Streptomyces sp. CS392 균주에서 분리한 항균, 면역 조절물질의 이화학적 특성 및 생물학적

Abstract

한국 토양 방선균에서 항균, 면역조절 물질을 개발 하고자 국내 전남 등지에서 채취한 토양 및 식품유래 미생물 약 900여종을 대상으로 항균, 면역조절 물질을 생산하는 균주를 스크리닝 하였다. 수 종의 후보 균주 중 항균효과를 가지는 CS392균주의 에틸 아세테이트 추출물이 LPS (lipopolysaccharide)로 자극한 면역세포에서 nitric oxide생성 억제효과를 가지며, 항산화 효과가 있음을 확인하였다. 선별한 CS392 균주는 유전학적, 배양학적, 생화학적 분석을 통하여 신규한 균주임을 확인하고 Streptomyces sp. CS392라 명명하였다. 이 균주의 최적 배양조건은 탄소원으로써 glucose(1% w/v), 질소원으로써 peptone(1% w/v), magnesium chloride(0.01% w/v), pH 7.0, 28℃ 였다. 최적배지에서 배양 후 얻은 발효액은 유기용매로 추출 후 silica gel, RP-18 silica gel column을 수행하여 3종의 물질을 정제하여 구조분석을 수행하였다. 구조분석 결과 주 물질은 manumycin group metabolite인 manumycin이었다. C1, C2는 구조분석 중에 있다. Manumycin 과 C1, C2의 약리효과를 조사하였다. 세가지 물질은 그람 양성세균, 메티실린 내성 황색포도상 구균, 반코마이신 내성 장구균에 유효함을 확인하였다. 세가지 물질은nitric oxide소거능력과 환원력을 보여 항 산화능도 일부 가지고 있음을 확인하였다. Manumycin 및 C1, C2는 90℃, 4시간까지 처리시 활성이 거의 감소하지 않아 열에 안정함을 보였으며, 121℃, 15pound, 15분 처리시 약간의 활성이 감소하였다. 또한 CS392균주 배양액은 pH 3에서 9까지 안정함을 보였다. Murine macrophage인 Raw 264.7 cell을 LPS (lipopolysaccharide)로 자극하여 nitric oxide와 pro-inflammatory cytokine생성을 유도하였을 때 세가지 물질은 모두 nitric oxide와 pro-inflammatory cytokine생성을 1-20 μg/ml 농도 범위에서 유의하게 억제함을 확인하였다. Stimuli와 약물을 처리한 세포에서 얻은 단백질은 웨스턴 블럿을 수행하여 iNOS단백질 발현이 억제됨을 확인하였다. 또한NF-kB의 핵 내로의 이동을 저해함을 확인하여 결과적으로 nitric oxide나 염증성 cytokine의 생성 억제는 NF-kB와 연관이 있음을 확인하였다. Manumycin과 C1은 SNP(sodium nitroprusside)가 방출한 nitric oxide를 제거하였으며, iNOS 발현 억제를 통해 nitric oxide 생성을 억제하여 dual effector로써 NO생성 반응을 억제하여 면역세포에서 염증 반응을 효율적으로 감소시킬 것으로 생각되었다. Manumycin group metabolite들이 in vivo 에서 효과가 있는지 확인하기 위해 mouse ear edema model과 DSS (Dextran sodium sulfate) colitis model에서 항염증 효과를 조사하였다. 마우스 귀부종은phorbol ester인 TPA를 이용하여 염증을 유도하였으며, 세가지 물질은 같은 양(50 μg/ear)을 단회 도포시 manumycin>C1>C2의 순으로 염증 억제 효과를 보였다. 대조구 및 약물 처리군의 마우스 귀 조직의 iNOS단백질의 발현 양상을 확인한 결과 약물 처리군은 양성, 음성 대조군과 비교시 유의한 발현억제 양상을 보였다. DSS (Dextran sodium sulfate)로 유도된 장염모델은 IBU(intestinal bowel disease)의 대표적인 동물 모델이다. ICR mouse에 DSS (Dextran sodium sulfate)를 자유 급여하며, 복강 내 투여로 약물을 1일 1회 1주일간 투여하며 마우스를 관찰하며 DAI (Disease activity index)와 체중변화를 측정하였으며, 7일째에 colon을 적출하여 colon의 조직학적 변화를 관찰하고, colon길이 및 colon조직에서 pro-inflammatory cytokine, TNF-α 생성량을 측정하였다. 세 물질 중 C2, manumycin이 DSS에 의해 증가된 염증조직을 완화시켰고, DAI를 유의하게 감소시켰으며, DSS에 의해 짧아지는colon length의 길이도 1-10 mg/kg에서 농도에 의존하여 정상colon과 유사하게 회복시켰으며, colon tissue에 증가되었던pro-inflammatory cytokine, TNF-α도 감소시켰다. 또한 iNOS 단백질의 발현도 유의적으로 감소하였다. 본 연구를 통해 manumycin group metabolite가 항생제 내성 세균에 광범위한 항균 효과를 가지며, 항 산화효과로써 환원력, NO 소거능을 가지는 것을 확인하였다. 또한 in vitro macrophage cell에서 manumycin과 유도체들의 iNOS 발현억제, NF-kB translocation억제 및 pro inflammatory cytokine 생성 억제 효과와 급성 귀 부종, 장염을 유발시킨in vivo 염증 모델에서 항 염증 효과를 가져 in vitro, in vivo 모델에 모두 효과적임을 설명하였다. 향후 이들의 작용기전을 보다 더 자세하게 연구하면, manumycin group metabolite의 소염작용과 그 구조와의 상관성을 이해할 수 있고, 과량의 nitric oxide 및 pro-inflammatory cytokine의 생성과 관련한 면역질환의 이해와 치료에 도움을 줄 것이라 사료된다.|With the goal of searching potent immonomodulatory substance with antimicrobial activity against various resistant pathogens, such as methicillin resistant Staphylococcus aureus (MRSA), strain CS392 was screened from several hundreds of actinomycetes strains preserved in our laboratory. Ethylacetate extract of fermentation broth of CS392 strain showed antimicrobial activity and suppressed the nitric oxide production in LPS (lipopolysaccharide) stimulated macrophage cells. Additionally ethylacetate extract showed anti-oxidative activities such as nitric oxide scavenging and reducing activity. Therefore we selected this strain for the detailed study. Based on the morphological and biochemical characteristic, CS392 was found related to the genus Streptomyces. Further, the 16S rRNA sequence analysis revealed that the strain was closely related to Streptomyces lanatus with 98.22% homology; therefore the strain was classified as Streptomyces sp. CS392. The strain was cultivated at 28ºC and 180 rpm for 3 days and the paper disc assay technique was employed to determine the anti-MRSA effect. Antimicrobial compounds production from the strain was optimized by using several nutritional parameters. Glucose, peptone and MgCl2 were found the best carbon, nitrogen and mineral source, respectively for the production of the compounds. After cultivation of CS392 strain under optimized condition for 2 days, fermentation broth was harvested, the antimicrobial compounds were extracted using ethyl acetate (1:1 v/v), and then purified using column chromatography. These compounds were finally purified and identified with reverse-phase HPLC with 60% acetonitrile (0.01% formic acid) linear gradient. Eventually, three different antimicrobial compounds (C1, C2 and C3) were achieved from the strain. Based on NMR, IR, MASS analysis, those compounds were identified as manumycin (C3) and its derivatives (C1 and C2, manumycin group metabolites). Antimicrobial activity of these compounds against different pathogenic strains was assessed using the minimum inhibitory concentrations (MICs). It was found to be active against eight different strains, including MRSA 693E , MRSA (2-32), MRSA (4-5), MRSA (4-21), Enterococcus faecalis ( ATCC 29212), VRE 82, VRE 89, VRE 98, Bacillus subtilis (ATCC 1928), Staphylococcus aureus (KCTC 1928) and Mycobacterium smegmatis (ATCC 9341). Antimicrobial activity of these compounds were stable in desirable pharmaceutical processing conditions such as heat (up to 90 ℃/ 4 hr, 121℃ /15 pound, 15 min), freezing (-20℃) and pH treatment (pH 3 to 9). To evaluate the chemical stability of compounds, a study was designed to assess the in vitro endurance and effects of media and storage conditions. These compounds were found to inhibit the production of tumor necrosis factor-a (TNF-α), interleukin-1 β (IL-1β) and interleukin-6 (IL-6). Manumycin derivatives (C1 and C2) dose dependently inhibited NO production and inducible NO synthase (iNOS) expression in Raw 264.7 macrophages activated with LPS. Besides, C1 and manumycin showed nitric oxide-scavenging effect concentration-dependently. To investigate the signaling pathway for NO inhibition by these compounds, we examined nuclear factor-κB (NF-κB) activation in Raw 264.7 cells. We found that manumycin derivatives inhibited the synthesis of pro-inflammatory cytokines and suppressed LPS-induced NF-κB activation in Raw 264.7 cells. These compounds, hence, deserved to be developed as promising anti-inflammatory drugs after further preclinical and pharmaceutical studies. Furthermore, these compounds were examined for their antioxidative activities with 2,2-diphenyl-1-pikryl hydrazyl (DPPH) radical scavenging assay, total phenolic content, Superoxide dismutase-like activity, super oxide scavenging activity and reducing power. These compounds showed nitric oxide scavenging activity but with slight reducing power. Among three, C3 showed the strongest activity. Since these compounds inhibited the production of pro-inflammatory cytokines, NO production, and iNOS expression in Raw 264.7 macrophages upon in vitro experiments, it was felt necessary to investigate in vitro and in vivo relationship. Firstly, these compounds were assayed for topical anti inflammatory activity by TPA(12-O-tetradecanoylphorbol-13-acetate)-induced ear edema model in ICR mice. The anti inflammatory activities (% inhibition) in the TPA-induced ear edema test shown by C1, C2 and C3 were 22.1%, 16.7% and 29.2%, respectively. These compounds thus showed slight inhibition in edema formation without any side effects. Using each ear punch biopsies, protein samples were taken from ear whole cell lysate and further effect of these compounds on iNOS expression was investigated by western blot analysis. Three compounds were found to inhibit TPA-induced expression of iNOS. In conclusion, these compounds have topical anti inflammatory activity mediated via inhibition of iNOS expression. These findings thus to gives a clear reason for the use of these compounds in the treatment of inflammation. Secondly, we investigated protective effect of these compounds on a mice model of colitis. Colitis was induced in ICR mice by administration of dextran sulfate sodium (DSS) (3%, w/v) in drinking water. DSS colitis model was well characterized morphologically and biochemically. DSS produced decrease in colon length accompanied by mucosal edema and bloody stool. To assess the dose effect on DSS-induced colitis, 1, 3 and 10 mg/kg/day of compounds were administered by intraperitoneal injection. Besides measuring colon length, we have estimated disease activity index (DAI) for a week, taking into account of body weight, stool consistency and gross bleeding. Pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1 β), and interleukin-6 (IL-6), were determined using immunoassays. Among three compounds, manumycin and C2 notably prevented shortening of colon length and reduced DSS-induced DAI scores including decrease in weight loss, diarrhea and gross bleeding. Manumycin found to suppress iNOS expression and abnormal secretions of pro-inflammatory cytokines, such as TNF-α, interleukin-1β and IL-6. In conclusion, three immunomodulatory compounds with strong antimicrobial activity were purified from a new Streptomyces strain isolated from Korean soil. One of the purified compounds was identified as manumycin and two others were as its derivatives. The results drawn from this study provide a scientific basis for the application of these compounds as antimicrobial anti-inflammatory agent. Further studies should/will be undertaken to explain the mechanism of action and roles of immune response by which these compounds exert their biological activity.