【作者】 杨洁；

【导师】 李国才； 郭停停；

【作者基本信息】 扬州大学 ， 病原生物学， 2023， 硕士

【摘要】 鲍曼不动杆菌(Acinetobacter baumannii,A.baumannii)是导致各种院内感染的重要病原菌,CRISPR-Cas系统是细菌的适应性免疫系统,与细菌的耐药和毒力密切相关。本课题通过构建I-Fa型cas3基因缺失回补株研究I-Fa型CRISPR-Cas系统对鲍曼不动杆菌ATCC19606的毒力及调控机制,然后全基因组测序分析I-Fb型CRISPR-Cas系统阳性菌株AB43,进一步分析不同CRISPR-Cas系统对毒力的调控差异,最后,为从毒力抑制角度探究耐药细菌感染治疗策略,筛选出能够减少鲍曼不动杆菌生物膜形成的毒力因子抑制剂桑黄酮,其与粘菌素联合使用可有效恢复耐药鲍曼不动杆菌的敏感性。一、I-Fa型cas3基因上调鲍曼不动杆菌ATCC19606的毒力机制探究课题组前期利用I-Fa型CRISPR-Cas系统阳性菌株ATCC19606构建了cas3基因敲除株,本研究构建了该缺失株的回补株(ATCC19606Δcas3/pcas3),并研究了 I-Fa型cas3基因对细菌毒力的调控机制。结果发现,cas3缺失株的生物膜形成能力显著降低,感染cas3缺失株的大蜡螟生存率显著提高,菌血症小鼠模型中感染缺失株的小鼠脏器载菌量显著降低,肺部炎症改变较轻微,血清中炎症因子水平也较低,提示I-Fa型cas3基因缺失能够显著降低鲍曼不动杆菌的毒力。分析其机制与cas3基因缺失后下调生物膜形成相关因子、外膜蛋白A等毒力因子有关,此外cas3还参与调控鲍曼不动杆菌的碳代谢和氧化磷酸化途径。二、全基因组分析探究鲍曼不动杆菌AB43株I-Fb型CRISPR-Cas系统与耐药性及毒力的关系课题组前期通过基因敲除和回补发现,鲍曼不动杆菌临床分离株AB43中的I-Fb型CRISPR-Cas系统可有效抑制细菌的毒力与耐药性。本研究分析了 AB43株的基因组特征,并探讨该菌株的CRISPR-Cas系统与耐药和毒力之间的关系。使用Illumina和PacBio测序对AB43菌株进行全基因组测序(WGS),结果发现其完整基因组由3854806 bp的染色体和104309 bp的质粒组成,所含CRISPR-Cas系统的具体特征主要包括:(1)AB43株携带完整的I-Fb型CRISPR-Cas系统;(2)CRISPR的105个间隔序列均未发现可与NCBI数据库中的抗生素耐药基因相匹配,且AB43株中抗生素耐药基因的数量不低于无CRISPR-Cas系统的菌株,提示CRISPR-Cas系统可能通过内源基因调控抑制耐药基因表达来使细菌对抗生素敏感;(3)105个CRISPR间隔序列中共有28个匹配噬菌体基因组数据库和质粒数据库中的基因,其中间隔序列20、21靶向毒力蛋白VirB4,可能与AB43的低毒力表型相关;(4)同源性分析证实AB43中的Cas1和Cas3蛋白与同亚型的Cas蛋白同源性较高;不同亚型的Cas蛋白序列差异较大,这可能是导致I-Fa和I-Fb型CRISPR-Cas系统对毒力调控差异的主要原因。三、鲍曼不动杆菌毒力因子抑制剂的筛选耐药鲍曼不动杆菌感染已成为世界范围内亟需解决的公共卫生问题,急需研究人员开发新的防治策略。联合用药是目前临床常用的简便、经济的治疗方法,以毒力因子作为作用靶点是一种新型的研究思路。本研究中我们筛查了多种潜在联合药物,发现桑黄酮能有效抑制毒力基因的表达,显著降低鲍曼不动杆菌毒力相关的表面运动能力和生物膜形成能力,但是不影响鲍曼不动杆菌的正常生长。为发掘其抗菌潜力,使用桑黄酮与多种抗生素联用,发现桑黄酮与粘菌素联用具有很好的抗菌活性,恢复耐药菌株ATCC19606R对粘菌素的敏感性,并经体外实验初步证明其安全性较强。为揭示其作用机制,检测了药物作用下ATCC19606R生物膜形成能力、细胞膜完整性和外膜渗透性、膜电位、ROS和ATP水平,结果显示桑黄酮协同粘菌素通过破坏细胞膜完整性,降低细胞内ATP水平等来发挥抗菌作用。因此,桑黄酮对鲍曼不动杆菌感染的治疗具有很大的应用价值。更多还原

【Abstract】 Acinetobacter baumannii(A.baumannii)is an important pathogen causing various nosocomial infections.CRISPR-Cas system is the adaptive immune system of bacteria,which is closely related to the drug resistance and virulence of bacteria.In this study,we studied the virulence and regulatory mechanism of type I-Fa CRISPR-Cas system against A.baumannii strain ATCC19606 by constructing a I-Fa cas3 gene deletion mutant and complemented strain,then analyzed A.baumannii strain AB43 containing type I-Fb CRISPR-Cas system by whole genome sequencing to further analyze the differences in the regulation of virulence by different type CRISPR-Cas systems.Finally,in order to explore the treatment strategy for drug-resistant bacterial infection from the perspective of virulence inhibition,we screened out the virulence factor inhibitor,mulberrin,which can reduce the formation of biofilm of A.baumannii.The combination of mulberrin and colistin can effectively restore the antibiotic sensitivity of A.baumannii.1.Mechanism of type I-Fa cas3 gene up-regulating virulence of A.baumannii ATCC19606Our research group constructed the cas3 gene knockout strain using the type I-Fa CRISPR-Cas system positive strain ATCC19606.In this study,we constructed a complemented strain(ATCC19606Δcas3/pcas3)with the deletion mutant and studied the regulatory mechanism of type I-Fa cas3 gene on bacterial virulence.The results showed that deletion of cas3(type I-Fa)gene could significantly reduce the biofilm formation ability,virulence and pathogenicity to mice,the organ bacterial load of mice infected with cas3 deletion strain was significantly reduced;the lung inflammation was slightly changed;and the serum cytokine level was also low.We analyzed that the mechanism was related to the downregulation of virulence factors such as biofilm formation related factors and outer membrane protein A(ompA)by deletion of cas3 gene.In addition,cas3 was also involved in the regulation of carbon metabolism and oxidative phosphorylation pathway of A.baumannii.2.Whole-Genome Analysis of A.baumannii Strain AB43 Containing a Type Ⅰ-Fb CRISPR-Cas System:Insights into the Relationship with Drug Resistance and virulenceOur group’s preliminary study has found that type I-Fb CRISPR-Cas system in clinical isolate A.baumannii AB43 can effectively inhibit the virulence and drug resistance of bacteria.This study aimed to analyze the genomic characteristics of the A.baumannii strain AB43 containing type I-Fb CRISPR-Cas system,and to investigate the relationship between the CRISPR-Cas system and antibiotic resistance and virulence in this strain.The wholegenome sequencing(WGS)of the AB43 strain was performed using Illumina and PacBio sequencing.The complete genome of AB43 consisted of a 3854806 bp chromosome and a 104309 bp plasmid.The specific characteristics of the CRISPR-Cas system in AB43 are described as follows:(1)The strain AB43 carries a complete type I-Fb CRISPR-Cas system;(2)None of the CRISPR spacers in A.baumannii AB43 were matched with antimicrobial resistance genes in the NCBI database.In addition,we found that the number of antibiotic resistance genes in AB43 was not lower than in the "no CRISPR-Cas system" strain.It suggested that CRISPR-Cas system may inhibit drug-resistance gene expression via endogenous gene regulation;(3)A total of 28 of 105 A.baumannii AB43 CRISPR spacers matched genes in the bacteriophage genome database and the plasmid database,implying that the CRISPR-Cas system in AB43 provides immunity against invasive bacteriophage and plasmids;the spacer 20 and 21 targeted the virulence protein VirB4,which might be related to the low virulence phenotype of AB43;(4)Homology analysis confirmed that Casl and Cas3 in AB43 share high sequence similarity with the same subtype Cas ptotein,the sequence differences of Cas proteins among different subtypes may be the main reason for the differences in the virulence regulation of the I-Fa and I-Fb-type CRISPR-Cas systems.3.Screening of virulence factor inhibitors of A.baumanniiA.baumannii infection has become a worldwide problem because of A.baumannii were resistant to a variety of antibiotics,it is urgent to develop new therapeutic methods.Combined medication is a simple and economic treatment currently commonly used in clinical practice.Taking virulence factors as action targets is a novel treatment method.In the present study,we screened multiple potential combinations of drugs and we found that the mulberrin can effectively inhibit the expression of virulence genes,and inhibit the virulence-related surfaceassociated motility and biofilm formation ability of A.baumannii,but does not affect them normal growth.In order to explore its antibacterial potential,we used mulberrin combined with a variety of antibiotics,and found that the combination of mulberrin and colistin had good antibacterial activity,and restored the sensitivity of the drug-resistant strain ATCC19606R to colistin.We evaluated its safety in vitro experiment.To understand its mechanism,we tested the biofilm formation ability,cell membrane integrity and outer membrane permeability,membrane potential,ROS and ATP levels of ATCC19606R under the action of drugs.The results showed that mulberrin cooperated with colistin to exert antibacterial effect by destroying cell membrane integrity and reducing intracellular ATP level.Therefore,mulberrin has great application value in the treatment of A.baumannii infection.更多还原