Cyclic dipeptides and cyclic dipeptide synthetase of Lactobacillus plantarum LBP-K10

Abstract

Lactic acid bacteria (LAB) have been recognized to be a probiotic microorganism for human health. Cyclic dipeptides (CDPs) produced by LAB are important for their antibiotic activities. CDPs were a group of molecules with several kinds of diketopiperazine structure and have served as attractive scaffolds for drug design. However, the entire pool of CDPs from a given lactic acid bacterial culture is not fully understood. The present study identified the CDPs produced by Lactobacillus plantarum LBP-K10, which was the most potent antimicrobial isolates from Korean kimchi. Totally, seven CDPs were identified through gas chromatography-mass spectroscopy. Additionally this study provided a new method for purifying a massive amount of the complete set of CDPs from culture filtrates (CFs) of Lb. plantarum LBP-K10 by using the anion exchange resin Amberlite IRA-67 or Purolite A420S. The set of all CDPs showed remarkable potency with bioactivities against multidrug-resistant bacteria, pathogenic fungi and influenza virus compared with the activities of single CDP. Recently, studies revealed lactic acid bacteria or its products might capable of inhibiting cancer progression by several mechanisms. However, most of studies focus on gastrointestinal (GI) tract related cancer, especially colon cancer. The effect of lactic acid bacteria or its related products on non-GI tract cancer was rarely examined. Since studies proved the effectively absorbing of CDPs, the cancer prevention effect lactic acid bacteria on a kind of non-GI tract cancer, breast cancer was investigated. A special methylene chloride extracted fraction (MC-K10) was identified, which mainly contained several CDPs from Lb. plantarum LBP-K10 culture filtrates (CF-K10). In this study, it was firstly proved that oral applying CF-K10 inhibited the progression of breast cancer growth in animal model. We further discovered that CF-K10 and MC-K10 might induce apoptosis in MDA-MB-231 cells. Moreover, it was demonstrated MC-K10 and cyclo(Phe-Pro) can impair the stemness and self-renew of cancer stem cells. These studies provide a new insight for the probiotic actives of LAB and its products, as well as give a hint for developing breast cancer prevention strategy. The importance and benefit of the CDPs on human heath encouraged us to investigate the cyclic dipeptide biosynthesis process. By ninhydrin activity staining, it was confirmed bioactive cyclic dipeptides synthesized in by Lb. plantarum LBP-K10 through cyclic dipeptide synthase (CDPS) dependent pathway. Then this CDPS were purified and characterized by 2D LC/MS analysis as a 26.1 kDa novel protein (accession number: gi311821850). The 690 bp gene sequence of this 26.1 kDa protein was also obtained from NCBI genome database. Interestingly, after transfected this gene into E. coli, the content of several proline base cyclic dipeptides was increased. When subjected to in vitro reaction conditions, CDPS helped to generate several cyclic dipeptides. To better understand that the mechanism of cyclic dipeptides synthesis in molecular level, X-ray analysis of CDPS was performed. CDPS was composed as a tetramer structure with 4 identical subunit showed in the ribbon model. This study for the first time identified a novel CDPS and defined in vitro reaction conditions based on CDPs for generating CDPs. This study will help to understand cyclic dipeptides synthesis mechanism and producing nature antibiotic cyclic dipeptides in vitro through biosynthesis reactor. Above of all, these studies identified the entire CDPs profile in MC extract fraction of Lb. plantarum LPB-K10, proved the cancer prevention effect of LAB and its produced CDPs, and for the first identified a novel CDPS. This study provided new insight on the probiotic LAB and benefit for understanding CDPs biosynthesis mechanisms.

Key words: Cyclic dipeptides

Cyclic dipeptide synthase

Antibiotic activity

Lactobacillus plantarum

Breast cancer

Cancer prevention

Cancer stem cells

Multidrug-resistant bacteria, Pathogenic fungi, Influenza A virus

Cyclo(Phe-Pro)