| Coxiella burnetii(Cb for short),a gram-negative and an obligate intracellular parasitism,is the pathogen that causes human Q fever.Cb,which transmits between human beings and animals mainly through aerosol and tick-bited approaches,is one of the most infectious pathogenes that have been known and is a potential bio-warfare or bio-terrorism.So far,all Cb isolates have been found to contain plasmids or plasmid-homologous sequences integrated into the chromosome.These plasmids encode several Type IVB secretion system(T4BSS)effector proteins,which would be secreted into cytosol and regulated eucaryotic functions.For a long time,plasmids and IPS are generally thought to be the key factor for nomal growth and host cell-parasiting of Cb organism,or could possibly allow Cb to survive in adverse environments or could be corelated with pathogenicity of Cb(acute Q fever or chronic Q fever).Owing to hard-culturing and the lack of motheds for Cb plasmid genetic manipulation,the disease pathogenesis and the biological functions of Cb plasmid or encoded by Cb plasmid genes are poorly understood.Recently years,the establishing of host cell-free growth of Cb has promoted the process of genetic research,which creates an opportunity to explore the pathogenic mechanism of Cb gene.According to abroad existing genetic operations of Cb,this paper constructed the shuttle vector thought genetic engineering technologies,established the genetic transformation techniques of Cb,and structured Cb endogenous plasmid deletion mutant by using principle of Cb similarity and incompatibility.The phenotype and virulence change of Cb plasmid deletion mutant were further analyzed by in vivo and in vitro experiments.Moreover,the replication and partitioning region of Cb endogenous plasmid were further analyzed by gene knockout technology.Chapter ?,Based on E.coli plasmid,pMMB207,both Cb autonomous shuttle vector with RSF1010 ori and Cb transformation system were established.First of all,four short flagments of green fluorescence protein gene(eGFP),kanamycin resistance gene(KanR),Cb constitutive gene expression promoter P311 and P1169,and vector backbone p207 with RSF1010 ori were respectively amplified by using PCR technique.Then,the four short flagments was fused into cassette sequence “P311-eGFP-P1169-KanR” by overlapping PCR technique.Finally,the cassette sequence “P311-eGFP-P1169-KanR” was digested by restriction endonuclease,Kpn I and Xho I,and was connected with vector backbone p207 that was double digested in the same way,in order to establish the shuttle vector p207 GK.The shuttle vector p207 GK was performed electrotransformation into Cb,and was continuously subcultured with host cell-free medium ACCM-2.The inverted fluorescence microscope was applied to observe the expression of eGFP gene.When the passage went down to high expression of eGFP gene,the semisolid with single layer “ACCM-2/agarose” slab was adopted to clone and segregate Cb transformants.The results successfully constructed autonomous Cb shuttle vector,obtained Cb transformants that steadily expressed eGFP,and completed clonal segregation,which indicated that we successfully built the Cb transformation system,laying the foundation for subsequently studying Cb with genetic method.Chapter ?,Bioinformatics was employed to analyze the full-length sequence of plasmid Grita of Cb phase II strain in our laboratory,and confirmed the plasmid as QpH1(GeneBank Accession No.AE016829).BLAST comparison analysis found the deletion of the sequence fragment CBUA0036~CBUA0039a of the plasmid in IPS,but the fragment was observed to be highly conservative in four plasmids(QpH1,QpRS,QpDG and QpDV),implying that this sequence was likely be the functional region mediating the replication and partition of Cb plasmid and contained the sequence replicating initiation site and relevant regulatory protein genes.As the backbone of homologous plasmid(named pQ),the fragment containing the sequence CBUA0036 ~ CBUA0039 a was obtained from plasmid QpH1 using PCR amplification technique.Meanwhile,overlapping PCR amplification technique was adopted to fuse eGFP gene,KanR gene,promoters of P311 and P1169,and the sequence pUC ori so as to form a cassette sequence “P311-eGFP-P1169-KanR-pUC ori” which was then sub-cloned onto vector backbone pQ,thus constructing novel shuttle vector pQGK.The novel shuttle vector pQGK was electro-transformed into Cb,undergone serial passage cultivation by abiosis culture medium ACCM-2 containing Kan,and observed via fluorescence microscope.The observation results showed that the novel shuttle vector p QGK could stably generate in Cb,and that Cb transformants presented green fluorescence under the stimulation from fluorescence,all of which indicated that the sequence fragment of CBUA0036~CBUA0039a on the vector pQGK contained the replication initiation region of plasmid QpH1.The transformants were cloned,separated and purified,but PCR identification on Cb clone strain by primers specific to plasmid QpH1 failed to detect any QpH1,suggesting that Cb clone strain had lost plasmid QpH1 and gained mutant strain Cb/pQGK which lacked Cb endogenous plasmid QpH1.SYBR Green absolute quantification Q-PCR technique was implemented to determine the relative copy numbers of the plasmids QpH1 and pQGK in wild strain Cb and mutant one Cb/pQGK,respectively,and the results revealed only single copy for these two plasmids.These phenomenons illustrated that the sequence CBUA0036 ~ CBUA0039 a could strictly control the replication,division and incompatibility when similar of the plasmid QpH1,possibly being the replicon of this plasmid.Q-PCR method was chosen to measure the proliferation ability of mutant strain Cb/pQGK in(ACCM-2)and outside(BGM)cells,and found no distinct differences in the formation or growth of parasitophorous vacuole or in the proliferation of Cb bacteria between mutant and wild strains,stating that the majority of the sequences of the plasmid QpH1 in Cb(CBUA0001~CBUA0034a,>85%)were not implicated in normal growth of Cb in or outside cells.In vivo(immunocompromised SCID mice)and in vitro(human macrophage THP-1 cell)experiments were carried out and found that the virulence of mutant strain Cb/pQGK has declined,which suggested that QpH1 plasmid is a virulence factor.Chapter ?,Based on shuttle vector pQGK,the sequence CBUA0036 ~CBUA0039a was deeply explored for the mechanism of its controls over the replication and division of the plasmid QpH1.Receiving gene knockout or sequence drop,the sequence CBUA0036~CBUA0039a on the shuttle plasmid pQGK was pruned in different degrees to establish a series of mutants of the shuttle vector pQGK,named pQGK-D1 ~ pQGK-D17.These shuttle vectors were separately electro transformed into Cb,experienced series passage cultivation in Kan-containing ACCM-2 culture medium,and checked using fluorescence microscope.When the gene eGFP showed high expression,Cb transformation bacteria were cloned,separated and purified,and PCR identification analysis was then implemented on Cb clone strain.As a consequence,only 14 shuttle vectors(pQGK-D1~pQGK-D14)could be successfully transformed into Cb,and demonstrated stable passages.The analysis of sequence comparison detected that the minimum threshold of the sequence length to let shuttle vector pQGK-D14 steadily generate in Cb was 600 bp(between CBUA0036 and CBUA0037)while shuttle vectors(pQGK-D15~pQGK-D17)with shorter sequence fragments were not able to obtain Cb transformant,denoting that this sequence of 600 bp covered the sequence(ori)of Cb plasmid QpH1 which replicated initiation site.Subsequently,PCR evaluation on Cb clone bacteria after its clone,separation and purification displayed that both shuttle vectors pQGK-D1 and pQGK-D5 could knockout plasmid QpH1,while the coexistence between shuttle vectors pQGK-D7 ~ D8 and pQGK-D11 ~ pQGK-D14 and the plasmid QpH1,manifesting that the replication,division and incompatibility when similar of the plasmid QpH1 were also governed by other unknown factors(the experiment is underway,and detailed results have yet to be supplemented).Chapter ?,On the basis of preliminary studies,we replaced the gene KanR on shuttle vector pQGK with the gene RifR using gene cloning technology to create novel shuttle vector pQGR.Cb was electro-transformed before cloning,separation and purification,and then was used to infect the cells BGM and THP-1.Consequently,after infected by BGM cells,Cb transformants of shuttle vector pQGR could only form weeny vacuoles which were not able to develop into classical big ones,and their proliferation ability was declined significantly;following the infection by THP-1 cells,no vacuoles were generated,and no intracellular proliferation of Cb was spotted either.All of these findings specified that the expression product of the RifR gene,ADP-ribosylase,might possess the function of ribosylation in Cb,affect normal biosynthesis of Cb,make the toxicity of Cb attenuated,and exert noticeable suppressing effects on vacuole development and bacterium proliferation after Cb being infected.Therefore,the gene RifR is not a suitable marker for drug screening in studies on Cb genetic transformation.In conclusion,the study successfully built an autonomous shuttle vector p207 GK based on RSF1010 ori,and set up a genetic transformation system for Cb utilizing this shuttle vector.Next,we also established a novel shuttle plasmid pQGK with homologous sequences(CBUA0036 ~ CBUA0039a)of Cb plasmid QpH1,and applied this shuttle vector to knock out QpH1,thus procuring the mutant without Cb plasmid QpH1.Further examination on the sequence of CBUA0036~CBUA0039a revealed the sequence area of plasmid Qp H1 replicating initiation site.In addition,during this research,we also found significant impacts of ADP-ribosylation enzyme,the encoding product of the RifR gene,on Cb biosynthesis,and hence concluded that it was not a proper marker of drug screening for Cb genetic transformation.The current study offered original genetics tool and approach for further exploring the biological functions and pathogenic mechanism of Cb plasmid. |
PDF Full Text Request