抗菌肽cecropin B对副猪嗜血杆菌诱导耐受机制的研究

Antimicrobial peptides (AMPs) represent an abundant and diverse group of molecules that are produced by many tissues and cell types from bacteria to mammals. Their general properties such as amphipathicity and cationicity allow them to directly ineract with cell membranes, resulting in microbial death. The differences in membrane structure between microbes and higher eukaryotes help AMPs in selectively targeting the microbial membranes, thereby making it less toxic for therapeutic use, and AMPs are also effective against multidrug resistant bacteria. The elucidation of the characteristic of AMPs is essential to allow these substances to develop a new generation of antibiotics to control infections of both people and animals. Cecropin B (CB) is a cationic α-helical antimicrobial peptide originally isolated from the giant silk moth, Hyalophora cecropia. CB displays antimicrobial effects through disruption of the anionic cell membranes of gram-negative bacteria. Previous work by our laboratory demonstrated that CB shows good activity against Haemophilus parasuis. As the development and spread of antimicrobial resistance in pathogenic bacteria, the potential for H. parasuis SH0165 to develop resistance to cecropin B was addressed in this study. CB was synthesized by solid-phase method applying Fmoc (9-fluorenylmethyloxycarbonyl) active ester chemistry. To generate CB-resistant H. parasuis, a step pressure method was used. Isolated colonies of H. parasuis SH0165 were inoculated into TSB, containing CB within the concentration range of 0.5-2μg/ml (0.25MIC-1MIC). When microbial population growing to log phase, the microbial culture was used to inoculate TSB containing higher concentration of CB. Like this, after several series, generated CB-resistant subpopulation. Measuring bacterial growth was performed and a slower growth rate of CB-resistant strain was observed.Transmission electron microscopy will be used to investigate the morphological difference between CB-resistant subpopulations and control bacteria.A DNA microarray will be used to investigate the changes in the gene expression levels of CB-resistant subpopulations compared to SH0165. Gene knockout technique and prokaryotic expression will be used in gene function verification, providing useful candidates for investigating the specific mechanisms of antimicrobial peptide resistance in bacteria.

抗菌肽是机体中重要的免疫分子，具有抗菌和抗病毒、抗真菌、抗寄生虫及抗肿瘤等生物活性，是一类具有广阔前景的、有望作为候选药物的生物分子。然而，细菌能否对抗菌肽产生耐受也必然成为人们关注的问题。Cecropin B是从罗普斯蚕蛾的免疫血淋巴中发现的一种抗菌肽，初步研究发现，cecropin B对副猪嗜血杆菌具有较强的抗菌活性。但是，副猪嗜血杆菌在cecropin B的频繁使用下能否产生耐受及产生耐受的分子机制还未见报道。本项目拟采用一系列浓度梯度的cecropin B对副猪嗜血杆菌临床分离株SH0165进行诱导，筛选耐受菌株。通过基因芯片技术获得耐受菌株基因组转录水平的差异表达谱，基因敲除和过表达差异显著的疑似耐受基因，初步探索副猪嗜血杆菌对cecropin B耐受的分子机制。为cecropin B在临床中合理使用提供指导依据，并为进一步研究细菌对抗菌肽的耐受机制奠定理论基础。

随着大量的抗生素在临床上广泛的应用，病原菌对其产生耐药性，尤其是产生多重耐药。开发新型的抗菌药物是人类解决上述问题的有效途径。抗菌肽是生物机体内产生的一种具有生物活性的小分子多肽，这类活性多肽多数具有强碱性、热稳定性以及广谱抗菌、抗病毒等特点，是开发新型抗菌药物的最有潜力的方向。Cecropin B是从罗普斯蚕蛾中发现的一种抗菌肽，随着cecropin B的抗菌活性逐渐被人们重视，细菌对其耐药性的研究也日渐重要。本团队的前期研究发现，副猪嗜血杆菌对抗菌肽cecropin B非常敏感，其最小抑菌浓度可低至2ug/ml。本项目采用一系列浓度的cecropin B对副猪嗜血杆菌临床分离株进行诱导，筛选耐药菌株，通过基因芯片获得转录水平差异表达谱，并构建基因缺失株考察基因的耐药功能。三年来，课题组围绕计划书的总体目标，积极地开展工作，采用基因芯片技术、RT-PCR、western-blot、免疫共沉淀、基因敲除等分子生物学技术及生物信息学平台，证实了基因HAPS_2096在副猪嗜血杆菌对cecropin B耐药中的确起到了重要作用。HAPS_2096的互补株对cecropin B的敏感性比缺失株显著降低。本项目的完成，不仅可以更深入地了解副猪嗜血杆菌对抗菌肽耐受的分子机制，而且将指导抗菌肽的临床应用提供理论参考。