Unveiling the Potent Antimicrobial Activity and Mechanism of Action of Novel Peptide YS12 from Bacillus velezensis against Drug-Resistant Strains: A Promising Strategy for Combating Antibiotic Resistance

Abstract

항생제의 남용으로 다제 내성 균주가 증가하는 가운데, 항균 펩타이드(AMPs)는 전통적인 항생제 대안으로 큰 주목을 받고 있다. 본 연구에서는, 한국의 전통식품인 김치에서 분리한 Bacillus velezensis CBSYS12 균주로부터 유래된 항균 펩타이드 YS12의 항균성, 항 바이오필름 작용 및 작용 메커니즘을 규명하였다. CBSYS12 균주는 김치로부터 분리 후, 최적배지를 결정하여 대량배양 하였고, 배양액으로부터 ultrafiltration 및 sequential chromatographic 기법을 사용하여 정제하였다. Tricine-SDS PAGE를 통해 대략 3.3kDa의 단일 단백질 밴드를 확인하였고, 또한 같은 위치에서 anti-biofilm을 확인할 수 있었다. 비슷한 분자량(~3348.4 Da)의 단백질이 MALDI-TOF에서도 나타나, 펩타이드 YS12의 순도와 균일성을 확인하였다.

AMPs는 다제 내성 감염성 균에 대한 넓은 항균 스펙트럼과 미생물이 쉽게 내성을 개발하기 어렵다는 특성 때문에, 전통적인 항생제의 잠재적인 대체재로 간주된다. 본 연구의 결과는 E. coli, P. aeruginosa, MRSA 4-5, VRE 82, 그리고 M. smegmatis와 같은 그램 양성 및 그램 음성 균에 대해 6-12 μg/ml 범위의 최소 억제 농도(MIC) 값을 가진 강력한 항균 활성을 보여주었다. 펩타이드 YS12는 상용 항생제와 시너지 효과를 보여주어, 다제 내성 균에 대한 효과적인 치료 방법을 제시하였다. 더불어, 항 바이오필름 검사에서는 펩타이드 YS12가 80 µg/ml에서 E. coli와 P. aeruginosa의 바이오필름 형성을 약 80% 억제하였다. 특히, YS12는 상용 항생제보다 더 뛰어난 바이오필름 제거 활성을 보였다. 또한, 펩타이드 YS12의 마우스 대식세포인 Raw 264.7에 대한 세포독성도 측정하였다. 그러나, 펩타이드 YS12는 잘 알려진 항균 화합물인 멜리틴에 비해 적은 수준의 적혈구 용해작용을 보였다. |Nowadays, the abuse of antibiotics has led to the rise of multi-drug-resistant bacteria. Antimicrobial peptides (AMPs) have attracted considerable attention as possible alternatives to traditional antibiotics. In this work, we aimed to evaluate the antimicrobial, anti-biofilm activity with the mechanism of action of an antimicrobial peptide designed as YS12 derived from Bacillus velezensis CBSYS12. The strain CBSYS12 was isolated from Korean food kimchi and purified, followed by ultrafiltration and sequential chromatographic methodology. Hereafter, Tricine-SDS PAGE revealed a single protein band of around 3.3kDa that further confirmed insitu inhibitory activity of the gel. A similar molecular weight (~3348.4 Da) protein also appeared in MALDI- TOF confirming the purity and homogeneity of the peptide YS12. AMPs are considered potential substitutes for traditional antibiotics due to their broad antimicrobial spectrum against multidrug-resistant (MDR) pathogenic bacteria and the inability of microorganisms to easily develop resistance to them. Intriguingly, our data revealed a strong antimicrobial activity with a minimum inhibitory concentration (MIC) value ranging from 6-12 μg/ml for different bacteria h as E. coli, P. aeruginosa, MRSA 4- 5, VRE 82, and M. smegmatis. Peptide YS12 provides a synergistic action with commercial antibiotics and thus creates a more effective approach to the treatment of multi-drug resistant bacteria. In addition, the anti-biofilm assay demonstrated that peptide YS12 was able to inhibit biofilm formation around 80% for both bacterial strains E. coli and P. aeruginosa at 80 µg/ml. Notably, YS12 exhibited a greater biofilm eradication activity than commercial antibiotics. Cytotoxicity towards mouse macrophage Raw 264.7 of peptide YS12 was measured. However, peptide YS12 causes a low level of hemolysis of red blood cells compared with the standard well-known antimicrobial compound melittin. In terms of mechanism of action, most AMPs do not target specific molecular receptors of pathogens, which is different from the mechanisms of traditional antibiotics. The results from fluorescent measurement revealed that peptide YS12 bound with LPS through electrostatic interaction. The principal mechanism of AMPs is that can act directly, interacting with cytoplasmic membranes and intracellular molecules. We carried out a membrane depolarization assay, N- phenyl-1-naphthyl amine (NPN) assay (which measures the permeability of the outer membrane), and propidium iodide assay (inner membrane permeability) to elucidate the possible mechanisms underlying the peptide’s mode of action using different fluorescent dyes.

Peptide YS12 exhibited direct antimicrobial activity by damaging the integrity of the bacterial membrane. Finally, confocal microscopy confirmed that the peptide carried out an intracellular targeting mechanism by cell penetration. In summary, our study proposed that the antimicrobial peptide YS12 may be a promising therapeutic agent against antibiotic-resistant bacteria.