鲍曼不动杆菌AbaI/AbaR群体感应系统对其毒力及宿主反应的调控作用研究

Acinetobacter baumannii is an important nosocomial pathogen which usually cause the clinical infection difficult to control. The research about its resistance has been widely reported, however, the regulatory mechanisms of virulence and host response are still poorly understood. The current studies show that quorum sensing (QS) system plays an important role in the regulation of virulence and pathogenicity of bacteria, also mediates the crosstalk between bacteria and host cells. The presence of quorum sensing systems in A. baumannii was recently confirmed. The mechanism of its quorum sensing is mediated by the AbaI /AbaR two-component system. However, the regulation effect of QS system on virulence and host response has not been clarified. In this study, molecular biology techniques will be used to construct deficient and retro-complement strains of abaI and abaR genes which encode the key molecules of QS system of A.baumannii. The impact of QS system on bacterial virulence phenotypes and the host cells response will be determined. On the basis of this, RNA-seq transcriptome analysis techniques will be used to compare the gene expressions of the wild-type strain, mutant strain and infected host cells. Finally, the omics data and experimental data will be integrated and global gene regulatory networks of Acinetobacter baumannii QS system will be constructed in order to clarify the interactive regulation effects of Acinetobacter baumannii QS system on its virulence and host response. This study will lay the foundation for further application of QS system on the control of Acinetobacter baumannii infections.

鲍曼不动杆菌是院内感染的重要致病菌之一，常引起难以控制的临床感染。以往研究主要关注其耐药机制，致病性研究非常有限，毒力及宿主反应调控机制尚不明晰。群体感应（Quorum Sensing，QS）系统在细菌毒力调控和致病过程中发挥重要作用,并介导细菌与宿主细胞的交互对话。由AbaI/AbaR二组分系统所介导的鲍曼不动杆菌QS系统新近发现，其是否参与细菌毒力及宿主反应调控尚不明确。本项目拟利用分子生物学技术构建abaI及abaR基因突变株和回补株，检测其对鲍曼不动杆菌关键毒力因子表型及对宿主细胞反应的调控作用，在此基础上，采用RNA-seq 技术从整体水平上对野生株、突变株以及感染的宿主细胞进行比较转录组分析，最终整合组学及实验数据，构建鲍曼不动杆菌QS系统的全局性基因调控网络，明晰QS系统对鲍曼不动杆菌毒力和宿主的交互调控作用，为进一步应用QS系统控制鲍曼不动杆菌感染奠定基础。

多重耐药甚至泛耐药鲍曼不动杆菌是目前我国乃至世界范围内重要的“超级细菌”之一，其导致的院内感染的爆发流行以及临床抗感染治疗失败给人类健康造成极大威胁。群体感应（Quorum sensing，QS）是细菌通过感知群体密度来协调自身群体行为的一种机制，参与包括细菌毒力和致病过程在内的多种生物学功能的调节。目前关于鲍曼不动杆菌AbaI/AbaR群体感应系统对细菌毒力和耐药的调控作用和机制尚不明晰。本研究从院内感染鲍曼不动杆菌临床分离株入手，分析QS系统在鲍曼不动杆菌临床株中的存在状况，探究其与细菌毒力和耐药的关系。构建abaI及abaR基因缺失突变株，检测其对鲍曼不动杆菌耐药、关键毒力因子表型及宿主细胞反应的影响。在此基础上，采用RNA-seq 技术从整体水平上对野生株、突变株以及感染的宿主细胞进行比较转录组分析，筛选可能受QS系统调控的靶基因，揭示鲍曼不动杆菌QS系统的全局性调控作用。结果表明：临床菌株广泛携带AbaI/AbaR群体感应系统，且该系统与细菌耐药、细菌对A549细胞的侵袭能力及大蜡螟感染毒性显著相关。基于QS基因缺失突变株的研究结果显示，与野生株相比，ΔabaI、ΔabaR及ΔabaIR缺失突变株表现出对某些抗菌药物的敏感性增加和显著的表面运动缺陷，感染的A549细胞早期凋亡比率显著下降；与野生株相比，ΔabaI和ΔabaIR突变株生物膜形成增加，对血清杀灭作用更敏感，对大蜡螟和小鼠的毒力作用显著下降；而ΔabaR突变株则表现出更强的血清抵抗力以及对大蜡螟和小鼠的毒力作用。RNA-seq结果表明，在ΔabaI、ΔabaR和ΔabaIR突变体中，群体感应高丝氨酸内酯合酶基因（abaI-AUO97-RS06645）和附近位点（AUO97-RS06600-06630）基因转录显著降低。在ΔabaR突变体中，脂类运输和代谢、苯乙酸（PAA）分解代谢途径和细菌分泌蛋白PKF表达上调。在ΔabaI突变体中，14个参与呼吸链中NADH脱氢酶的NADH醌氧化还原酶亚基表达下调，CsuA/BABCDE chaperone-usher菌毛组装系统高表达。以AHL合成酶基因abaI为靶点是一种有效的毒力减弱策略。通过本研究进一步明晰了AbaI/AbaR群体感应系统对鲍曼不动杆菌耐药和毒力的调控作用，为进一步应用QS系统控制鲍曼不动杆菌感染奠定了基础。