Yohei Ishiyama

α‐Amylase is a potential growth inhibitor of Porphyromonas gingivalis, a periodontal pathogenic bacterium

BACKGROUND AND OBJECTIVE Porphyromonas gingivalis is a major etiological agent in the development and progression of periodontal diseases. In this study, we isolated a cell growth inhibitor against P. gingivalis species from rice protein extract. MATERIAL AND METHODS The cell growth inhibitor active against P. gingivalis was purified from polished rice extract using a six-step column chromatography process. Its antimicrobial properties were investigated through microscope analysis, spectrum of activity and general structure. RESULTS The inhibitor was identified as AmyI-1, an α-amylase, and showed significant cell growth inhibitory activity against P. gingivalis species. Scanning electron microscopy micrograph analysis and bactericidal assay indicated an intriguing possibility that the inhibitor compromises the cell membrane structure of the bacterial cells and leads to cell death. Moreover, α-amylases from human saliva and porcine pancreas showed inhibitory activity similar to that of AmyI-1. CONCLUSIONS This is the first study to report that α-amylases cause cell death of periodontal pathogenic bacteria. This finding highlights the potential importance and therapeutic potential of α-amylases in treating periodontal diseases.

Effect of substituting arginine and lysine with alanine on antimicrobial activity and the mechanism of action of a cationic dodecapeptide (CL(14‐25)), a partial sequence of cyanate lyase from rice

The antimicrobial activity of analogs obtained by substituting arginine and lysine in CL(14‐25), a cationic α‐helical dodecapeptide, with alanine against Porphyromonas gingivalis, a periodontal pathogen, varied significantly depending on the number and position of cationic amino acids. The alanine‐substituted analogs had no hemolytic activity, even at a concentration of 1 mM. The antimicrobial activities of CL(K20A) and CL(K20A, K25A) were 3.8‐fold and 9.1‐fold higher, respectively, than that of CL(14‐25). The antimicrobial activity of CL(R15A) was slightly lower than that of CL(14‐25), suggesting that arginine at position 15 is not essential but is important for the antimicrobial activity. The experiments in which the alanine‐substituted analogs bearing the replacement of arginine at position 24 and/or lysine at position 25 were used showed that arginine at position 24 was crucial for the antimicrobial activity whenever lysine at position 25 was substituted with alanine. Helical wheel projections of the alanine‐substituted analogs indicate that the hydrophobicity in the vicinity of leucine at position 16 and alanines at positions 18 and/or 21 increased by substituting lysine at positions 20 and 25 with alanine, respectively. The degrees of diSC3‐5 release from P. gingivalis cells and disruption of GUVs induced by the alanine‐substituted analogs with different positive charges were not closely related to their antimicrobial activities. The enhanced antimicrobial activities of the alanine‐substituted analogs appear to be mainly attributable to the changes in properties such as hydrophobicity and amphipathic propensity due to alanine substitution and not to their extents of positive charge (cationicity).

Crystal structure of α-amylase from Oryza sativa: molecular insights into enzyme activity and thermostability

AmyI-1 is an α-amylase from Oryza sativa (rice) and plays a crucial role in degrading starch in various tissues and at various growth stages. This enzyme is a glycoprotein with an N-glycosylated carbohydrate chain, a unique characteristic among plant α-amylases. In this study, we report the first crystal structure of AmyI-1 at 2.2-Å resolution. The structure consists of a typical (β/α)8-barrel, which is well-conserved among most α-amylases in the glycoside hydrolase family-13. Structural superimposition indicated small variations in the catalytic domain and carbohydrate-binding sites between AmyI-1 and barley α-amylases. By contrast, regions around the N-linked glycosylation sites displayed lower conservation of amino acid residues, including Asn-263, Asn-265, Thr-307, Asn-342, Pro-373, and Ala-374 in AmyI-1, which are not conserved in barley α-amylases, suggesting that these residues may contribute to the construction of the structure of glycosylated AmyI-1. These results increase the depths of our understanding of the biological functions of AmyI-1. Graphical Abstract Crystal structure of α-amylase AmyI-1 from rice.

Contribution of cationic amino acids toward the inhibition of Arg‐specific cysteine proteinase (Arg‐gingipain) by the antimicrobial dodecapeptide, CL(14–25), from rice protein

CL(14–25), a dodecapeptide, exhibits antimicrobial activity against Porphyromonas gingivalis with the 50% growth‐inhibitory concentration (IC50) value of 145 µM, and arginine‐specific gingipain (Rgp)‐inhibitory activity. Kinetic analysis revealed that CL(14–25) is a mixed‐type inhibitor, with inhibition constants (Ki and Ki′ values) of 1.4 × 10−6 M and 4.3 × 10−6 M, respectively. To elucidate the contributions of four cationic amino acid residues at the N‐ and C‐termini of CL(14–25) toward Rgp‐inhibitory activity, we investigated the Rgp‐inhibitory activities of truncated and alanine‐substituted analogs of CL(14–25). Rgp‐inhibitory activities significantly decreased by truncated analogs, CL(15–25) and CL(16–25), whereas those of CL(14–24) and CL(14–23) were almost as high as that of CL(14–25). Rgp‐inhibitory activities of alanine‐substituted analogs, CL(R14A) and CL(R14A, R15A) also significantly decreased, whereas those of CL(K25A) and CL(R24A, K25A) were higher than that of CL(14–25). These results suggest that the arginine residue at position 15 substantially contributes to the Rgp‐inhibitory activity and that the arginine residue at position 14 plays important roles in exerting Rgp‐inhibitory activity. In this study, we demonstrated that CL(K25A) was a potent, dual function, peptide inhibitor candidate, exhibiting Rgp‐inhibitory activity with Ki and Ki′ of 9.6 × 10−7 M and 1.9 × 10−6 M, respectively, and antimicrobial activity against P. gingivalis with an IC50 value of 51 µM.

Pyrrhocoricin, a proline-rich antimicrobial peptide derived from insect, inhibits the translation process in the cell-free Escherichia coli protein synthesis system

Previous studies have shown that pyrrhocoricin, a proline-rich antimicrobial peptide (PrAMP), killed sensitive species in a dose-dependent manner by specifically binding to DnaK. Here, on the basis of the finding that DnaK-deficient Escherichia coli strains are susceptible to PrAMPs, we used pyrrhocoricin to investigate internal targets other than DnaK. Using conventional antibiotics (bleomycin, streptomycin, and fosfomycin) that have known modes of action, first, we validated the availability of an assay using a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on E. coli lysate, for evaluating inhibition of protein synthesis. We found that, similarly to bleomycin and streptomycin, pyrrhocoricin inhibited GFP synthesis in RTS in a concentration-dependent manner. In addition, blockage of transcription and translation steps in RTS was individually estimated using RT-PCR after gene expression to determine mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that this inhibition of GFP synthesis by pyrrhocoricin did not occur at the transcription step but rather at the translation step, in a manner similar to that of GFP synthesis by streptomycin, an inhibitor of the translation step by causing misreading of tRNA. These results suggest that RTS is a powerful assay system for determining if antimicrobial peptides inhibit protein synthesis and its transcription and/or translation steps. This is the first study to have shown that pyrrhocoricin inhibited protein synthesis by specifically repressing the translation step.

Growth-inhibition of hiochi bacteria in namazake (raw sake) by bacteriocins from lactic acid bacteria

The bacteriocins produced by Lactococcus lactis subsp. lactis C101910 (C101910) and NBRC 12007 (NBRC 12007) were used to prevent the growth of sake spoiling hiochi bacteria (Lactobacillus hilgardii, Lactobacillus fructivorans, and Lactobacillus paracasei) in namazake, which is raw (unpasteurized) sake. The bacteriocin concentrations required for decreasing the viable cell concentrations of L. hilgardii and L. fructivorans below the detection limit (1.0 x 10(2) cells/ml) in 24 h from the initial concentration of 4.0-9.5 x 10(5) cells/ml in the namazake at pH 4.5 and at 4 degrees C, were 18-35 U/ml and 5.6 U/ml for the bacteriocin from C101910 and NBRC 12007, respectively. To decrease the viable cell concentration of L. paracasei from the initial concentration of 7.5 x 10(5) cells/ml to below the detection limit (1.0 x 10(2) cells/ml) in 24 h, 350 U/ml bacteriocin from C101910 and 140 U/ml bacteriocin from NBRC 12007 were required. In experiments using McIlvaine buffer (pH 4.5) with 15% ethanol instead of namazake as the medium, the viable cell concentrations of L. hilgardii and L. paracasei decreased to less than 1.0 x 10(2) cells/ml, whereas those of L. fructivorans decreased to less than 1.0 x 10(3) cells/ml, when bacteriocins were added at the concentrations that had proven effective in namazake. The membrane depolarization assay using a fluorescent probe showed that the presence of ethanol stimulated the collapse of the membrane potential induced by bacteriocins. The ethanol induced collapse of the membrane potential suggests that the application of bacteriocins at the storage stage of namazake is more beneficial than when used in other stages of the sake brewing process.

Evaluation of positive interaction for cell growth between Bifidobacterium adolescentis and Propionibacterium freudenreichii using a co-cultivation system with two microfiltration modules

Using a co-cultivation system developed previously, positive interaction for cell growth between Bifidobacterium adolescentis and Propionibacterium freudenreichii was evaluated. The total dry cell weight (DCW) of these two strains obtained in the co-cultivation system was 1.5-1.7-fold of the sum of the DCWs obtained in two single cultivations of each bacterium.

Protective effect of composite earthworm powder against diabetic complications via increased fibrinolytic function and improvement of lipid metabolism in ZDF rats

Thrombosis is the leading cause of mortality globally. It is not only a complication but also a risk factor for progression of diabetes. However, alternative oral therapies and prophylaxis with less adverse effect for thrombosis have not been well studied. In this study, composite powder containing earthworm (CEP) was used and its fibrinolytic activity was measured. CEP was found to have a high urokinase-type plasminogen activator like activity in an in vitro assay. It also had significantly shortened euglobulin clot lysis time (ECLT) at 4 and 24 h after ingestion in Sprague Dawley rats. Zucker Diabetic Fatty rats were used to assess the effect of CEP on diabetes and diabetic nephropathy. After 10 weeks of feeding, CEP significantly shortened ECLT and attenuated HbA1c, hepatic lipid accumulation, and urinary albumin excretion and improved glomerular mesangial matrix score. Therefore, CEP may have beneficial effects on diabetes and diabetic nephropathy. Graphical abstract CEP improved ECLT and suppressed plasma PAI-1, HbA1c, and renal mesangial matrix score in ZDF rats after 10 weeks feeding.