an enzyme that catalyzes the decomposition of H2O2, inhibited S. oralis cytotoxicity, and H2O2 alone was capable of eliciting epithelial cell death. Moreover, S. oralis mutants lacking the spxB gene encoding pyruvate oxidase, which are deficient in H2O2 production, exhibited reduced cytotoxicity toward Detroit 562 epithelial cells. In addition, enzyme-linked immunosorbent assays revealed that both S. oralis and H2O2 induced interleukin-6 production in Detroit 562 epithelial cells. These results suggest that streptococcal H2O2 is cytotoxic to epithelial cells, and promotes bacterial evasion of the host defense systems in the oral cavity and upper respiratory tracts

Competence-induced protein Ccs4 facilitates pneumococcal invasion into brain tissue and virulence in meningitis

Streptococcus pneumoniae is a major pathogen that causes pneumonia, sepsis, and meningitis. The candidate combox site 4 (ccs4) gene has been reported to be a pneumococcal competence-induced gene. Such genes are involved in development of S. pneumoniae competence and virulence, though the functions of ccs4 remain unknown. In the present study, the role of Ccs4 in the pathogenesis of pneumococcal meningitis was examined. We initially constructed a ccs4 deletion mutant and complement strains, then examined their association with and invasion into human brain microvascular endothelial cells. Wild-type and Ccs4-complemented strains exhibited significantly higher rates of association and invasion as compared to the ccs4 mutant strain. Deletion of ccs4 did not change bacterial growth activity or expression of NanA and CbpA, known brain endothelial pneumococcal adhesins. Next, mice were infected either intravenously or intranasally with pneumococcal strains. In the intranasal infection model, survival rates were comparable between wild-type strain-infected and ccs4 mutant strain-infected mice, while the ccs4 mutant strain exhibited a lower level of virulence in the intravenous infection model. In addition, at 24 hours after intravenous infection, the bacterial burden in blood was comparable between the wild-type and ccs4 mutant strain-infected mice, whereas the wild-type strain-infected mice showed a significantly higher bacterial burden in the brain. These results suggest that Ccs4 contributes to pneumococcal invasion of host brain tissues and functions as a virulence factor

Construction of <i>S. oralis</i> spxB deletion mutant.

<p>(A) Black arrow indicates the gene encoding pyruvate oxidase (SMSK23_0092 spxB). A targeted deletion mutant lacking this region was constructed by allelic exchange using the temperature-sensitive shuttle vector pSET4s. (B) S. oralis ATCC35037 wild-type (WT), spxB-deletion mutant (KO), or reverse mutant (Rev) was cultured in BHI broth or 5% RPMI1640 medium at 37°C for 18 h in a 5% CO₂ atmosphere. Concentrations of H₂O₂ in culture supernatants were quantitatively determined using a hydrogen peroxide colorimetric detection kit. Data are shown as the mean ± SD of triplicate samples. *p<0.05 as compared with concentration of wild-type strain.</p