J Bacteriol Virol.  2014 Jun;44(2):152-161. 10.4167/jbv.2014.44.2.152.

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

• Danshiitsoodol N
• Yamashita
• Noda M
• Kumagai T
• Matoba Y
• Sugiyama M

• KMID: 2168690
• DOI: http://doi.org/10.4167/jbv.2014.44.2.152

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 100degrees 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.

Keyword

Apergillus oryzae; Gingipain; Inhibitor; Koji

MeSH Terms

Aflatoxins
Anti-Bacterial Agents
Aspergillus
Aspergillus oryzae*
Bacteria
Connective Tissue
Genes, Regulator
Hot Temperature
Oryza
Periodontitis
Porphyromonas
Porphyromonas gingivalis*
Soybeans
Virulence*
Aflatoxins
Anti-Bacterial Agents

Figure

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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

• 

  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.

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  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.

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  Figure 4. Effect of cultivation time on maximal protease production by A. oryzae S-03.

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  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.

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