本文選題：基因組島 + 肺炎克雷伯菌��； 參考：《復(fù)旦大學(xué)》2009年博士論文

【摘要】：目前院內(nèi)感染在臨床感染病人中所占的比例日益增高,已經(jīng)引起人們?cè)絹碓蕉嗟闹匾�。院�?nèi)感染多由耐藥的條件致病菌感染引起,抵抗力低下的病人如發(fā)生院內(nèi)感染,將給治療帶來更大的困難。因此找到院內(nèi)感染的傳播機(jī)制,從而有效控制院內(nèi)感染是臨床治療的一個(gè)重要方面。條件致病菌對(duì)醫(yī)院抗生素壓力環(huán)境適應(yīng)能力的增強(qiáng)以及臨床菌株中抗生素抗性基因的播散是導(dǎo)致院內(nèi)感染傳播的重要原因。先前已有大量研究發(fā)現(xiàn),一些抗生素抗性基因正在臨床條件致病菌中廣泛播散。而一些特殊的基因元件可以在不同菌株之間水平轉(zhuǎn)移,在這些元件中可以攜帶有抗生素抗性基因,這些基因元件的轉(zhuǎn)移導(dǎo)致了抗性基因在菌株之間的傳播。目前研究較多的可轉(zhuǎn)移基因元件包括:接合性質(zhì)粒、整合子、轉(zhuǎn)座子等。這些基因元件中大多攜帶有各種抗生素抗性基因,它們可以通過不同的分子機(jī)制在不同的菌株之間進(jìn)行轉(zhuǎn)移,例如:接合性質(zhì)粒通過接合作用,整合子通過att識(shí)別位點(diǎn)特異性重組,轉(zhuǎn)座子通過兩端攜帶的重復(fù)序列來達(dá)到重組的目的。這些基因元件的傳播在一定程度上導(dǎo)致了臨床耐藥菌株的播散。 另一方面,在臨床條件致病菌株感染過程中,抵抗抗生素抗性壓力只是其中的一個(gè)方面,除此之外,這些臨床菌株還需要適應(yīng)各種各樣的生存環(huán)境壓力,包括溫度、pH、營養(yǎng)等等方面。而如何適應(yīng)這些惡劣的環(huán)境,完成自身的生存,需要這些臨床菌株能夠快速的適應(yīng)外界環(huán)境(包括抗生素抗性)多種方面的改變。近年來人們通過生物信息學(xué)分析發(fā)現(xiàn)在微生物的全基因組中,大部分的基因高度保守,稱為“基因骨架”,另外一部分基因則變異性很大,稱為“可變基因池”,而這部分“可變基因池”主要是由于基因的水平轉(zhuǎn)移形成。近來有研究發(fā)現(xiàn),一些大的基因片段可以在不同菌株之間水平移動(dòng),這些基因片段不同于接合性質(zhì)粒、整合子或者轉(zhuǎn)座子,它們自身具有一些典型的結(jié)構(gòu)特點(diǎn):常位于tRNA基因位點(diǎn)后面;常攜帶有編碼整合酶或轉(zhuǎn)座酶的基因;GC含量與基因組的保守骨架不同;攜帶有一些重復(fù)序列;基因片段大小不等,大的可達(dá)100 kb以上等。這些片段攜帶的基因也是多種多樣的,可攜帶有與抗生素抗性相關(guān)的基因,也可攜帶有致病相關(guān)因子或者代謝相關(guān)基因等等�，F(xiàn)在將具有以上特征的這些基因片段統(tǒng)稱為“基因組島(Genomic Island,GI)”�；蚪M島的水平轉(zhuǎn)移是形成“可變基因池”的主要來源之一,對(duì)于微生物在短時(shí)間內(nèi)獲得新特性快速適應(yīng)環(huán)境從而實(shí)現(xiàn)在短時(shí)間內(nèi)的進(jìn)化具有重要意義。 目前基因組島已經(jīng)在多種菌株中發(fā)現(xiàn),研究較多的菌株包括Escherichiacoli、Salmonella enterica等。肺炎克雷伯菌(Klebsiella pneumoniae)是臨床常見的條件致病菌之一,目前發(fā)現(xiàn)在肺炎克雷伯很多臨床菌株可以產(chǎn)生β-內(nèi)酰氨酶,有的還攜帶有超廣譜β-內(nèi)酰氨酶,導(dǎo)致對(duì)常用的β-內(nèi)酰胺類抗生素耐藥。目前在K.pneumoniae菌株中也發(fā)現(xiàn)一個(gè)來源于耶爾森菌屬與致病相關(guān)的基因組島。但是是否在K.pneumoniae臨床菌株中存在著其它的基因組島對(duì)于K.pneumoniae臨床菌株的生存以及致病具有重要意義,目前還沒有相關(guān)研究,因此本課題的目的是挖掘K.pneumoniae臨床耐藥菌株中的基因組島,探討K.pneumoniae臨床耐藥菌株播散的相關(guān)分子,為臨床K.pneumoniae臨床耐藥菌株播散的檢測(cè)以及控制提供一定的線索。 由于基因組島大小不等,變異較大,因此很難找到一個(gè)簡(jiǎn)單高效的篩選基因組島的方法。有人曾采用基因芯片的方法對(duì)基因組島進(jìn)行篩選,但此方法費(fèi)用較為昂貴,難以用于臨床菌株基因組島的普篩。由于基因組島的一個(gè)典型特征就是位于tRNA基因位點(diǎn)后面,歐z延畹熱瞬捎蒙鐨畔⒀Х椒ǘ苑窩卓死撞甑膖RNA基因位點(diǎn)進(jìn)行分析,確定了一些可能為基因組島插入熱點(diǎn)的tRNA基因位點(diǎn)。同時(shí)進(jìn)一步對(duì)這些位點(diǎn)的上下游保守序列進(jìn)行分析,基于該位點(diǎn)上下游的保守序列設(shè)計(jì)特異性的引物對(duì)該位點(diǎn)進(jìn)行篩選,根據(jù)PCR結(jié)果來確定是否有基因組島插入。 (1)K.pneumoniae臨床耐藥菌株中基因組島的篩選 針對(duì)生物信息學(xué)分析確定的基因組島可能的插入熱點(diǎn)進(jìn)行PCR篩選,我們發(fā)現(xiàn)arg6 tRNA、asn34 tRNA、phe55 tRNA以及met56 tRNA這四個(gè)基因位點(diǎn)確實(shí)是基因組島的插入熱點(diǎn)。我們發(fā)現(xiàn)51株臨床菌株在這四個(gè)位點(diǎn)上有83個(gè)位點(diǎn)上可能有基因組島的插入,其中arg6 tRNA基因位點(diǎn)16個(gè),asn34 tRNA基因位點(diǎn)9個(gè),met56 tRNA基因位點(diǎn)7個(gè),而phe55 tRNA基因位點(diǎn)則全部沒有得到空位點(diǎn)長度的PCR產(chǎn)物。51株K.pneumoniae臨床菌株phe55 tRNA位點(diǎn)處有44株臨床菌株得到了3.7 kb的PCR擴(kuò)增產(chǎn)物,我們將這個(gè)片段命名為KpGI-1,1株臨床菌株(HS04160)得到了6.4 kb的PCR擴(kuò)增產(chǎn)物,我們將這個(gè)片段命名為KpGI-2,6株臨床菌株沒有得到PCR擴(kuò)增產(chǎn)物,懷疑有大的基因組島插入。在K.pneumoniae MGH78578基因組中,在這個(gè)位點(diǎn)有一個(gè)12.6 kb的基因組島插入,我們將它命名為KpGI-3�；蚪M島KpGI-3中攜帶了1個(gè)整合酶基因和7個(gè)鞭毛編碼相關(guān)基因。通過PCR篩選,我們發(fā)現(xiàn)菌株HS04053擴(kuò)增得到了KpGI-3中7個(gè)鞭毛編碼相關(guān)基因,但是沒有整合酶基因,而是一個(gè)更長的基因片段取代了這個(gè)整合酶基因。因此我們可以判斷在HS04053攜帶有一個(gè)變異了的KpGI-3基因組島。 (2)KpGI-1和KpGI-2確定為新的基因組島 我們將3.7 kb的PCR產(chǎn)物KpGI-1和6.4 kb的PCR產(chǎn)物KpGI-2進(jìn)行測(cè)序,我們對(duì)KpGt-1和KpGI-2的基因序列分析發(fā)現(xiàn),KpGI-1的GC含量為46.75%,KpGI-2的GC含量為38.03%,與K.pneumoniae MGH78578基因組57.5%的GC含量存在明顯不同。同時(shí)KpGI-1和KpGI-2基因序列中,存在著163 bp的重復(fù)序列(DR),這個(gè)163 bp的DR序列與K.pneumoniae MGH78578中的KpGI-3中的163 bp的DR序列完全一致。KpGI-1的orf2中攜帶有一個(gè)與轉(zhuǎn)座酶一致的保守的結(jié)構(gòu)域(pfam01527),KpGI-2的orf1中攜帶有一個(gè)不完整的整合酶基因(CAD06800)。KpGI-1和KpGI-2的這些特點(diǎn)完全符合基因組島的特征,因此可以將KpGI-1和KpGI-2定義為兩個(gè)新的基因組島。 通過對(duì)新基因組島KpGI-1和KpGI-2的DNA序列進(jìn)行分析,KpGI-1中的orfs與目前已知的蛋白同源性均較低,但是有的基因中含有與已知基因一致的保守的結(jié)構(gòu)域。KpGI-1中的orf3含有的結(jié)構(gòu)域與乙�；D(zhuǎn)移酶的結(jié)構(gòu)域(pfam00583)一致,KpGI-1中的orf4含有一個(gè)未知的結(jié)構(gòu)域。與KpGI-1相似,KpGI-2中的大部分orfs與目前已知的蛋白同源性均較低,KpGI-2中的orf4含有結(jié)構(gòu)域DEXDc(DEAD-like helicases superfamily)(CDD accession no.cd00046)和HELICc(Helicase superfamily c-terminal domain)(pfam00271)。KpGI-2中的ORF5與Salmonella enterica Weltevreden HI_N05-537菌株中的Fic蛋白具有高度同源性,并且ORF5同時(shí)含有Fic(filamentation induced by cAMP,Fic)蛋白家族的結(jié)構(gòu)域(pfam02661)。 (3)基因組島KpGI-2來源分析 fic基因最初在E.coli菌株中發(fā)現(xiàn),被認(rèn)為參與了cAMP誘導(dǎo)下細(xì)菌生長的調(diào)節(jié)。我們發(fā)現(xiàn)fic基因不僅在E.coli菌屬中高度保守,同時(shí)在K.pneumoniae菌屬和S.enteria菌屬中也高度保守。與E.coli菌屬和S.enteria菌屬不同的是,在臨床菌株來源的已測(cè)序菌株K.pneumoniae MGH78578基因組中含有兩個(gè)fic基因(kpn_03747和kpn_03553)。kpn_03747在K.pneumoniae菌屬中高度保守且與E.coli菌屬和S.enteria菌屬中的fic基因具有高度相似性,而另一個(gè)fic基因kpn_03553則與保守的fic基因kpn_03747相似性較低。 在51株K.pneumoniae臨床菌株中,所有的菌株都含有基因kpn_03747,且與鄰近的基因也都具有高度的連鎖性。而51株K.pneumoniae臨床菌株中有3株失去了基因kpn_03553,其中一株為HS04160,失去了基因kpn_03553但攜帶有另一個(gè)fic基因,該fic基因位于基因組島KpGI-2中。同時(shí)我們進(jìn)一步分析發(fā)現(xiàn)KpGI-2中的fic基因與Salmonella Weltevreden HI N05-537中的fic基因Sew_A3907具有高度的相似性,而我們分析發(fā)現(xiàn)Sew_A3907也位于SalmonellaWeltevreden HI_N05-537菌株中phe tRNA基因后的一個(gè)14.6 kb的基因組島上。由此我們推測(cè)HS04160中的基因組島KpGI-2可能由S.enteria菌株中的基因Sew_A3907水平轉(zhuǎn)移而來。 (4)KpGI-2基因組島的功能研究 在E.coli K-12菌株中,cAMP可以誘導(dǎo)攜帶有fic基因的菌株發(fā)生絲化,并且使其生長也受到抑制,而fic基因突變株和cAMP受體(CRP)突變株在cAMP誘導(dǎo)后則沒有細(xì)菌絲化現(xiàn)象以及生長抑制現(xiàn)象出現(xiàn)。因此,fic基因參與了cAMP對(duì)細(xì)菌絲化和生長的調(diào)節(jié)過程。我們將KpGI-2中攜帶的基因分別進(jìn)行克隆,觀察這些基因以及KpGI-2在cAMP作用下對(duì)細(xì)菌形態(tài)和生長的影響,我們發(fā)現(xiàn)這些基因在cAMP作用下對(duì)細(xì)菌絲化的誘導(dǎo)為非特異性的,但是對(duì)細(xì)菌生長卻具有不同的作用�；騩rf2+orf3在cAMP作用下明顯抑制了細(xì)菌的生長,而orf4和orf5則與之相反,在cAMP作用下明顯促進(jìn)了細(xì)胞的生長,而整個(gè)基因組島KpGI-2在cAMP的作用下對(duì)細(xì)胞的生長的影響與含有空質(zhì)粒的對(duì)照菌株相比沒有明顯差異。由于Fic蛋白被認(rèn)為是毒素/抗毒素系統(tǒng)中Doc/PhD的前體,Doc蛋白有殺細(xì)胞作用,而PhD蛋白可以拮抗Doc蛋白的殺細(xì)胞作用,因此我們推測(cè)KpGI-2可能也是毒素/抗毒素系統(tǒng)的一種,攜帶的基因?qū)?xì)胞的生長具有相反的作用。
[Abstract]:At present, the proportion of nosocomial infection in patients with clinical infection is increasing, and people have paid more and more attention. The infection of nosocomial infection is caused by the infection of resistant conditional pathogenic bacteria. The patients with low resistance, such as hospital infection, will bring more difficulties to treatment. The control of nosocomial infection is an important aspect of clinical treatment. The enhanced adaptability of the pathogenic bacteria to the hospital antibiotic pressure environment and the dissemination of antibiotic resistance genes in clinical strains are important reasons for the transmission of nosocomial infection. Some special gene elements can be transferred horizontally between different strains, and antibiotic resistant genes can be carried in these components. The transfer of these genes leads to the spread of resistant genes among the strains. Most of these gene elements carry a variety of antibiotic resistance genes, which can be transferred among different strains through different molecular mechanisms, such as conjugative plasmids through conjugation, integrons reorganized through att identification sites, and transposons through repeated sequences carried on both ends of the transposons to achieve the restructured order. The spread of these gene elements to some extent leads to the spread of clinical drug-resistant strains.
On the other hand, resistance to antibiotic resistance is only one aspect of the infection process in clinical conditions. In addition, these clinical strains need to adapt to a variety of environmental pressures, including temperature, pH, nutrition and so on. And how to adapt to these harsh environments and complete their own survival, need these Clinical strains can quickly adapt to a variety of changes in the environment (including antibiotic resistance). In recent years, it is found that most of the genes are highly conserved in the whole genome of microbes by bioinformatics analysis. The other part of the gene is called "gene skeleton", and the other part is called "variable gene pool", and this is called "variable gene pool", and this is called "variable gene pool". Some of the "variable gene pools" are mainly due to the horizontal transfer of genes. Recent studies have found that some large gene fragments can move between different strains. These genes are different from conjugative plasmids, integrons or transposons. They have some typical structural characteristics: often located at the tRNA gene site. Faces; often carrying genes with encoding integrase or transposable enzymes; GC content is different from the conservative skeleton of the genome; carries a number of repeated sequences; the gene fragments vary in size and can be larger than 100 kb. These fragments carry a variety of genes that carry genes associated with resistance to antibiotic resistance and may also be associated with pathogenicity. These genes, such as factors or metabolic related genes, are now known as "Genomic Island (GI)". The horizontal transfer of the genome island is one of the main sources of the "variable gene pool", which can quickly adapt to a short time to adapt to the environment and achieve a short time. The internal evolution is of great significance.
At present, genomic islands have been found in a variety of strains, including Escherichiacoli, Salmonella enterica and so on. Klebsiella pneumoniae (Klebsiella pneumoniae) is one of the common clinical pathogenic bacteria. At present, many clinical strains of klebber have been found to produce beta acylase, and some also have super broad-spectrum. Beta lactamase, which causes resistance to commonly used beta lactam antibiotics, is also found in the strain of the K.pneumoniae strain associated with the pathogenic genome of the genus Jerson. But whether other genomic islands exist in the K.pneumoniae clinical strain are important for the survival and pathogenesis of K.pneumoniae clinical strains. The purpose of this project is to explore the genomic island of K.pneumoniae clinically resistant strains, to explore the related molecules of the spread of clinical resistant strains of K.pneumoniae, and to provide some clues for the detection and control of clinical K.pneumoniae resistant strains.
It is difficult to find a simple and efficient method of screening genomic island because of the size and variation of genome island. At the back of the tRNA gene site, the Z gene loci of the steamer RNA locus are analyzed to determine some tRNA loci which may be the hot spots of the genome Island, and the conservative sequence of the upper and lower reaches of these loci is further analyzed, based on the conservative sequence design of the upper and lower reaches of the site. Specific primers were used to screen the locus and determine whether genomic islands were inserted according to the PCR results.
(1) screening of genomic islands in K.pneumoniae drug-resistant strains
We found that the four gene loci of arg6 tRNA, asn34 tRNA, phe55 tRNA and met56 tRNA are indeed the hot spots of genomic islets. We found that 51 clinical strains may have genomic islets inserted at 83 loci on these four loci. We have found that the four gene loci of tRNA, asn34, tRNA and met56 tRNA are the hot spots of the genome island. There are 16 arg6 tRNA gene loci, 9 asn34 tRNA loci and 7 met56 tRNA loci, while the phe55 tRNA gene loci have not obtained the PCR product.51 strain of the.51 strain of the.51 strain of the.51 strain of the.51 strain. The 1,1 strain clinical strain (HS04160) obtained the PCR amplification product of 6.4 KB. We named the fragment of the KpGI-2,6 strain the clinical strain of the KpGI-2,6 strain without a PCR amplification product and suspected a large genomic island insertion. In the K.pneumoniae MGH78578 genome, there was a 12.6 KB genomic island insertion at this site, and we named it the KpGI-3. base. 1 integrase genes and 7 flagellate coding related genes were carried in the group island KpGI-3. Through PCR screening, we found that strain HS04053 amplified 7 flagellum encoding genes in KpGI-3, but there was no integrase gene, but a longer gene fragment that replaced the integrase gene. So we can judge in HS04053 Carry a mutant KpGI-3 genome island.
(2) KpGI-1 and KpGI-2 are identified as new genome Islands
We sequenced the PCR product KpGI-1 of 3.7 KB and KpGI-2 of the PCR product of 6.4 KB. The sequence analysis of KpGt-1 and KpGI-2 found that the GC content of KpGI-1 was 46.75%, the KpGI-2 GC content was 38.03%, which was significantly different from the 57.5% of the 57.5% genome. The repeating sequence (DR), the 163 BP DR sequence and the DR sequence of the 163 BP in the KpGI-3 in K.pneumoniae MGH78578, carries a conservative domain (pfam01527) consistent with the transposable enzyme in ORF2. It accords with the characteristics of genomic islands. Therefore, KpGI-1 and KpGI-2 can be defined as two new genomic islands.
By analyzing the DNA sequence of the new genomic island KpGI-1 and KpGI-2, the ORFs in KpGI-1 is lower than the known protein, but some genes contain the domain of ORF3 in the conserved domain.KpGI-1 that is consistent with the known genes, which is consistent with the domain of acetyltransferase (pfam00583), and ORF4 contained in KpGI-1. There is an unknown domain. Similar to KpGI-1, most of the ORFs in KpGI-2 is lower than the present known protein, and ORF4 in KpGI-2 contains the domain DEXDc (DEAD-like helicases superfamily) (CDD accession no.cd00046) The Fic protein in the nterica Weltevreden HI_N05-537 strain is highly homologous, and ORF5 also contains the domain of the Fic (filamentation induced by cAMP, Fic) protein family (pfam02661).
(3) analysis of the source of genomic island KpGI-2
The FIC gene was first found in the E.coli strain and was considered to be involved in the regulation of bacterial growth induced by cAMP. We found that the FIC gene is highly conserved not only in the genus E.coli, but also in the genus K.pneumoniae and S.enteria bacteria. The sequenced bacteria from the genus E.coli and S.enteria are the sequenced bacteria derived from the clinical strains. The genome of K.pneumoniae MGH78578 containing two FIC genes (kpn_03747 and kpn_03553).Kpn_03747 is highly conserved in the genus K.pneumoniae and is highly similar to FIC genes in the genus E.coli and S.enteria, while the other FIC gene kpn_03553 is less similar to the conservative gene.
Of 51 strains of K.pneumoniae clinical strains, all the strains had gene kpn_03747 and had a high linkage with adjacent genes, and 3 of the 51 K.pneumoniae clinical strains lost the gene kpn_03553, one of which was HS04160, lost the gene kpn_03553 but carried another FIC gene, and the FIC gene was located in the gene. At the same time, we further analyzed that the FIC gene in KpGI-2 was highly similar to the FIC gene Sew_A3907 in Salmonella Weltevreden HI N05-537, and we found that Sew_A3907 was also located on a 14.6 genomic island after the SalmonellaWeltevreden HI_N05-537 strain. It is speculated that genomic KpGI-2 in HS04160 may be transferred from the level of Sew_A3907 in S.enteria strain.
(4) study on the function of KpGI-2 genome Island
In the E.coli K-12 strain, cAMP can induce filagenesis and inhibit the growth of the strains carrying FIC gene, while the FIC gene mutant and the cAMP receptor (CRP) mutant have no bacterial filamentary and growth inhibition after cAMP induction. Therefore, the FIC gene participates in the regulation of cAMP on the filamentary and growth of bacteria. We cloned the genes carried in KpGI- 2 to observe the genes and the effects of KpGI-2 on the morphology and growth of bacteria under the action of cAMP. We found that these genes were not specific to the induction of bacterial filaments under the action of cAMP, but they have different effects on the growth of bacteria. The gene orf2+orf3 is under the action of cAMP. The growth of bacteria was obviously inhibited, while ORF4 and ORF5, in contrast, obviously promoted cell growth under the action of cAMP, while the effect of the whole genome island KpGI-2 on the growth of the cells was not significantly different from that of the control strain containing the empty plasmid. Because Fic protein was considered to be the Doc/PhD in the toxin / antitoxin system. The precursor, Doc protein has cell killing effect, and PhD protein can antagonize the cytotoxicity of Doc protein, so we speculate that KpGI-2 may also be one of the toxin / antitoxin system, which has the opposite effect on cell growth.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R378

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 張維;尿道致病性大腸桿菌新基因R049的染色體定位及其缺失株構(gòu)建的研究[D];天津醫(yī)科大學(xué);2012年

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本文編號(hào)：1786487