类鼻疽伯克霍尔德菌（Bp）所致的类鼻疽误诊率和病死率极高，其致病机制不清，缺乏可靠的疫苗，被美国CDC归为B类生物恐怖菌，海南是我国类鼻疽病例数最多的地区。细菌非编码小RNA(sRNA)多数可结合分子伴侣蛋白Hfq与靶RNA互作，调控细菌环境耐受、毒力产生和代谢等生命活动，但在Bp中被发现和阐明生物学功能的sRNA极少。我们前期完成了Bp的全基因组测序，通过以Hfq蛋白为基础的免疫共沉淀技术和RNA-seq测序分析，发现并鉴定了9种新的sRNA。本项目拟构建sRNA缺失株和回复株，观察它们的相关表型和环境适应力的变化；通过细胞和动物试验来研究sRNA对毒力的影响；采用差异转录组和差异蛋白谱分析，寻找差异表达的基因，获得转录水平的sRNA调控元，并对基因功能进行分类。项目将阐明Bp新sRNA的重要生物学功能、致病和耐药机制，为抗Bp感染提供新的分子靶标，也为类鼻疽的防治提供新思路和理论依据。

Melioidosis results from infection with Burkholderia pseudomallei and is associated with high mortality and misdiagnosis rates. The organism can infect any organ system, although the lung is the most common organ affected. Hainan island is regarded as a main endemic area in China. Currently, our understanding of the pathogenesis about the pathogen is limited, there is no reliable vaccine for melioidosis, and therefore, B. pseudomallei is considered a potential bioterrorism threat and is listed as a category B agent by the U.S. Centers for Disease Control and Prevention. In most cases, small noncoding RNAs (sRNAs) require the RNA chaperone Hfq for optimal regulation by promoting sRNA-mRNA pairing and by stabilizing some sRNAs in vivo and act as important regulators in response to stress, virulence and metabolism in bacteria. However, the presence and roles of sRNAs in B. pseudomallei remain largely unclear. We used a method that took advantage of the conserved inherent sRNAs-binding capability of Hfq to enrich sRNAs from B. pseudomallei and validated 9 novel sRNAs. To study the function of these nine sRNAs on accommodated environment stress and virulence, we will use the λ-Red-mediated recombination method to construct a B. pseudomallei sRNAs deletion mutant and observe the effect of growth status under various environment stress, we perform a growth curve on wild type, sRNAs mutant and complemented strains. To determine ascertain these sRNAs are essential for virulence, invasion assays will be performed with the sRNAs mutant and wild-type strains both in vitro and vivo. To further explore the specific mechanism of sRNAs, differential target mRNA and protein regulated by the sRNAs mutant to the wild-type will be identified by the microarray and proteomics technology from mRNA and protein levels and be classified by biological function. The study will provide pathogenicity mechanism and function of sRNAs for further research, and find a new strategy for melioidosis prevention and cure.