Identification and functional analysis of glutamine transporter inStreptococcus mutans

Background Streptococcus mutans, a biofilm-forming bacterium, possesses several transporters that function as import/export molecules. Among them, the PII protein family is composed of members that regulate glutamine synthesis in bacterial species. Objective In this study, we characterized the function of the glutamine transporter in S. mutans MT8148. Methods The SMU.732 gene, corresponding to glnP in S. mutans, is homologous to the glutamine transporter gene in Bacillus subtilis. We constructed a glnP-inactivated mutant strain (GEMR) and a complement strain (comp-GEMR) and evaluated their biological functions. Results Growth of GEMR was similar in the presence and absence of glutamine, whereas the growth rates of MT8148 and comp-GEMR were significantly lower in the presence of glutamine as compared to its absence. Furthermore, biofilms formed by MT8148 and comp-GEMR were significantly thicker than that formed by GEMR, while the GEMR strain showed a significantly lower survival rate in an acidic environment than the other strains. Addition of n-phenyl-2-naphthylamine, used to label of the membrane, led to increased fluorescence intensity of MT8148 and GEMR, albeit that was significantly lower in the latter. Conclusions These results suggest that glnP is associated with glutamine transport in S. mutans, especially the import of glutamine involved in biofilm formation

Contribution of collagen-binding protein Cnm of Streptococcus mutans to induced IgA nephropathy-like nephritis in rats

IgA nephropathy (IgAN), the most common primary glomerulonephritis, is considered an intractable disease with unknown pathogenic factors. In our previous study, Streptococcus mutans, the major causative bacteria of dental caries, which expresses Cnm, was related to the induction of IgAN-like nephritis. In the present study, the Cnm-positive S. mutans parental strain, a Cnm-defective isogenic mutant strain, its complementation strain, and recombinant Cnm (rCnm) protein were administered intravenously to Sprague Dawley rats, and the condition of their kidneys was evaluated focusing on the pathogenicity of Cnm. Rats treated with parental and complement bacterial strains and rCnm protein developed IgAN-like nephritis with mesangial proliferation and IgA and C3 mesangial deposition. Scanning immunoelectron microscopy revealed that rCnm was present in the electron-dense deposition area of the mesangial region in the rCnm protein group. These results demonstrated that the Cnm protein itself is an important factor in the induction of IgAN in rats

Cnm of Streptococcus mutans is important for cell surface structure and membrane permeability

Streptococcus mutans, a Gram-positive facultative anaerobic bacterium, is a major pathogen of dental caries. The protein Cnm of S. mutans is involved in collagen binding, but its other biological functions are unknown. In this study, a Cnm-deficient isogenic mutant and a complementation strain were generated from a Cnm-positive S. mutans strain to help determine the properties of Cnm. Initially, comparison of the cell surface structure was performed by electron microscopy, which demonstrated that Cnm appears to be localized on the cell surface and associated with a protruding cell surface structure. Deep RNA sequencing of the strains revealed that the defect in Cnm caused upregulated expression of many genes related to ABC transporters and cell-surface proteins, while a few genes were downregulated. The amount of biofilm formed by the Cnm-defective strain increased compared with the parental and complemented strains, but the biofilm structure was thinner because of elevated expression of genes encoding glucan synthesis enzymes, leading to increased production of extracellular polysaccharides. Particular antibiotics, including bacitracin and chloramphenicol, had a lower minimum inhibitory concentration for the Cnm-defective strain than particular antibiotics, including bacitracin and chloramphenicol, compared with the parental and complemented strains. Our results suggest that S. mutans Cnm is located on the cell surface, gives rise to the observed protruding cell surface, and is associated with several biological properties related to membrane permeability

Elimination of Hepatitis C Virus from Hepatocytes by a Selective Activation of Therapeutic Molecules

To eliminate hepatitis C virus (HCV) from infected hepatocytes, we generated two therapeutic molecules specifically activated in cells infected with HCV. A dominant active mutant of interferon (IFN) regulatory factor 7 (IRF7) and a negative regulator of HCV replication, VAP-C (Vesicle-associated membrane protein-associated protein subtype C), were fused with the C-terminal region of IPS-1 (IFNβ promoter stimulator-1), which includes an HCV protease cleavage site that was modified to be localized on the ER membrane, and designated cIRF7 and cVAP-C, respectively. In cells expressing the HCV protease, cIRF7 was cleaved and the processed fragment was migrated into the nucleus, where it activated various IFN promoters, including promoters of IFNα6, IFNβ, and IFN stimulated response element. Activation of the IFN promoters and suppression of viral RNA replication were observed in the HCV replicon cells and in cells infected with the JFH1 strain of HCV (HCVcc) by expression of cIRF7. Suppression of viral RNA replication was observed even in the IFN-resistant replicon cells by the expression of cIRF7. Expression of the cVAP-C also resulted in suppression of HCV replication in both the replicon and HCVcc infected cells. These results suggest that delivery of the therapeutic molecules into the liver of hepatitis C patients, followed by selective activation of the molecules in HCV-infected hepatocytes, is a feasible method for eliminating HCV

Identification and functional analysis of glutamine transporter inStreptococcus mutans

Background Streptococcus mutans, a biofilm-forming bacterium, possesses several transporters that function as import/export molecules. Among them, the PII protein family is composed of members that regulate glutamine synthesis in bacterial species. Objective In this study, we characterized the function of the glutamine transporter in S. mutans MT8148. Methods The SMU.732 gene, corresponding to glnP in S. mutans, is homologous to the glutamine transporter gene in Bacillus subtilis. We constructed a glnP-inactivated mutant strain (GEMR) and a complement strain (comp-GEMR) and evaluated their biological functions. Results Growth of GEMR was similar in the presence and absence of glutamine, whereas the growth rates of MT8148 and comp-GEMR were significantly lower in the presence of glutamine as compared to its absence. Furthermore, biofilms formed by MT8148 and comp-GEMR were significantly thicker than that formed by GEMR, while the GEMR strain showed a significantly lower survival rate in an acidic environment than the other strains. Addition of n-phenyl-2-naphthylamine, used to label of the membrane, led to increased fluorescence intensity of MT8148 and GEMR, albeit that was significantly lower in the latter. Conclusions These results suggest that glnP is associated with glutamine transport in S. mutans, especially the import of glutamine involved in biofilm formation

Distribution of periodontopathic bacterial species in Japanese children with developmental disabilities

Abstract Background Recent developments in molecular biological techniques have enabled rapid detection of periodontopathic bacterial species in clinical specimens. Accumulated evidence suggests that detection of specific bacterial species enables identification of subjects at high risk for the onset of periodontitis. We investigated the distribution of 10 selected periodontopathic bacterial species in dental plaque specimens obtained from children with disabilities who were attending daycare centers. Methods A total of 187 children (136 boys, 51 girls) aged 1-6 years old and diagnosed with such disabilities as mental retardation, cerebral palsy, and autism, participated in the study. Subgingival dental plaque specimens were collected from the buccal side of the maxillary left second primary molar after a clinical examination. Bacterial DNA was extracted from the specimens and PCR analyses were carried out to detect 10 selected periodontopathic species using specific primers for each. In addition, statistical analyses were performed to analyze the correlations among clinical parameters and the detected species. Results The most frequently detected species was Capnocytophaga sputigena (28.3%), followed by Aggregatibacter actinomycetemcomitans (20.9%) and Campylobacter rectus (18.2%). Eikenella corrodens, Capnocytophaga ochracea, and Prevotella nigrescence were detected in approximately 10% of the specimens, whereas Treponema denticola, Tannerella forsythia, and Prevotella intermedia were rarely found, and Porphyromonas gingivalis was not detected in any of the subjects. The total numbers of detected species were positively correlated with the age of the subjects. There were 10 subjects with positive reactions for T. denticola and/or T. forsythia, in whom the total number of bacterial species was significantly higher as compared to the other subjects. Furthermore, subjects possessing C. rectus showed significantly greater values for periodontal pocket depth, gingival index, and total number of species. Conclusion We found that approximately one-fourth of the present subjects with disabilities who possessed at least one of T. denticola, T. forsythia, and C. rectus were at possible risk for periodontitis. Follow-up examinations as well as preventive approaches should be utilized for such individuals.</p

Relationship between IgA Nephropathy and Porphyromonas gingivalis; Red Complex of Periodontopathic Bacterial Species

IgA nephropathy (IgAN) has been considered to have a relationship with infection in the tonsil, because IgAN patients often manifest macro hematuria just after tonsillitis. In terms of oral-area infection, the red complex of periodontal bacteria (Porphyromonas gingivalis (P. gingivalis), Treponema denticol (T. denticola) and Tannerella forsythia (T. forsythia)) is important, but the relationship between these bacteria and IgAN remains unknown. In this study, the prevalence of the red complex of periodontal bacteria in tonsil was compared between IgAN and tonsillitis patients. The pathogenicity of IgAN induced by P. gingivalis was confirmed by the mice model treated with this bacterium. The prevalence of P. gingivalis and T. forsythia in IgAN patients was significantly higher than that in tonsillitis patients (p &lt; 0.001 and p &lt; 0.05, respectively). A total of 92% of tonsillitis patients were free from red complex bacteria, while only 48% of IgAN patients had any of these bacteria. Nasal administration of P. gingivalis in mice caused mesangial proliferation (p &lt; 0.05 at days 28a nd 42; p &lt; 0.01 at days 14 and 56) and IgA deposition (p &lt; 0.001 at day 42 and 56 after administration). Scanning-electron-microscopic observation revealed that a high-density Electron-Dense Deposit was widely distributed in the mesangial region in the mice kidneys treated with P. gingivalis. These findings suggest that P. gingivalis is involved in the pathogenesis of IgAN