Lakshmaiah Narayana Jayaram, Golla Radha, Mishra Biswajit, Wang Xiuqing, Lushnikova Tamara, Zhang Yingxia, Verma Atul, Kumar Vikas, Xie Jingwei, Wang Guangshun
Department of Pathology and Microbiology, College of Medicine, University of Nebraska Medical Center, 985900 Nebraska Medical Center, Omaha, Nebraska 68198-5900, United States.
Mass Spectrometry and Proteomics Core Facility, University of Nebraska Medical Center, Omaha, Nebraska 68198, United States.
ACS Infect Dis. 2021 Jun 11;7(6):1795-1808. doi: 10.1021/acsinfecdis.1c00101. Epub 2021 Apr 23.
This study aims to push the frontier of the engineering of human cathelicidin LL-37, a critical antimicrobial innate immune peptide that wards off invading pathogens. By sequential truncation of the smallest antibacterial peptide (KR12) of LL-37 and conjugation with fatty acids, with varying chain lengths, a library of lipopeptides is generated. These peptides are subjected to antibacterial activity and hemolytic assays. Candidates (including both forms made of l- and d-amino acids) with the optimal cell selectivity are subsequently fed to the second layer of filters, including salts, pH, serum, and media. These practices lead to the identification of a miniature LL-37 like peptide (d-form) with selectivity, stability, and robust antimicrobial activity against both Gram-positive and negative bacteria. Proteomic studies reveal far fewer serum proteins that bind to the d-form than the l-form peptide. C10-KR8d targets bacterial membranes to become helical, making it difficult for bacteria to develop resistance in a multiple passage experiment. , C10-KR8d is able to reduce bacterial burden of methicillin-resistant (MRSA) USA300 LAC in neutropenic mice. In addition, this designer peptide prevents bacterial biofilm formation in a catheter-associated mouse model. Meanwhile, C10-KR8d also recruits cytokines to the vicinity of catheters to clear infection. Thus, based on the antimicrobial region of LL-37, this study succeeds in identifying the smallest anti-infective peptide C10-KR8d with both robust antimicrobial, antibiofilm, and immune modulation activities.
本研究旨在拓展人类杀菌肽LL-37工程的前沿领域，LL-37是一种关键的抗菌天然免疫肽，可抵御入侵的病原体。通过对LL-37最小抗菌肽（KR12）进行连续截短，并与不同链长的脂肪酸偶联，生成了一个脂肽文库。对这些肽进行抗菌活性和溶血试验。随后，将具有最佳细胞选择性的候选物（包括由l-和d-氨基酸制成的两种形式）置于第二层筛选条件下，包括盐、pH值、血清和培养基。这些操作导致鉴定出一种具有选择性、稳定性且对革兰氏阳性和阴性细菌均具有强大抗菌活性的微型LL-37样肽（d型）。蛋白质组学研究表明，与d型肽结合的血清蛋白比l型肽少得多。C10-KR8d靶向细菌膜并形成螺旋结构，使得细菌在多次传代实验中难以产生耐药性。此外，C10-KR8d能够降低中性粒细胞减少小鼠中耐甲氧西林金黄色葡萄球菌（MRSA）USA300 LAC的细菌载量。此外，这种设计肽可在导管相关小鼠模型中防止细菌生物膜形成。同时，C10-KR8d还能将细胞因子募集到导管附近以清除感染。因此，基于LL-37的抗菌区域，本研究成功鉴定出了具有强大抗菌、抗生物膜和免疫调节活性的最小抗感染肽C10-KR8d。
