首页|基于CRISPR/Cas9技术构建鲍曼不动杆菌lpxC缺失株及其表型研究

基于CRISPR/Cas9技术构建鲍曼不动杆菌lpxC缺失株及其表型研究

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脂多糖(lipopolysaccharides，LPS)是革兰阴性菌细胞膜外膜重要组成成分，与多数革兰阴性菌不同，鲍曼不动杆菌在LPS缺失后仍可存活并获得对多黏菌素的耐药性。有关LPS缺失鲍曼不动杆菌的研究多是针对多黏菌素诱导产生的LPS缺失株，背景复杂且不稳定。为深入研究LPS缺失介导的多黏菌素耐药鲍曼不动杆菌，本研究通过双质粒CRISPR/Cas9系统敲除鲍曼不动杆菌ATCC 19606的lpxC基因，构建稳定且背景清晰的LPS缺失株，并对缺失株的形态、生长速率、抗生素的敏感性、毒力、膜通透性和膜电势进行分析。动物实验经中国医学科学院、北京协和医学院医药生物技术研究所实验动物管理与动物福利委员会批准(批准号:IMB-20240119D9)。结果显示，鲍曼不动杆菌ATCC 19606的lpxC基因被成功敲除，lpxC缺失后菌株失去外膜LPS，形态由杆状变为球状，细菌生长速率变慢，膜通透性升高，膜电势降低，对β-内酰胺类、喹诺酮类、氨基糖苷类、大环内酯类、糖肽类抗生素的敏感性增强，并且体内毒力大幅下降。lpxC缺失引起鲍曼不动杆菌的膜稳态、适应性发生明显改变，了解LPS缺失介导的多黏菌素耐药鲍曼不动杆菌的特征变化有利于探究鲍曼不动杆菌耐药机制、寻找治疗新策略。
Construction and characterization of lpxC deletion strain based on CRISPR/Cas9 in Acinetobacter baumannii
Lipopolysaccharides(LPS)are major outer membrane components of Gram-negative bacteria.Unlike most Gram-negative bacteria,Acinetobacter baumannii can still survive and acquire polymyxin resistance after complete loss of LPS.Previous studies of LPS-deficient Acinetobacter baumannii mostly focused on LPS-deficient strains induced by polymyxin,whose background was complex and unstable.To investigate LPS loss-mediated polymyxin resistant-Acinetobacter baumannii,this study constructed a stable and clear background LPS-deficient strain by knocking out lpxC gene in Acinetobacter baumannii ATCC 19606 with CIRSPR/Cas9,and then studied the phenotypic changes of the lpxC-deficient strain including morphology,growth rate,antibiotic susceptibility,virulence,membrane permeability,and membrane potential.Animal experiments were approved by the Animal Care and Welfare Committee Institute of Medicinal Biotechnology,CAMS and PUMC(approval number:IMB-20240119D9).The results indicated that the lpxC gene of Acinetobacter baumannii ATCC 19606 was successfully knocked out.After losing the lpxC,the strain underwent the morphological change from rod-shaped to spherical.Furthermore,it leads to reduced growth rate,enhanced membrane permeability,decreased membrane potential,lower virulence,and increased antibiotic susceptibility to β-lactams,quinolones,aminoglycosides,macrolides,glycopeptides.The lpxC deletion results in significant changes in membrane homeostasis and adaptability of Acinetobacter baumannii.Understanding the phenotypic changes of colistin-resistant Acinetobacter baumannii mediated by LPS loss is useful for exploring the resistance mechanism of Acinetobacter baumannii and developing new therapeutic strategies.

Acinetobacter baumanniigene editingCRISPR/Cas9 systemlpxC genepolymyxinresistancelipopolysaccharide

孙宗倜、张友文、李海滨、王秀坤、于洁、解晋茹、庞蓬勃、胡辛欣、聂彤颖、卢曦、庞晶、侯磊、杨信怡、李聪然、孙琅、游雪甫

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中国医学科学院、北京协和医学院医药生物技术研究所,抗感染药物研究北京市重点实验室,北京 100050

中国医学科学院病原微生物菌(毒)种保藏中心药用微生物相关菌(毒)种保藏分中心,北京 100050

中国医学科学院、北京协和医学院医药生物技术研究所实验动物中心,北京 100050

鲍曼不动杆菌 基因编辑 CRISPR/Cas9系统 lpxC基因 多黏菌素 耐药 脂多糖

国家自然科学基金国家自然科学基金科技部国家科技资源共享服务平台国家病原微生物资源库项目中国医科院医学与健康科技创新工程项目

3214100382104249NPRC-322022-12M-2-002

2024

10.16438/j.0513-4870.2024-0044

药学学报
中国药学会 中国医学科学院药物研究所

药学学报

CSTPCD北大核心
影响因子：1.274
ISSN：0513-4870
年,卷(期)：2024.59(5)