|
Sign In to gain access to subscriptions and/or personal tools.
|
Protein Antigen in Serotype k Streptococcus mutans Clinical Isolates
K. Nakano1,
R. Nomura1,
H. Nemoto1,
J. Lapirattanakul1,
N. Taniguchi1,
L. Grönroos2,
S. Alaluusua2,3 and
T. Ooshima1,*
1 Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry, 1-8 Yamada-oka, Suita, Osaka 565-0871, Japan;
2 Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, and
3 Department of Pediatric and Preventive Dentistry, Institute of Dentistry, University of Helsinki, Helsinki, Finland
View larger version (74K):
[in this window]
[in a new window]
|
Figure 1. GTF and PA expression in S. mutans oral isolates. Test strains were grown overnight, then washed and adjusted to OD550 = 1.0 with phosphate-buffered saline. Whole bacterial cells or the culture supernatants concentrated by ammonium sulfate precipitation (50%) were then dissolved with SDS gel loading buffer. An equal amount of each protein (1 µg) was separated by 10% SDS polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane. The transferred protein bands were reacted with rabbit antibodies against cell-associated GTF for detection of GTFB and GTFC, those against cell-free GTF for GTFD detection, and those against PA for PA detection. Finally, the bands were visualized with an alkaline-phosphatase-conjugated antirabbit immunoglobulin G antibody and 5-bromo-4chloro-3-indolylphosphate-nitroblue tetrazolium substrate.
|
|
View larger version (77K):
[in this window]
[in a new window]
|
Figure 2. Detection of transcription of genes encoding PA by RT-PCR. Total RNA was prepared from the tested strains, then treated for 30 min at 37°C with 0.1 U of RQ1 RNase-Free DNase per µL, to remove contaminating DNA, after which amplification of cDNA synthesized from mRNA was carried out. Successive PCR assays were performed under the following conditions: 30 cycles at 94°C for 30 sec, 50°C for 30 sec, and 72°C for 30 sec, using the following primers: PAtF, 5'-TAG TAA AAC ACT GTG TGG TGC TGT-3', and PAtR, 5'-CCA GCT TGG TTT GAC TTT GTT CAG-3' (Appendix Fig. A). Amplification of 16S rRNA was performed using the following sets of primers: 5'-GTG GGA CGC AAG GAA ACA CAC TGT GC-3', and 5'-CGT CGC CTT GGT AAG CTC TTA CCT TAC C-3' to confirm that cDNA was successfully extracted.
|
|
View larger version (16K):
[in this window]
[in a new window]
|
Figure 3. Biological properties of clinical isolates of S. mutans. Cellular hydrophobicity rates are shown for each serotype (A) and based on the presence or absence of PA expression (B). Phagocytosis rates for each serotype are shown (C) and based on the presence or absence of PA expression (D). The phagocytosis rate was calculated as follows. Tested strains were grown overnight, then washed and adjusted with PBS to 1.0 x 108 CFU/mL. Next, a 500-µL quantity of each suspension was mixed with human peripheral blood (500 µL) and incubated for 10 min at 37°C. Interactions between polymorphonuclear leukocytes (PMNs) and the bacteria were observed under a microscope following Giemsa staining. The phagocytosis rate is expressed as mean ratio of PMNs with bacterial phagocytosis of per 100 PMNs, with 500 PMNs examined. PA+ indicates strains with PA expression similar to the reference strains, and PA- indicates those with an absence of PA expression as compared with the reference strains. Statistical analysis was carried out by ANOVA (A,C) or a t test (B,D) (*P < 0.05, ***P < 0.001).
|
|
Journal of Dental Research, Vol. 87, No. 10,
964-968 (2008)
DOI: 10.1177/154405910808701001
 CiteULike  Complore  Connotea  Del.icio.us  Digg  Reddit  Technorati  Twitter What's this?
|
|
