act通过对荚膜多糖乙酰化修饰增强ST11-KL64型碳青霉烯耐药肺炎克雷伯菌毒力的机制研究

碳青霉烯耐药肺炎克雷伯菌（CRKP）感染形势严峻，近年ST11-KL64型CRKP成为流行克隆且毒力增强，但毒力增强机制尚不明确，亟待阐明。申请人前期通过比较基因组学、基因敲除和小鼠试验发现乙酰转移酶基因act是ST11-KL64型CRKP重要毒力因子；乙酰化检测发现act可对荚膜多糖（CPS）乙酰化修饰，但修饰后分子结构不明；细菌刺激试验提示CPS乙酰化后可增强巨噬细胞促炎因子分泌；分子模拟提示CPS乙酰基与巨噬细胞TLR4共价结合。据此提出act通过对CPS乙酰化修饰增强ST11-KL64型CRKP毒力的假说。我们拟通过酶活测定、核磁共振等，揭示乙酰化修饰CPS分子结构及抗原性改变；通过CPS刺激巨噬细胞等试验，明确CPS乙酰化对巨噬细胞分泌促炎因子影响及TLR4在其中的作用；通过分子流行病学分析act在CRKP中流行特点。研究成果将有助于CRKP感染防治，为临床抗感染治疗提供新思路。

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are critical in clinical settings. Recent studies showed that the CRKP belonging to ST11-KL64 have become a major clone in China and their virulence has enhanced. However, the mechanisms behind the virulence enhancement are poorly understood and therefore need to be illuminated. In preliminary studies, we identified an acyltransferase encoding gene act, which plays important roles in virulence enhancement of ST11-KL64 CRKP, by comparative genomics, gene knockouts and mouse lethality studies. By acetylation analysis, we found that act could acetylate the capsular polysaccharide (CPS) of ST11-KL64 CRKP, however, the molecular structure of acetylated CPS remained to be investigated. Furthermore, experiments from co-culture of bacteria and macrophages indicated that acetylated CPS could enhance the secretion of proinflammatory factors in macrophages and the molecular modeling showed that the acetyl group in CPS can covalently bound to TLR4. Taken together, we hypothesized that act enhances the virulence of ST11-KL64 CRKP by acetylating the CPS. To validate our hypothesis, we will determine the molecular structure and antigenic changes of acetylated CPS by enzymatic experiments and NMR, the effects of acetylated CPS and TLR4 in the secretion of proinflammatory factors of macrophages by stimulation experiments, and epidemiology features of act in CRKP by epidemiology analyses. Our findings will facilitate and provide new insights in the treatment of CRKP infections in clinical practices.