【作者】 李敏；

【导师】 张炜；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2022， 博士

【摘要】 肺炎克雷伯菌(Klebsiella Pneumoniae,KP)属于肠杆菌科,可感染人和多种动物,对公共卫生和动物养殖业具有较大威胁。目前,通常使用抗生素来治疗肺炎克雷伯菌感染,抗生素的广泛使用导致了多重耐药菌的产生。在多重耐药菌,尤其是泛耐药菌的威胁下,噬菌体及其衍生物重新被重视,并应用于防控肺炎克雷伯菌感染。本研究从污水中分离到169株肺炎克雷伯菌噬菌体,建立了小型肺炎克雷伯菌噬菌体库,通过噬菌体基因组分析和基本生物学特性分析确定了噬菌体疗法的候选株,同时发现肺炎克雷伯菌具有荚膜型特异性;通过基因替换证实了肺炎克雷伯菌噬菌体的宿主特异性与TFP(tail fiberprotein,TFP)基因有关,而且进一步锁定了宿主谱决定区;通过基因缺失技术证明了肺炎克雷伯菌的荚膜是噬菌体P560的宿主受体。此外,发现了分别针对KL47和KL64型KP的荚膜多糖(capsular polysaccharides,CPS)有解聚活性的噬菌体源解聚酶,也证明了其在体内外的抗菌效果;解析了其中KL47型解聚酶P560dep的三维结构,鉴定了催化域以及关键催化氨基酸位点。本研究为利用合成生物学定制噬菌体和开发新型解聚酶提供了理论基础。1肺炎克雷伯菌噬菌体的分离鉴定及生物学特性分析收集肺炎克雷伯菌和分离噬菌体是进行噬菌体相关研究的前提。因此,本研究首先对收集的KP进行了肺炎克雷伯菌烈性噬菌体的分离鉴定、基本生物学特性分析及全基因组测序分析。从生活污水和医疗污水中共分离到169株KP噬菌体,选取其中8株进行了透射电镜观察,选取17株进行了噬菌体全基因组的提取、测序和分析,结果表明有5株噬菌体属于Przondovirus。噬菌体的宿主谱分析表明KP噬菌体具有荚膜型特异性。我们从烈性噬菌体中选取P560、P509和P545进行了基本生物学特性分析,为肺炎克雷伯菌噬菌体库提供了新的备选噬菌体株,为噬菌体疗法提供了具有良好特性的候选噬菌体。2噬菌体决定宿主谱关键蛋白的鉴定及改造噬菌体由于其特异性而作为一种很有前途的抗菌剂,但也由于其宿主范围窄而受到限制。为了揭示KP噬菌体宿主谱形成的分子基础,以更好的应用合成生物学重定制基因工程噬菌体,本研究利用传统的噬菌体同源重组技术,通过将噬菌体P510中编码TFP蛋白的基因(ORF38)分别与噬菌体P560中编码TFP蛋白的基因(ORF42或ORF43)替换,分别获得基因工程噬菌体RPA3和RPB1,RPA3和RPB1仅因改变了 TFP而完全改变了其宿主谱。基因工程噬菌体RPA3不仅对浮游态CRKP Kp30具有显著的抑菌作用,还对抑制生物被膜的形成和清除成熟生物被膜具有显著的效果。我们的研究揭示了 TFP蛋白是KP噬菌体宿主特异性形成的重要决定因素,并提示基因工程噬菌体可能是未来噬菌体治疗的有效途径。此外,我们研制的噬菌体鸡尾酒制剂JS-KPP3可有效抑制KL47型和KL64型CRKP,具有作为环境杀菌剂的潜力;随着JS-KPP3噬菌体鸡尾酒制剂配比的不断优化,有望广泛应用于养殖业和临床环境净化。3噬菌体耐受菌和宿主受体的相关研究目前报道的噬菌体的宿主受体有LPS、外膜蛋白、荚膜、鞭毛和脂类等。为了鉴定KP噬菌体的宿主受体和揭示KP对噬菌体的耐受机制,首先分离到30株对噬菌体P560耐受的Kp42变异株,通过PCR和测序分析,鉴定到wbap基因突变的耐受菌Kp42-P560R。然后对野生株Kp42和耐受菌株Kp42-P560R进行了全基因组测序及比较基因组学分析,未发现外膜蛋白有突变,而影响CPS的形成的wbap基因发生了插入突变,并引起翻译移码。为验证荚膜是噬菌体的宿主受体,我们构建了缺失株Kp42:△wbaP和互补株Kp42 pwbaP,发现wbaP基因影响荚膜的形成、而荚膜影响噬菌体P560对其的裂解性和吸附力。研究表明肺炎克雷伯菌Kp42的荚膜参与了噬菌体P560对其的吸附,是噬菌体P560侵染宿主Kp42必不可少的受体之一。细菌对噬菌体的耐受机制和宿主受体的研究为噬菌体疗法的应用奠定了理论基础。4重组解聚酶的表达、鉴定及抗菌研究噬菌体源的抗菌蛋白主要包括裂解酶、解聚酶、穿孔素、转运信号多肽和跨膜素等。我们预测到两个分别针对KL47型和KL64型肺炎克雷伯菌荚膜多糖的噬菌体源解聚酶P560dep和P510dep,进行了重组解聚酶的原核表达与纯化、酶活性分析以及特异性分析,评估了体内外的抗菌效果。结果表明重组解聚酶P560dep能够降解KL47型肺炎克雷伯菌表面的CPS,且P560dep的解聚谱与噬菌体P560的宿主谱相同;P510dep能够降解KL64型CPS,解聚谱与P510的宿主谱相同。解聚酶P560dep和P510dep均可显著抑制生物被膜的形成。体内试验表明,P560dep可预防和治疗耐碳青霉烯类肺炎克雷伯菌所致小鼠模型中的菌血症感染。在KL47 CRKP感染前后,腹腔注射单剂量的解聚酶(50 μg/只),可对90%-100%的小鼠有保护作用,可减轻小鼠肺和肝的病理变化。可见,解聚酶P560dep是一种有潜力的抗菌剂,可作为一种抗耐药菌治疗方案。5解聚酶P560dep结构与功能的研究目前关于解聚酶的抗菌研究很多,但关于解聚酶结构的研究却很少。为了揭示解聚酶结构与功能的关系,我们通过晶体衍射法和硒代甲硫氨酸法得到解聚酶P560dep的3D结构,其分辨率为1.6 ?;包括四个结构域:N端结构域、催化结构域、凝集素样(lectin-like)结构域和C端结构域。与其他解聚酶相比,P560dep具有一种新型的模块结构,即在其右旋平行β螺旋结构域内插入两个串联lectin-like结构域。对预测的6个候选催化域的18个氨基酸位点进行定点突变,结合酶活试验表明,共有4个区域和6个残基在其催化功能中起主要作用,其中区域5中的残基Asp298、Asp311和Glu314以及区域6中的残基Glu554在其催化功能中起主要作用。目前,这种单一尾丝蛋白中存在多个催化口袋的现象尚未见报道。lectin-like结构域中的疏水口袋可能促进与多糖的结合能力。高效解聚酶结构与功能的分析,为进一步设计抗肺炎克雷伯菌的广谱性酶提供了理论基础。总之,针对肺炎克雷伯菌耐药严重的问题,本研究建立了小型肺炎克雷伯菌噬菌体库,通过噬菌体基本生物学特性分析和基因组序列分析确定了噬菌体候选株;通过基因替换证实了肺炎克雷伯菌噬菌体的宿主特异性与TFP基因有关;通过噬菌体耐受菌分析和基因缺失分析证明了基于wbaP基因突变的噬菌体耐受机制,同时证明了肺炎克雷伯菌的荚膜是噬菌体P560的重要宿主受体。本研究也发现了分别针对KL47和KL64型荚膜的噬菌体源解聚酶,证明了其在体内外的抗菌效果;解析了 KL47型解聚酶P560dep的同源三聚体结构,鉴定了两个催化域的关键氨基酸催化位点。本研究为噬菌体应用提供了肺炎克雷伯菌噬菌体候选株,也为利用合成生物学定制噬菌体和开发新型解聚酶提供理论基础。更多还原

【Abstract】 Klebsiella Pneumoniae(KP)belongs to Enterobacteriaceae,which can infect human and many kinds of animals,posing a great threat to public health and animal breeding industry.Currently,antibiotics are commonly used to treat K.pneumoniae infections,and their widespread use has led to the development of multidrug-resistant bacteria.Under the threat of multidrug-resistant bacteria,especially pan-drug-resistant bacteria,bacteriophages and their derivatives have been reemphasized and applied to prevent and control K.pneumoniae infection.In this study,169 strains of K.pneumoniae phage were isolated from sewage,and a small library of K.pneumoniae phage was established.Candidate phages were identified by phage genome analysis and basic biological characteristics analysis.Meanwhile,K.pneumoniae phage was found to have capsular type specificity.Tail fiber protein(TFP)replacement confirmed that the host specificity of K.pneumoniae phage was related to the TFP gene,and further locked the host range determination region.Besides,the capsule of K.pneumoniae is one of the host receptors of phage P560.Phage-derived depolymerase was found to have polysaccharides-degrading activity against capsular polysaccharides(CPS)of KL47 and KL64 KP respectively,and its antibacterial effect was also proved in vitro and in vivo.Three-dimensional structure of KL47 depolymerase P560dep was analyzed,and the catalytic domain and key catalytic amino acid sites were identified.This study provides a theoretical basis for customizing phages with synthetic biology and developing novel depolymerases.1 Isolation and characterization of K.pneumoniae phageCollection of K.pneumoniae and phage isolation are the premise of phage related researches.Therefore,this study firstly carried out isolation and identification,basic biological characteristics analysis and whole genome sequencing analysis of K.pneumoniae lytic phages.A total of 169 strains of KP phages were isolated from domestic sewage and medical sewage,8 of which were observed by transmission electron microscopy,and 17 phage genomes of which were extracted,sequenced and analyzed.The results showed that 5 strains belonged to Przondovirus.Host range analysis of the phage showed that KP phage had KL-type specificity.We selected P560,P509 and P545 from the lytic phages for basic biological characteristics analysis,providing new candidate phages for the bank of K.pneumoniae phage and candidate phages with good characteristics for phage therapy.2 Identification and modification of host range-determining protein of phagePhages are a promising antibacterial agent because of their specificity,but they are also limited by their narrow host range.In order to reveal the molecular basis of KP phage host range formation and apply synthetic biology to recustomize genetically engineered phages,this study used traditional phage homologous recombination technology.Genetically engineered phages RPA3 and RPB1 were obtained by replacing the TFP genes(ORF42 and ORF43)in phage P560 with the TFP gene(ORF38)in phage P510,respectively.RPA3 and RPB1 completely obtained the host range of P510,only by changing TFP gene.Engineered phage RPA3 not only had significant bacteriostasis effect on planktic CRKP Kp30,but also had significant inhibitory effect on the formation of biofilm and the removal of mature biofilm.Our study revealed that TFP protein is an important determinant of KP phage host-specific formation and suggested that engineered phages may be an effective approach for phage therapy in the future.In addition,the phage cocktail JS-KPP3 developed by us can effectively inhibit pan-resistant KL47 and KL64 CRKP,which has the potential as an environmental antimicrobials.JS-KPP3 phage cocktail is expected to be widely used in breeding industry and clinical environment purification with the continuous optimization of the component of JS-KPP3 phage cocktail.3 Studies on phage-resistant bacteria and host receptorThe host receptors of phages included LPS,outer membrane proteins,capsules,flagella and lipids.In order to identify host receptors and reveal the mechanism of phage resistance,30 Kp42 mutants resistant to phage P560 were firstly isolated,and the wbaP mutants Kp42-P560R was identified by PCR and sequencing analysis.Then,the whole genome sequencing and comparative genomic analysis of the wild strain Kp42 and the phage-resistant mutants Kp42-P560R showed that no mutation of outer membrane protein was found,however,insertion mutation of wbaP gene affecting the formation of CPS,which caused translation frameshift.To verify that the capsule is the host receptor of phage,we constructed the deletion mutant Kp42:ΔwbaP and the complementary strain Kp42 pwbaP,and found that the wbaP gene affected the formation of capsule,while capsule affects the lysis ability and adsorption capacity of phage P560 to it.This study have shown that capsule of K.pneumoniae Kp42 participated in the adsorption of phage P560,and was one of the essential receptors for phage P560 to infect Kp42 strain.The study of phage resistance mechanism and host receptor laid a theoretical foundation for the application of phage therapy.4 Expression,identification and antibacterial study of recombinant depolymerasePhage-derived antimicrobial proteins mainly include lysin,depolymerase,holin,transport signal polypeptide and transmembrane peptide,etc.We predicted two phage-derived depolymerases P560dep and P510dep,targeting KL47 and KL64 K.pneumoniae capsular polysaccharides,respectively,and conducted prokaryotic expression and purification,enzyme activity analysis and specificity analysis of recombinant depolymerases,and evaluated their antibacterial effect in vitro and in vivo.The results showed that depolymerase P560dep can degrade the CPS on the surface of KL47 K.pneumoniae,and the depolymerization spectrum of P560dep was the same as the host range of phage P560.P510dep can degrade KL64 CPS,and the depolymerization spectrum matches the host range of P510.Both depolymerase P560dep and P510dep significantly inhibited biofilm formation.In vivo experiments,P560dep can prevent and treat bacteremia infection caused by carbapenem-resistant K.pneumoniae in mouse models.Before and after KL47 CRKP infection,intraperitoneal injection of a single dose of depolymerase(50 μg/mouse)can protect 90%-100%of the mice and reduce the pathological changes of lung and liver.Therefore,depolymerase was an attractive antibacterial agent and can be used as an antimicrobial therapy schedule.5 Study on structure and function of depolymerase P560depAt present,there are many researches on antibacterial activity of depolymerases,but few researches on structure of depolymerases.In order to reveal the relationship between structure and function of depolymerase,3D structure of depolymerase P560dep was obtained by crystal diffraction method and selenomethionine method with a resolution of 1.6 ?.It consists of four domains:N-terminal domain,catalytic domain,lectin-like domain and C-terminal domain.Compared with other depolymerases,P560dep has a novel modular structure in which two tandem lectin-like domains are inserted into its dextral parallel β helix domain.Site-directed mutations were carried out on 18 amino acid sites in the 6 predicted candidate catalytic domains,and enzyme activity tests showed that 4 regions and 6 residues played a role in the catalytic function,among which residues Asp298,Asp311 and Glu314 in region 5 and Glu554 in region 6 played a major role in the catalytic function.At present,the existence of multiple catalytic pockets in a single tail fiber protein has not been reported.Hydrophobic pockets in the lectin-like domain may promote the ability to bind to polysaccharides.The analysis of the structure and function of high-efficiency depolymerase with structural framework provides a theoretical basis for further design of broad-spectrum enzyme against K.pneumoniae.In conclusion,aiming at the serious problem of drug resistance of K.pneumoniae,this study established a small K.pneumoniae phage library,and identified candidate phages for phage therapy through biological characterization and genome sequence analysis.The host specificity of K.pneumoniae phage was confirmed to be related to the TFP gene by gene replacement.The phage-resistance mechanism based on wbaP gene mutation was demonstrated by phage resistance analysis and gene deletion analysis,and the capsule of K.pneumoniae was proved to be an important host receptor for phage P560.In this study,phage-derived depolymerases targeting KL47 and KL64-type capsules were also found,demonstrating their antibacterial effects in vitro and in vivo;the homotrimeric structure of KL47-type depolymerase P560dep was analyzed,and 6 key amino acid catalytic sites of two catalytic domains.This study provides a K.pneumoniae phage candidates for phage application,and also provides a theoretical basis for the use of synthetic biology to customize phage and develop novel depolymerases.更多还原