Antimicrobial and anti-inflammatory peptides of Bacillus strains isolated from Korean foods

Abstract

국문초록

한국전통 식품에서 분리한 바실러스 균주가 생산하는 항균, 항염 펩타이드에 관한 연구

최 윤 희 지도교수: 유 진 철 조선대학교 약학대학 약학과

바실러스 속CS61 한국 전통 식품에서 분리한 바실러스 속 균주 CS61은 다약제 내성균주에 항균 효과를 보이는 펩타이드를 생산하는 것을 확인하였다. CS61 항균 펩타이드는 컬럼 크로마토 크래피를 통하여 분리정제 하여 분자량이 1100Da 정도임을 확인하였으며, 이 펩타이드는 pH2.0-10.0사이, 온도는 80℃까지 활성이 유지되었다. 또한 단백질 분해효소인 lipase, proteinase K, α-chymotrypsin에 안정하였다. 다양한 chemical에 대한 안정성을 확인한 결과 EDTA, TCA, 계면활성제를 제외한 유기용매에 의해 활성이 감소하였다. CS61 항균펩타이드의 다약제 내성균인 메티실린 저항성 황색 포도상구균(MRSA), 반코마이신 저항성 황색 포도상 구균(VRSA), 반코마이신 저항성 장알균(VRE)에 대한 최소 저해 농도값은 0.625-20μg/ml 이었다. N-말단 아미노산 염기서열 분석 결과 A-I-N-X-D-A-A-Y-L의 염기서열로 구성되어 있었으며, 네번째 자리에 읽히지 않은 아미노산을 20가지 다양한 아미노산으로 치환하여 치환체의 항균활성을 비교하였을 때 시스테인으로 치환된 치환체에서 항균 활성을 보임을 알 수 있었다. CS61 항균 펩타이드의 항산화 활성은 DPPH 라디칼 소거능과 환원력을 통해 분석하였다. 이 펩타이드의 DPPH 소거능은 1000 μg/ml 의 농도에서 77.789±1.8의 소거능을 보였으며 대조군인 아스코르브산과 비교하였을 때 거의 비슷한 수준의 활성을 나타내었다. 환원력은 대조군인 아스코르브산 보다는 낮지만 농도 의존적으로 증가하였다. Raw264.7 대식세포는 LPS에 의해 일산화 탄소의 생성이 증가하는데 CS61 항균 펩타이드를 처리하였을 때 이의 생산을 억제하는 효과를 보였다. 또한 LPS에 의해 유도되는 염증 단백질인 iNOS, COX-2 단백질의 발현을 농도 의존적으로 감소 시켰으며 RT-PCR를 통해 유전자 수준에서의 발현 또한 감소함을 확인하였다. 염증반응에 관여하는 싸이토카인인 TNF-α, IL-6, IL-1β의 발현을 ELISA를 통해 확인한 결과 농도의존적으로 감소하였고 이 싸이토카인의 유전자 수준에서의 발현 또한 감소함을 확인하였다.

바실러스 속 CS32 한국 전통식품에서 분리한 바실러스 CS32 균주는 항균펩타이드를 생산함을 확인하였다. 항생제 저항성 균주인 MRSA, VRE, VRSA에 탁월한 효과를 보였으며, 이런 병원성 균주에 대한 최소저해농도는 0.156-80 μg/ml 이었다. 항균 펩타이드는 Sepharose CL-6B, Sephadex G50 컬럼을 통해 분리하였고 Tricine SDS-PAGE와 MALDI-TOF를 통해 5697.9Da 정도 크기를 보이는 펩타이드임을 확인하였다. N-말단 염기서열 분석을 통해12개의 아미노산 서열을 분석(APLEIXXIFHDN)하였고 이 서열은 기존에 보고 되었던 것과 다른 신규한 펩타이드임을 확인하였다. 이 펩타이드는 단백질 분해효소인 lipase, proteinase K, α-chymotrypsin, Trypsin에 대해 안정하였으며, 다양한 chemical에 대해서도 안정성을 보였다. 또한 pH5.0-10.0사이에서 활성이 유지되었고 온도에도 안정하였다. CS32 항균 펩타이드의 항산화 활성을 DPPH 라디칼 소거능과 환원력을 통해 시험하였다. DPPH 라디칼 소거능 시험 결과 CS32 펩타이드는 1000 μg/ml 의 농도에서 56.9±0.79의 소거능을 보였으며 대조군인 아스코르브산과 비교하였을 때 같은 농도에서 절반정도의 활성을 나타내었다. 환원력은 대조군인 아스코르브산 보다는 낮지만 농도 의존적으로 증가하였다. Raw264.7 대식세포는 LPS에 의해 일산화 탄소의 생성이 증가하는데 CS32 항균 펩타이드를 처리하였을 때 이의 생산을 억제하는 효과를 보였다. 또한 LPS에 의해 유도되는 염증 단백질인 iNOS, COX-2 단백질의 발현을 농도 의존적으로 감소 시켰으며 RT-PCR를 통해 유전자 수준에서의 발현 또한 감소함을 확인하였다. 염증반응에 관여하는 싸이토카인인 TNF-α, IL-6, IL-1β의 발현을 ELISA를 통해 확인한 결과 농도의존적으로 감소하였고 이 싸이토카인의 유전자 수준에서의 발현 또한 감소함을 확인하였다.|ABSTRACT

Antimicrobial and anti-inflammatory peptides of Bacillus strains isolated from Korean foods

Choi, Yun Hee Advisor: Prof. Jin Cheol Yoo, Ph.D. Department of Pharmacy, Graduate School of Chosun University

Bacillus sp. CS61 In an attempt to isolate effective antimicrobial peptides (AMPs) from a microbial source for the treatment of multidrug-resistant (MDR) bacteria, peptide was purified from Bacillus sp. CS61 newly isolated from the traditional Korean fermented foods. CS61 AMP (ca. 1100Da) was purified to homogeneity by using sequential chromatographic steps. It was found to be stable at pH 2.0–10.0 and up to 80 ℃. The AMP activity was resistant to the proteolytic action of lipase, proteinase K and α-chymotrypsin. The effect of several chemicals on the AMP was evaluated. With an exception to EDTA, TCA and detergent, the AMP was completely sensitive to organic solvents. CS61 AMP performed antimicrobial activity against MDR bacteria such as meticillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant S. aureus (VRSA) and vancomycin-resistant enterococci (VRE). Minimal inhibitory concentrations of CS61 AMP against MRSA, VRSA and VRE were in the range of 0.625–20μg/ml. The N-terminal amino acid sequence of CS61 AMP was A-I-N-X-D-A-A-Y-L, which differed from reported AMPs. The fourth unidentified amino acid was replaced and several peptides were synthesized. Among them, only cysteine replacement displayed antimicrobial activity. The antioxidative activities of the AMP were determined by using 2,2-diphenyl-a-picrylhydrazyl (DPPH) and reducing antioxidant power methods. The CS61 AMP has good antiradical potency against the DPPH. Scavenging effect of DPPH radicals was 77.789±1.8 at 1000μg/ml. The reducing power of CS61 AMP was increased in a concentration dependent manner. However, the DPPH scavenging effect and reducing power of ascorbic acid (control) were more effective than CS61 AMP. Bacillus sp. CS61 AMP was shown in this study to have potent inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages. Consistent with these findings, CS61 AMP was further shown to suppress the expression of LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxigenase-2 (COX-2) in a dose-dependent manner at both the protein and mRNA gene expression levels. In addition, the release of tumor necrosis factor- α (TNF-α) and interleukin-6 (IL-6), IL-1β the mRNA expression levels of these cytokines were reduced by CS61 AMP in a dose-dependent manner. These results suggest that the anti-inflammatory properties of CS61 AMP are caused by iNOS, COX-2, TNF-α, IL-6 and IL-1β in RAW 264.7.

Bacillus sp. CS32 In this study, an antibacterial peptide produced by an isolated Bacillus licheniformis CS32 from traditional Korean fermented foods, was identified as antimicrobial peptide. CS32 AMP showed antimicrobial activity against MDR bacteria such as meticillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant S. aureus (VRSA) and vancomycin-resistant enterococci (VRE). Minimal inhibitory concentrations of CS32 AMP against MRSA, VRSA and VRE were in the range 0.156–80μg/ml. The corresponding AMP was purified to homogeneity by ammonium sulfate precipitation, Sepharose CL-6B and Sephadex G50 column chromatography. Tricine Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the active peptide had an apparent molecular weight about 5697.9 Da. Complete amino acid sequence of the peptide yielded twelve amino acids from the N-terminal end (APLEIXXIFHDN), which differed from reported AMPs. The antimicrobial peptide activity was resistant to the proteolytic action of lipase, proteinase K, α-chymotrypsin and trypsin. The effect of several chemicals on the antimicrobial activity was evaluated. The antimicrobial activity was slightly decreased after its treatment with Triton X-100. Antimicrobial activity was not affected by treatment with organic solvents and EDTA. The antimicrobial activity was relatively heat resistant and also active over a wide range of pH 5-10. Such characteristics indicate that these AMPs may be potential candidate for alternative agents to control important clinical pathogens in animal diseases. The antioxidative activities of the AMP were determined by using the 2,2-diphenyl-a-picrylhydrazyl (DPPH) and reducing antioxidant power methods. The CS32 AMP has good antiradical potency against the DPPH. Scavenging effect of DPPH radicals was 56.9±0.79 at 1000μg/ml. The reducing power of CS32 AMP was increased in a concentration dependent manner. However, the DPPH scavenging effect and reducing power of ascorbic acid (control) were more effective than CS32 AMP. Bacillus sp. CS32 AMP was shown in this study to have potent inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages. The level of nitric oxide (NO) production in the CS32 AMP and LPS-treated RAW 264.7 cells were determined by western blotting, RT-PCR and ELISA. The cytotoxicity of the CS32 AMP and LPS was measured by an MTT assay. CS32 AMP was shown to suppress the expression of LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxigenase-2 (COX-2) in a dose-dependent manner at both the protein and mRNA gene expression levels. In addition, the release of tumor necrosis factor- α (TNF-α), interleukin-6 (IL-6) and IL-1β the mRNA expression levels of these cytokines were reduced by CS32 AMP in a dose-dependent manner. These results suggest that the anti-inflammatory properties of CS32 AMP are caused by iNOS, COX-2, TNF-α, IL-6 and IL-1β in RAW 264.7 cells.