Association of collagen-binding protein with the development of IgA
nephropathy-like nephritis in rat model
Shuhei Naka*1, Daiki Matsuoka1, Taro Misaki2, Yasuyuki Nagasawa3, Seigo Ito4, Ryota
Nomura5, Kazuhiko Nakano6, Michiyo Matsumoto-Nakano1
1

Department of Pediatric Dentistry, Okayama University Graduate School of Medicine,
Dentistry and Pharmaceutical Sciences, Japan
2
Division of Nephrology, Seirei Hamamatsu Genaral Hospital, Japan3
Department of General Internal Medicine, Hyogo College of Medicine, Japan
4
Department of Internal Medicine, Japan Seif-Defense Iruma Hospital, Japan
5
Department of Pediatric Dentistry, Graduate School of Biomedical and Health Sciences,Hiroshima University, Japan
6
Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry, Japan
Background: We previously reported that administration of collagen-binding protein(Cnm)-positive S. mutans caused IgA nephropathy-like nephritis in the jugular vein ofmodel rats. In the present study, the involvement of Cnm in development of IgA
nephropathy-like nephritis in a jugular vein model was investigated using a Cnm
deletion mutant strain and its complement strain.
Methods: An S. mutans SN74 strain, that with Cnm deletion, and a complement strain
were used in this study. The tested strain was administered through the jugular vein ofSprague-Dawley rats (4-week-old males). After 45 days, the rats were euthanized, thenurine and blood samples collected, and kidneys extracted. Renal tissue pathology in thekidneys was evaluated using periodic acid-schiff (PAS) staining and fluorescentimmunostaining with IgA antibodies.
Results: There were no significant differences in major serum measurements or urinary
protein levels among the groups. On the other hand, there was a greater rate of hematuria
positivity in the SN74 and complement groups than in the deletion group. Mesangial
proliferation scores, determined in images following PAS staining, for the SN74 andcomplement groups were significantly higher as compared to the deletion group (P<0.05,P<0.001, respectively). Furthermore, immunostaining evaluations demonstrated
significantly greater levels of IgA deposition in the mesangial region in the SN74 andcomplement groups, and IgA positivity than in the deletion group (P<0.05, P<0.01,respectively).
Conclusion: These results suggest that Cnm may be involved in IgA nephropathy-like
nephritis in rats induced by administration of S. mutans into the jugular vein.

Effects of Cyclodextran on Exfoliation of Biofilm Formed by
Streptococcus mutans
Kana Goto, Haruka Asaumi, Sakuya Matsuura, and Michiyo Matsumoto-Nakano
Department of Pediatric Dentistry, Okayama University Graduate School of Medicine,Dentistry and Pharmaceutical Sciences, Okayama, Japan
Background: Cyclodextran (CI), a cycloisomaltooligosaccharide, is known to function
as a dextran analogue. In our previous report, CI was shown to have a strong inhibitory
effect on glucosyltransferase B produced by S. mutans, a major pathogen of dental caries.
The present study was conducted to investigate the effects of CI on exfoliation of biofilm
formed by S. mutans.
Methods: S. mutans MT8148 bacteria were stained with SYTO® 9 and suspended usingchemically defined medium containing 0.5% sucrose, Alexa Fluor® 647-labeled dextran
conjugate, and 0~10% CI, then cultured for 24 hours to form biofilm. Next, MT8148
organisms were stained with hexidium iodide and cultured for 24 hours, then 0~10% CI
was added and incubation performed for three hours, and biofilm structures wereobserved using confocal laser scanning microscopy. Finally, to examine the antibacterial
effects of CI, cultured MT8148 organisms were adjusted to OD600 = 0.1, then 0-20% CI
was added and culturing performed at 37°C for six hours, followed by OD570
measurement.
Results: Cell amounts in biofilms were nearly the same with all CI concentrations used.
Biofilms formed without CI showed abundant extracellular polysaccharides covering thesurface as compared to those formed in its presence. Biofilm structures formed with added
CI had lower levels of density and thickness as compared to those without CI.
Antibacterial assay findings showed that OD570 was slightly decreased in a CI dose-dependent manner.
Conclusion: These results suggest exfoliation activity by CI in S. mutans-related biofilm.
Further studies are needed to clarify the detailed mechanism.

Streptococcus mutans Cell Surface Protein Expression Profilingin Non-alcoholic Steatohepatitis Patients
Keiko Tabata, Shuhei Naka, Michiyo Matsumoto-Nakano
Department of Pediatric Dentistry, Okayama University Graduate School of Medicine,Dentistry and Pharmaceutical Sciences, Japan
Background: Approximately 20% of non-alcoholic fatty liver disease (NAFLD) patientsare reported to progress from non-alcoholic fatty liver (NAFL) to non-alcoholicsteatohepatitis (NASH). NASH has become one of the most common hepatic diseases inchildren. We previously reported that Streptococcus mutans organisms with both cellsurface collagen-binding protein (Cnm) and 190-kDa cell surface protein antigen (PA)induced NASH aggravation. The present study was performed to analyze expression
profiling of these proteins in S. mutans organisms isolated from NAFLD patients.
Methods: Using saliva specimens collected from 40 biopsy-proven NAFLD patients(NASH n=20, NAFL n=20), S. mutans organisms were isolated. PCR was performed todetect the cnm gene encoding Cnm using genomic DNA extracted from those organisms,
while PA expression was confirmed by western blotting. Total RNA was extracted fromS. mutans and complementary DNA synthesized by reverse transcription. Real-timereverse transcription PCR was used to evaluate expression levels of cnm and the pac geneencoding PA.
Results: The percentage of Cnm- and PA-positive (Cnm+PA+) strains in samples from theNASH patients was significantly greater than in those from NAFL patients (P<0.05).
Although the expression level of cnm was not significantly different, that of pac wassignificantly higher in Cnm+PA+ S. mutans organisms isolated from NASH patients(P<0.05). Additionally, the expression level of pac in Cnm+PA+ S. mutans in NASH

patient samples was significantly higher as compared to that in Cnm-negative and PApositive (Cnm-PA+) S. mutans organisms (P<0.05).
Conclusion: These findings indicate an association of Cnm+PA+ S. mutans organisms
exhibiting a high level of pac expression with NASH aggravation.

Biofilm formation associated with oral bacterial dominance inpediatric patients affected by aggressive periodontitis
Yukiko Miyai*, Shuhei Naka, Michiyo Matsumoto-Nakano
Department of Pediatric Dentistry, Okayama University Graduate School of Medicine,
Dentistry and Pharmaceutical Sciences, Japan.
Background: Early primary tooth loss is caused by rapid alveolar bone destruction anddiagnosed as aggressive periodontitis. In our previous study, Actinomyces and Veillonellaspecies were frequently detected in affected patients. Actinomyces naeslundii, anindigenous oral bacterium, produces peptidoglycan, which induces production of
inflammatory cytokines and activates osteoclasts, leading to alveolar bone resorption. The
present study investigated biofilm formation by A. naeslundii isolated from patients with
rapid alveolar bone destruction.
Methods: Patients with aggressive periodontitis were divided into Group 1, which had agreater number of Actinomyces species as compared to Veillonella species detected, andGroup 2, with a greater number of Veillonella species detected. A total of 82 A. naeslundii
strains were isolated from Groups 1 and 2, and used in this study. Qquantitative analysesof biofilm formed by those strains were performed using Brain Heart Infusion (BHI)
containing 1% sucrose.
Results: There was no significant difference between Groups 1 and 2 for biofilm quantity
after 24 hours of incubation. In contrast, after 48 hours of incubation, that quantity wassignificantly greater in Group 2 (P<0.05).
Conclusion: These results indicate that the quantity of biofilm formed by A. naeslundii

from patients showing Veillonella species dominance was greater as compared to those inwhom Actinomyces species were dominant. Further studies are needed to clarify theassociation of these two species in regard to biofilm formation and periodontitis
manifestation.

Pathogenicity of collagen-binding protein in Streptococcus mutans
Daiki Matsuoka*1, Shuhei Naka1, Kana Goto1, Ryota Nomura2, Kazuhiko Nakano3
Michiyo Matsumoto-Nakano1
1

Department of Pediatric Dentistry, Okayama University Graduate School of Medicine,

Dentistry and Pharmaceutical Sciences, Japan
Department of Pediatric Dentistry, Graduate School of Biomedical and HealthSciences, Hiroshima University
3
Department of Pediatric Dentistry, Division of Oral infection and Disease Control,
Osaka University Graduate School of Dentistry, Suita, Japan
2

Background: The most common form of chronic glomerulonephritis is IgA nephropathy(IgAN) and accounts for half of all pediatric cases. Streptococcus mutans, a Grampositive, facultative anaerobic bacterium is known to be a major pathogen of dental caries.
We previously reported that S. mutans with collagen-binding protein (Cnm) wasfrequently detected in the oral cavity of IgAN patients. The present study was conducted
to investigate the pathogenicity of Cnm using silkworm and animal models infected withrecombinant Cnm (rCnm).
Methods: The cnm gene, which encodes Cnm of S. mutans TW871, was ligated topGEX6p-1, a protein expression vector. Using a mass culture of E. coli BL21, rCnm wasobtained and transformed by the constructed plasmid. Several different concentrations ofrCnm protein were injected through the dorsal surface of silkworms into the hemolymph,
after which survival was determined every 12 hours for 204 hours. In addition, rCnm wasinto administered the jugular vein of specific pathogen-free Sprague-Dawley rats (4week-old males), then the kidneys were evaluated following immunofluorescent staining
with IgA antibodies.

Results: Among the different concentrations of rCnm administered to the silkworms, asignificant difference for survival after 204 hours was noted between the group injectedwith PBS and that with rCnm at 50 μg. Furthermore, immunofluorescent staining of rat
kidneys with IgA showed significantly greater IgA deposition in the mesangial region in
the rCnm-treated as compared to the PBS-treated group.
Conclusions: These results suggest that Cnm is an S. mutans pathogenic factor related tooccurrence of IgAN.