Michiyo Matsumoto-Nakano

Non-alcoholic fatty liver disease (NAFLD) comprises non-alcoholic fatty liver (NAFL), which rarely progresses, and non-alcoholic steatohepatitis (NASH), which may progress to cirrhosis and hepatocellular carcinoma. Previous studies have shown that Streptococcus mutans positive for collagen-binding protein (Cnm) and the 190-kDa cell surface protein antigen (PA) may be involved in NASH progression. In this study, we analyzed saliva samples obtained from patients with NAFL and NASH, and investigated the involvement of these proteins in NASH development by administrating clinical isolates to a mouse model. There was no significant difference in the detection rate of S. mutans between the NAFL and NASH patient groups; however, Cnm ⁺ /PA ⁺ S. mutans strains were more frequently detected in patients with NASH. Furthermore, patients with NASH with Cnm ⁺ /PA ⁺ S. mutans showed significantly higher levels of type IV collagen 7S, a biomarker of liver fibrosis, compared to patients with NASH without these strains. Adiponectin, an anti-inflammatory cytokine mainly secreted by adipocytes, was significantly lower. The collagen-binding ability of Cnm ⁺ /PA ⁺ S. mutans strains was significantly higher than that of other S. mutans strains without both proteins. Cnm ⁺ /PA ⁺ S. mutans showed significantly higher binding ability to type IV collagen than to type I collagen. RT-qPCR analysis revealed that pac expression was significantly higher in Cnm ⁺ /PA ⁺ S. mutans isolated from the NASH group. Administration of Cnm ⁺ /PA ⁺ S. mutans into a NASH mouse model via the jugular vein caused NASH-like pathology in mouse liver tissues. These results suggest that Cnm ⁺ /PA ⁺ S. mutans is associated with NASH aggravation.

Shikonin is extracted from the roots of Lithospermum erythrorhizon, and shikonin extracts have been shown to have inhibitory effects on several bacteria. However, shikonin extracts are difficult to formulate because of their poor water solubility. In the present study, we prepared a shikonin dispersion, which was solubilized by the inclusion of β-1,3-1,6 glucan, and analysed the inhibitory effects of this dispersion on Streptococcus mutans and non-mutans streptococci. The shikonin dispersion showed pronounced anti-S. mutans activity, and inhibited growth of and biofilm formation by this bacterium. The shikonin dispersion also showed antimicrobial and antiproliferative effects against non-mutans streptococci. In addition, a clinical trial was conducted in which 20 subjects were asked to brush their teeth for 1 week using either shikonin dispersion-containing or non-containing toothpaste, respectively. The shikonin-containing toothpaste decreased the number of S. mutans in the oral cavity, while no such effect was observed after the use of the shikonin-free toothpaste. These results suggest that shikonin dispersion has an inhibitory effect on S. mutans and non-mutans streptococci, and toothpaste containing shikonin dispersion may be effective in preventing dental caries.

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.

Hydroxyapatite adsorbs various substances, but little is known about the effects on oral bacteria of adsorption onto hydroxyapatite derived from scallop shells. In the present study, we analyzed the effects of adsorption of Streptococcus mutans onto scallop-derived hydroxyapatite. When scallop-derived hydroxyapatite was mixed with S. mutans, a high proportion of the bacterial cells adsorbed onto the hydroxyapatite in a time-dependent manner. An RNA sequencing analysis of S. mutans adsorbed onto hydroxyapatite showed that the upregulation of genes resulted in abnormalities in pathways involved in glycogen and histidine metabolism and biosynthesis compared with cells in the absence of hydroxyapatite. S. mutans adsorbed onto hydroxyapatite was not killed, but the growth of the bacteria was inhibited. Electron microscopy showed morphological changes in S. mutans cells adsorbed onto hydroxyapatite. Our results suggest that hydroxyapatite derived from scallop shells showed a high adsorption ability for S. mutans. This hydroxyapatite also caused changes in gene expression related to the metabolic and biosynthetic processes, including the glycogen and histidine of S. mutans, which may result in a morphological change in the surface layer and the inhibition of the growth of the bacteria.

A relationship between IgA nephropathy (IgAN) and bacterial infection has been suspected. As IgAN is a chronic disease, bacteria that could cause chronic infection in oral areas might be pathogenetic bacteria candidates. Oral bacterial species related to dental caries and periodontitis should be candidates because these bacteria are well known to be pathogenic in chronic dental disease. Recently, several reports have indicated that collagen-binding protein (cnm)-(+) Streptococcs mutans is relate to the incidence of IgAN and the progression of IgAN. Among periodontal bacteria, Treponema denticola, Porphyromonas gingivalis and Campylobacte rectus were found to be related to the incidence of IgAN. These bacteria can cause IgAN-like histological findings in animal models. While the connection between oral bacterial infection, such as infection with S. mutans and periodontal bacteria, and the incidence of IgAN remains unclear, these bacterial infections might cause aberrantly glycosylated IgA1 in nasopharynx-associated lymphoid tissue, which has been reported to cause IgA deposition in mesangial areas in glomeruli, probably through the alteration of microRNAs related to the expression of glycosylation enzymes. The roles of other factors related to the incidence and progression of IgA, such as genes and cigarette smoking, can also be explained from the perspective of the relationship between these factors and oral bacteria. This review summarizes the relationship between IgAN and oral bacteria, such as cnm-(+) S. mutans and periodontal bacteria.

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 < 0.001 and p < 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 < 0.05 at days 28a nd 42; p < 0.01 at days 14 and 56) and IgA deposition (p < 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.

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-specific 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.