| 其他摘要 | Antimicrobial peptides (AMPs) are a group of molecules with potent antimicrobial activity and other activities such as iron regulation, immunity regulation, protease inhibition, free radicals clearance and so on. More than 1,200 AMPs have been identified from nearly all groups of organisms, including bacteria, fungi, plants and animals. AMPs are considered as the promising potent antimicrobial agents in the era of antibiotic resistance and many of these are conducting the preclinical or clinical trials. However, some studies suggested that the therapeutic use of AMPs might result in the resistance of microorganisms. In this study, we designed a selection protocol, where Micrococcus luteus, Bacillus subtilis and Candida albicans were grown for several hundred generations in media containing sole AMPs or combined AMPs identified from Odorrana grahami. The used AMPs are Brevinin 2E-OG1、Nigrocin-OG4 and Palustrin-OG1. And then, we studied the resistance mechanisms using protease assay and proteomics. We inoculated the selected strains in the culture media with low concentrations of AMPs, sole or combined, and transferred them daily. We increased the concentrations of AMPs every ten transfers cautiously. After 80 transfers, all strains of M. luteus remained sensitive to Palustrin OG-1. All the other tested strains, including those grown in combined AMPs, were resisted to the AMPs used in the selection protocol. However, the combinations of Palustrin-OG1 with Brevinin 2E-OG1 or Nigrocin-OG4 could decrease the resistance to some extent. After five transfers in the medium without AMPs, the tested strains could maintain the resistance, which means the induced resistance by AMPs can transmit to further generations stably. The secretion of extracellular proteases is one of the mechanisms that microorganism resist to AMPs. So we studied the extracellular proteases activity through casein degradation assay and MIC assay of AMPs pretreated by culture supernatant. The results indicated the extracellular proteases might play important roles in the resistance of B. subtilis and M. luteus, but not in C. albicans. Furthermore, we studied the resistance mechanism of B.subtilis to AMPs using proteomic technology and identified five proteins with differential expression. The up-regulated proteins are yraA with unknown function, Tpx belonging to Thiol peroxidase family and pdhD which is an indentified dihydrolipoamide dehydrogenase. The down-regulated proteins are spore coat-associated protein-cotN/tasA and Glyceraldehyde-3-phosphate dehydrogenase 1-gapA. Tpx and yraA are regulated by the Spx which is regulated by sigma-M factor. GapA and cotN/tasA can be down-regulated by increasing of free amino acids that may be produced from the degradation of peptides by yraA. We suggested that the sigma-M factor play an important role in the mechanisms of B. subtilis resist to AMPs. In conclusion, the combined application of AMPs can postpone the resistance of microorganism. Our studies provided a new thread to the therapeutic use of AMPs. At the same time, the research on resistance mechanisms can be a basis of the coming study. In addition, we designed a systematic nomenclature of antimicrobial peptides and established a database to exhibit it. |
修改评论