A novel antimicrobial peptide from Bacillus strain exhibiting therapeutic potential against vancomycin resistant Enterococcus group of organisms.

Abstract

Antimicrobial peptides are widely distributed and have been found in organisms ranging from prokaryotes to plants, insects and mammals. To protect themselves from microbes, microorganisms including bacteria secrete antimicrobial peptide. They are generally considered as a part of the innate immune system and rapidly increase in concentration in the host upon challenge by pathogens. Bacillus is an interesting genus to be investigated for antimicrobial activity because these species produce a large number of peptides with biological activities. Kimchi is found to be perfect for antimicrobial peptide production which is a traditional Korean food fermented by various organisms such as Bacillus and Lactobacillus. The aim of this work was to investigate the therapeutic potential of antimicrobial substances produced by Bacillus species. Bacillus strain K1R was amended from bacterial supernatant obtained from fermentation in culture media containing carbon and nitrogen sources which is therefore called optimized media for that particular strain and then by the application of ammonium sulfate precipitation, concentration (membrane filters), dialysis, sequential column chromatography, AMP was obtained. To determine the purity and molecular weight, the purified peptide was subjected to tricine-sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Protein band around 4.6 kDa was seen in tricine-SDS-PAGE which was confirmed by in situ inhibitory activity of the gel. K1R was found to be stable over a broad range of pH (6.5-9) and thermo tolerant up to 60°C and unaltered activity at low temperature of 0°C and 4°C. K1R showed better result than vancomycin and bacitracin against different MDR pathogens particularly on vancomycin resistant enterococcus group of organisms. Amino acid sequence of K1R was AVQGTLEDALNLSKGALNQVQKAIQNGDXLTVXGXLGTIXL AVSX. Its antagonistic effect against MDR pathogens like Salmonella typhimurium and Enterococcus sp. verified that K1R has potential application in treating infectious MDR cases. Antioxidant activity of K1R was also comparable to that of standard ascorbic acid.|항균펩타이드는원핵생물에서식물, 곤충과포유류에이르기까지다양한생명체에서발견된다. 병원균으로부터스스로를보호하기위하여박테리아를포함하는미생물은항균펩타이드를분비한다.이는선천면역시스템의일부로서병원균이침입하였을때, 숙주의체내에서그농도가급격 이증가한다. Bacillus 는항균활성연구에있어서흥미 로운 속에속하는데, 이는생물학적활성 을 가지는수많 은펩타이를생 산하기때문이다. 대표적인예로김치는, Bacillus 나 Lactobacillus와같은다양한미 생물에의해발 효된한국의전통식품으로서, 항균펩타이드생산에최적인 조건을 나타내는것 으로 밝혀졌다. 본연 구의목적은, Bacillus 종에의해생산된항미생물의치 료적효능을조사하고자하였다. Bacillus 속균 주인K1R은탄소와질소공급원을 포함하는세포 배양액의발효에의해얻어진박테 리아상등액으로부 터되었고이는이균주에최적화된배지조 건이라고할수있으 며황산알루미늄침전 법, 멤브레인필터 를통한농축, 여과, 일련의컬럼크로 마토그래피, AMP에의하여획득되었다. 순도와 분자량을결 정 하기위해서정제한펩타이 드를 Tricine SDS-PAGE겔에전기영동하였다. 단백질밴 드는Tricine SDS-PAGE겔에서약 4.6 kDa에서확인되었고, 이는전기 영동된겔의항균활 성검사 를통하여확 정되었다. K1R은넓 은범위의pH(6.5~9) 에서도안정적인것 으로밝혀졌으며, 60℃까지열내성을 가지며 0℃및 4℃의낮은온도에서도활성이변하지않았다. 특히, K1R은반코마이 신내성장내구 균 에서 MDR 병원균에대하여반코마이신이나바시트라신보다더나은결과를보여주었다. K1R 의아미 노산서 열은 AVQGTLEDALNLSKGALNQVQKAIQNGDXLTVXGXLGTIXLAVS X이었다.또한, K1R 은살모넬라나 장구균등의 MDR 병원균에대해서도저해효과를보였는데이는 K1R이감 염성 MDR 의치료에 잠재적 인응용 가치가있음 을의미 한다. 이와더불어, K1R의항산화활 성효과는아스코 르브산과비슷한수 준이였다.