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

Efficacy of FimA antibody and clindamycin in silkworm larvae stimulated with Porphyromonas gulae

Objective: Porphyromonas gulae, a major periodontal pathogen in animals, possesses fimbriae that have been classified into three genotypes (A, B, C) based on the diversity of fimA genes encoding fimbrillin protein (FimA). P. gulae strains with type C fimbriae were previously shown to be more virulent than other types. In this study, we further examined the host toxicity mediated by P. gulae fimbriae by constructing recombinant FimA (rFimA) expression vectors for each genotype and raised antibodies to the purified proteins. Methods and Results: All larvae died within 204 h following infection with P. gulae type C at the low-dose infection, whereas type A and B did not. Among fimA types, the survival rates of the larvae injected with rFimA type C were remarkably decreased, while the survival rates of the larvae injected with rFimA type A and type B were greater than 50%. Clindamycin treatment inhibited the growth of type C strains in a dose-dependent manner, resulting in an increased rate of silkworm survival. Finally, type C rFimA-speci?c antiserum prolonged the survival of silkworm larvae stimulated by infection with P. gulae type C strain or injection of rFimA type C protein. Conclusion: These results suggested that type C fimbriae have high potential for enhancement of bacterial pathogenesis, and that both clindamycin and anti-type C rFimA-specific antibodies are potent inhibitors of type C fimbriae-induced toxicity. This is the first report to establish a silkworm infection model using P. gulae for toxicity assessment

Potential Role of the Intratumoral Microbiota in Prognosis of Head and Neck Cancer

The tumor microbiome, a relatively new research field, affects tumor progression through several mechanisms. The Cancer Microbiome Atlas (TCMA) database was recently published. In the present study, we used TCMA and The Cancer Genome Atlas and examined microbiome profiling in head and neck squamous cell carcinoma (HNSCC), the role of the intratumoral microbiota in the prognosis of HNSCC patients, and differentially expressed genes in tumor cells in relation to specific bacterial infections. We investigated 18 microbes at the genus level that differed between solid normal tissue (n = 22) and primary tumors (n = 154). The tissue microbiome profiles of Actinomyces, Fusobacterium, and Rothia at the genus level differed between the solid normal tissue and primary tumors of HNSCC patients. When the prognosis of groups with rates over and under the median for each microbe at the genus level was examined, rates for Leptotrichia which were over the median correlated with significantly higher overall survival rates. We then extracted 35 differentially expressed genes between the over- and under-the-median-for-Leptotrichia groups based on the criteria of >1.5 fold and p < 0.05 in the Mann–Whitney U-test. A pathway analysis showed that these Leptotrichia-related genes were associated with the pathways of Alzheimer disease, neurodegeneration-multiple diseases, prion disease, MAPK signaling, and PI3K-Akt signaling, while protein–protein interaction analysis revealed that these genes formed a dense network. In conclusion, probiotics and specific antimicrobial therapy targeting Leptotrichia may have an impact on the prognosis of HNSCC.Hamada M., Inaba H., Nishiyama K., et al. Potential Role of the Intratumoral Microbiota in Prognosis of Head and Neck Cancer. International Journal of Molecular Sciences 24, 15456 (2023); https://doi.org/10.3390/ijms242015456

Roles of Porphyromonas gulae proteases in bacterial and host cell biology

Porphyromonas gulae, an animal-derived periodontal pathogen, expresses several virulence factors, including fimbria, lipopolysaccharide (LPS) and proteases. We previously reported that its invasive efficiency was dependent on fimbriae types. In addition, P. gulae LPS increased inflammatory responses via toll-like receptors. The present study was conducted to investigate the involvement of P. gulae proteases in bacterial and host cell biology. Porphyromonas gulae strains showed an ability to agglutinate mouse erythrocytes and also demonstrated co-aggregation with Actinomyces viscosus, while the protease inhibitors antipain, PMSF, TLCK and leupeptin diminished P. gulae proteolytic activity, resulting in inhibition of haemagglutination and co-aggregation with A. viscosus. In addition, specific proteinase inhibitors were found to reduce bacterial cell growth. Porphyromonas gulae inhibited Ca9-22 cell proliferation in a multiplicity of infection- and time-dependent manner. Additionally, P. gulae-induced decreases in cell contact and adhesion-related proteins were accompanied by a marked change in cell morphology from well spread to rounded. In contrast, inhibition of protease activity prevented degradation of proteins, such as E-cadherin, beta-catenin and focal adhesion kinase, and also blocked inhibition of cell proliferation. Together, these results indicate suppression of the amount of human proteins, such as gamma-globulin, fibrinogen and fibronectin, by P. gulae proteases, suggesting that a novel protease complex contributes to bacterial virulence

A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins

A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) κ locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igκ region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

ホンガク コウベ トキワ タンキ ダイガク ニ オケル ダイ53カイ リンショウ ケンサ ギシ コッカ シケン ケッカ ブンセキ ニ ツイテ

We have analyzed results of the 53rd National Examination by the same method as we did in the last year. The examination pass rate of the Kobe Tokiwa College students was 77.4%. This number is better than that of the national average pass rate (74.7%). However, this number is still low relative to the pass rate of the new graduates (88.8%). Therefore, the further discussion on the improvement in pass rates is required. It is essential to raise the percentage of the correct answers in the categories which the Kobe Tokiwa College students find difficult. The examination pass rate is expected to be improved by making students to repeatedly solve the questions of the past five years according to each student level