Identification of small molecular peptide for the detection of Salmonella typhimurium and Salmonella enteritidis

Abstract

Salmonella enteritidis and Salmonella typhimurium are the most common and severe food-borne pathogens responsible for causing salmonellosis in humans and animals. Salmonella is a genus of rod-shaped, Gram-negative bacteria. Salmonella infections can be fatal if not detected and treated at early stages. The development of ultrasensitive detection system is the first critical step in controlling this salmonella disease. We have screened novel diverse peptides displaying on M13 bacteriophage that bind to salmonella typhimurium, salmonella enteritidis with high affinity from a phage display. Alternative ligands such as peptides can be used for pathogen capture and detection and offer advantages over antibodies, including reduced cost, ease of production and modification, and improved stability. In addition, we make a versatile polyvalent directed peptide polymer(PDPP) to overcome limitation of peptide binding affinity. To accomplish this, we used phage display technique to identify peptide to be used as detection probes that can specifically bind to S. enteritidis and S. typhimurium. We identified two peptide for detection of the S. typhimurium and one peptide for detection S. enteritidis through each pahge display rounds. Each peptides demonstrated higher binding affinities and the dissociation constants (Kd) were found to be in the range of nanomolar levels(SEBP : 205± 70.7, STBP1 : 466±45.3, STBP2 : 388±44.2). Peptide specificity was performed using the gram-negative bacteria vibrio parahaemolyticus and was found to be much higher towards S. enteritidis and S. typhimurium. Single peptide have several limitation of binding affinity and sensitivity. To address this limitation, we conjugated onto polyvalent directed peptide polymer, which led to 10-fold increase in binding affinity compared to the single peptide (SEB-PDPP : 15.4± 1.85nM, STB-PDPP1 : 35.7±10.4nM, STB-PDPP2 : 37.9±5.48nM). The concept of polyvalent interactions can be explained as simultaneous binding of multiple ligands on one entity to multiple receptors on another. The polyvalent affinity is significantly higher than that for the binding of a single ligand to a single receptor. These interactions effect the high binding affinity of PDPP compared to single peptide. This study reports the identification of higher affinity and specificity peptide(12mer), which may be useful as capture and detection probes in biosensor detection system for salmonellosis. In addition, we used polyvalent directed peptide polymer(PDPP), and showed increased binding affinity and fluorescence intensity. This probe can detect 100 CFU/ml of S. enteritidis and S. typhimurium by fluorescence signal.

| 살모넬라균은 사람, 동물에 감염되며 또한 환경에도 흔히 존재할 수 있는 병원균으로서 식중독에 관련되는 주요 혈청형은 Salmonella typhimurium 및 S. enteritidis 등이다. 살모넬라는 막대 모양의 세균으로 그람 음성균으로 알려져 있다. 살모넬라균에 의한 식중독 사고와 같이 대규모 집단 발병의 위험도가 높은 전염병은 감염을 확인 하고 원인균을 판정할 때까지의 시간이 피해를 줄이는데 매우 중요하다. 하지만 기존의 살모넬라균 분석법은 5-7일의 시간이 소요되어 질병 발생 시 신속한 조기 진단 및 예방에 어려움이 있다. 따라서 신속한 식중독균 진단을 통한 치료 및 예방 방안이 필요하다. 고민감도 검출 시스템의 개발은 이러한 살모넬라 질병을 제어 할 수 있는 첫 번째 치료 단계이다. 본 연구는 페이지 디스플레이(phage display)기술을 이용하여 살모넬라 티피뮤리움과 엔테라이티디스에 높은 민감도와 선택도를 가지는 생체적합성 저분자 탐침인 펩타이드를 제시한다. 항체를 대신할 수 있는 대안물질인 펩타이드는 생산이 용이하고 높은 안정성을 가지고 있고 합리적인 가격측면에서 많은 장점을 제공한다. 스크리닝한 펩타이드를 이용하여 다중 인식 펩타이드 고분자(Polyvalent directed peptide polymer)를 제작하여 펩타이드가 가지고 있는 결합 친화성 (Binding affinity) 한계점을 극복할 수 있는 탐침자를 개발하였다. 본 연구를 통해서 파지 디스플레이 라이브러리 스크리닝을 통해 살모넬라 티피뮤리움과 엔테라이티디스에 결합하는 펩타이드 서열을 각각 2개, 1개를 확보하였다. 각각의 펩타이드는 나노몰(nanomoloar) 수준의 결합 친화도를 확인 하였고, 비브리오 파라해모라이티쿠스(vibrio parahaemolyticus)를 이용하여 결합 특이성(Specificity)을 규명하였다. 또한, 다중 인식 펩타이드 고분자를 제작하여 10배 높은 결합 친화성을 보였고 100 CFU/mL이라는 검출 한계점을 확인하였다. 이를 통해 현행 항체 기반으로 하는 진단법을 대체할 수 있는 차세대 식중독균 살모넬라 진단 탐침자를 선보인다. 현존하는 식중독균 검출 기술은 많은 시간이 소요될 뿐만 아니라 항체를 기반으로 하는 진단법은 항체의 안정성이 진단 시스템에 영향을 미치기 때문에 항체를 대신할 수 있는 진단 탐침자가 필요하다. 따라서 본 발명은 펩타이드와 다중 인식 펩타이드 고분자를 제작하여 극미량의 식중독균을 검출함으로써 식중독 예방이 가능할 것이다.; Salmonella enteritidis and Salmonella typhimurium are the most common and severe food-borne pathogens responsible for causing salmonellosis in humans and animals. Salmonella is a genus of rod-shaped, Gram-negative bacteria. Salmonella infections can be fatal if not detected and treated at early stages. The development of ultrasensitive detection system is the first critical step in controlling this salmonella disease. We have screened novel diverse peptides displaying on M13 bacteriophage that bind to salmonella typhimurium, salmonella enteritidis with high affinity from a phage display. Alternative ligands such as peptides can be used for pathogen capture and detection and offer advantages over antibodies, including reduced cost, ease of production and modification, and improved stability. In addition, we make a versatile polyvalent directed peptide polymer(PDPP) to overcome limitation of peptide binding affinity. To accomplish this, we used phage display technique to identify peptide to be used as detection probes that can specifically bind to S. enteritidis and S. typhimurium. We identified two peptide for detection of the S. typhimurium and one peptide for detection S. enteritidis through each pahge display rounds. Each peptides demonstrated higher binding affinities and the dissociation constants (Kd) were found to be in the range of nanomolar levels(SEBP : 205± 70.7, STBP1 : 466±45.3, STBP2 : 388±44.2). Peptide specificity was performed using the gram-negative bacteria vibrio parahaemolyticus and was found to be much higher towards S. enteritidis and S. typhimurium. Single peptide have several limitation of binding affinity and sensitivity. To address this limitation, we conjugated onto polyvalent directed peptide polymer, which led to 10-fold increase in binding affinity compared to the single peptide (SEB-PDPP : 15.4± 1.85nM, STB-PDPP1 : 35.7±10.4nM, STB-PDPP2 : 37.9±5.48nM). The concept of polyvalent interactions can be explained as simultaneous binding of multiple ligands on one entity to multiple receptors on another. The polyvalent affinity is significantly higher than that for the binding of a single ligand to a single receptor. These interactions effect the high binding affinity of PDPP compared to single peptide. This study reports the identification of higher affinity and specificity peptide(12mer), which may be useful as capture and detection probes in biosensor detection system for salmonellosis. In addition, we used polyvalent directed peptide polymer(PDPP), and showed increased binding affinity and fluorescence intensity. This probe can detect 100 CFU/ml of S. enteritidis and S. typhimurium by fluorescence signal.