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DANSHIITSOODOL, YAMASHITA, NODA, KUMAGAI, MATOBA, and SUGIYAMA: Aspergillus oryzae S-03 Produces Gingipain Inhibitors as a Virulence Factor for Porphyromonas gingivalis
Original Article

J Bacteriol Virol 2014;44(2):152-161.

Published online: 9 February 2014

DOI: https://doi.org/10.4167/jbv.2014.44.2.152

Aspergillus oryzae S-03 Produces Gingipain Inhibitors as a Virulence Factor for Porphyromonas gingivalis

Narandalai DANSHIITSOODOL, Hideyuki YAMASHITA, Masafumi NODA, Takanori KUMAGAI, Yasuyuki MATOBA, Masanori SUGIYAMA*

Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical and Health Sciences, Hiroshima University; Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan

*Corresponding author: Masanori Sugiyama, Ph.D., Professor. Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical and Health Sciences, Hiroshima University. Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan. Phone: +81-82-257-5280, Fax: +81-82-257-5283, e-mail: sugi@hiroshima-u.ac.jp

** We thank Mr. Matsunosuke Higuchi, the president of Higuchi Matsunosuke Shoten Co. Ltd, Japan, for his donation of A. oryzae strains. We thank Dr. Osamu Yamada, National Research Institute of Brewing, Japan for supporting analysis of aflatoxin-encoding gene cluster.

Received 10 March 201414 March 2014       Accepted 17 March 2014

Copyright © 2014 Journal of Bacteriology and Virology

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Oral infection with Porphyromonas (P.) gingivalis causes periodontitis that is manifested by the destruction of gingival connective tissues. Although a few types of antibiotics are effective against the infection, its use induces the appearance of drug-resistant bacteria. The present study shows that the fermented product of Aspergillus (A.) oryzae S-03, cultivated on the fat-removed soybean, inhibits the cell growth of the P. gingivalis. Likewise, the fermented product of the S-03 strain cultured for 26∼42 h displays an inhibitory activity to gingipain as a virulence factor of P. gingivalis. The activity is not lost even with heat treatment at 100°C for 15 min. We also demonstrate that the S-03 strain exhibits high protease activity. In addition, the strain does not produce aflatoxin because of the loss of a regulatory gene, aflR, necessary for the toxin biosynthesis.

Keywords: Apergillus oryzae, Gingipain, Inhibitor, Koji

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Figure 1.
PCR amplification specificity of the genomic DNA of A. oryzae S-03 using primers designed for the aflatoxin gene cluster. The primer sequences are shown in Table 1. Lanes: 1, aflT; 2, nor-1; 3, aflR; 4, norA; 5, avnA; 6, verB; 7, vbs
jbv-44-152f1.tif
Figure 2.
Southern blot analysis of the PstI-digested chromosomal DNA from Aspergillus species. The aflR fragment was used as a probe. The arrow indicates the molecular mass of the signals. Lanes 1, A. oryzae M-01; 2, A. oryzae W-52; 3, A. oryzae S-03. The M-01 and W-52 strains are classified into group 2 and group 1, respectively. The aflR gene found in the W-52 strain, but not in the M-01 and S-03 strains.
jbv-44-152f2.tif
Figure 3.
Activities of α-amylase and protease produced by A. oryzae. The detailed measurement method is described in the Materials and Methods section. Hatched rectangle, A. oryzae S-03.
jbv-44-152f3.tif
Figure 4.
Effect of cultivation time on maximal protease production by A. oryzae S-03.
jbv-44-152f4.tif
Figure 5.
Antibacterial activity produced by A. oryzae S-03 cultivated on the fat-removed soybeans. The supernatant fluid was obtained extraction of koji and applied on paper disc. 1, non-heat treated supernatant fluid; 2, the fluid treated with 100°C for 10 min; 3, the fluid treated with 60°C for 30 min.
jbv-44-152f5.tif
Table 1.
Primers used in this study
Name Sequence Tm
ITS1 5′-TCCGTAGGTGAACCTGCGG-3′ 60.4
ITS4 5′-TCCTCCGCTTATTGATATGC-3′ 54.3
ITS5 5′-GGAAGTAAAAGTCGTAACAAGG-3′ 54.8
aflT(1) 5′-GCACCAAATGGGTCTTTCTCGT-3′ 58.6
aflT(2) 5′-ATCCACGGTGAAGAGGGTAAGG-3′ 60.4
nor1-A 5′-CGGACGAGGTCTCATTGAAGCTTT-3′ 60.5
nor1-B 5′-ATCGATGATGAAGGCCGTGA-3′ 56.3
new aflR(1) 5′-CCGGCGCATAACACGTACTC-3′ 60.4
new aflR(2) 5′-GGCGCTTGGCCAATAGGTTC-3′ 60.4
norA(1) 5′-GGCTGGAAAGGGGTAATGGG-3′ 60.4
norA(2) 5′-TCTTGCGACCCTCACGAGAA-3′ 58.4
avnA(1) 5′-AATCGCACCCAATGAGCTGTCT-3′ 58.6
avnA(2) 5′-ATGGCCCGGGTTCTTTAGCAAC-3′ 60.4
verB-A 5′-GATGCACCATGACCTCATGCGTTA-3′ 60.5
verB-B 5′-CACGGCAGCGTTATTGATCATCTC-3′ 60.4
vbs(1) 5′-TGCGAATGCTACGGCTCTCA-3′ 58.4
vbs(2) 5′-CAACCGCCATCTCCTGGTCT-3′ 60.4
no homology(1) 5′-CCTCGGAGACCGAAATATCTGAATACC-3′ 62.0
no homology(2) 5′-CGGCGTACCAAATTCATTGCTTAC-3′ 58.8
Tubulin(1) 5′-CCAAGAACATGATGGCTGCT-3′ 56.3
Tubulin(2) 5′-CTTGAAGAGCTCCTGGATGG-3′ 58.4
Table 2.
Activity of Rgp and Kgp proteases of P. gingivalis W83. The values are expressed as percentage of the activity determined in the absence of inhibitor.
Sample Concentration (μg/ml) Activity with non-treated samples (%)
 Activity with heat treated samples (%)
RgpA- Kgp RgpB RgpA Kgp RgpB
Control 100 100 100 100 100 100
400 24.5 72.2 56.5 26.6 71.4 59.5
LMWF extract 200 65.4 91.2 74.4 81.3 92.0 75.7
100 94.5 100 95.3 100 100 95.4

The purified RgpB or RgpA-Kgp protease complex were preincubated with the given concentrations of inhibitor for 15 min at 25°C.

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