肺炎克雷伯临床菌株基因组岛的识别与鉴定

发布时间：2018-04-22 08:55

本文选题：基因组岛 + 肺炎克雷伯菌　；参考：《复旦大学》2009年博士论文

【摘要】：目前院内感染在临床感染病人中所占的比例日益增高,已经引起人们越来越多的重视。院内感染多由耐药的条件致病菌感染引起,抵抗力低下的病人如发生院内感染,将给治疗带来更大的困难。因此找到院内感染的传播机制,从而有效控制院内感染是临床治疗的一个重要方面。条件致病菌对医院抗生素压力环境适应能力的增强以及临床菌株中抗生素抗性基因的播散是导致院内感染传播的重要原因。先前已有大量研究发现,一些抗生素抗性基因正在临床条件致病菌中广泛播散。而一些特殊的基因元件可以在不同菌株之间水平转移,在这些元件中可以携带有抗生素抗性基因,这些基因元件的转移导致了抗性基因在菌株之间的传播。目前研究较多的可转移基因元件包括:接合性质粒、整合子、转座子等。这些基因元件中大多携带有各种抗生素抗性基因,它们可以通过不同的分子机制在不同的菌株之间进行转移,例如:接合性质粒通过接合作用,整合子通过att识别位点特异性重组,转座子通过两端携带的重复序列来达到重组的目的。这些基因元件的传播在一定程度上导致了临床耐药菌株的播散。另一方面,在临床条件致病菌株感染过程中,抵抗抗生素抗性压力只是其中的一个方面,除此之外,这些临床菌株还需要适应各种各样的生存环境压力,包括温度、pH、营养等等方面。而如何适应这些恶劣的环境,完成自身的生存,需要这些临床菌株能够快速的适应外界环境(包括抗生素抗性)多种方面的改变。近年来人们通过生物信息学分析发现在微生物的全基因组中,大部分的基因高度保守,称为“基因骨架”,另外一部分基因则变异性很大,称为“可变基因池”,而这部分“可变基因池”主要是由于基因的水平转移形成。近来有研究发现,一些大的基因片段可以在不同菌株之间水平移动,这些基因片段不同于接合性质粒、整合子或者转座子,它们自身具有一些典型的结构特点:常位于tRNA基因位点后面;常携带有编码整合酶或转座酶的基因;GC含量与基因组的保守骨架不同;携带有一些重复序列;基因片段大小不等,大的可达100 kb以上等。这些片段携带的基因也是多种多样的,可携带有与抗生素抗性相关的基因,也可携带有致病相关因子或者代谢相关基因等等。现在将具有以上特征的这些基因片段统称为“基因组岛(Genomic Island,GI)”。基因组岛的水平转移是形成“可变基因池”的主要来源之一,对于微生物在短时间内获得新特性快速适应环境从而实现在短时间内的进化具有重要意义。目前基因组岛已经在多种菌株中发现,研究较多的菌株包括Escherichiacoli、Salmonella enterica等。肺炎克雷伯菌(Klebsiella pneumoniae)是临床常见的条件致病菌之一,目前发现在肺炎克雷伯很多临床菌株可以产生β-内酰氨酶,有的还携带有超广谱β-内酰氨酶,导致对常用的β-内酰胺类抗生素耐药。目前在K.pneumoniae菌株中也发现一个来源于耶尔森菌属与致病相关的基因组岛。但是是否在K.pneumoniae临床菌株中存在着其它的基因组岛对于K.pneumoniae临床菌株的生存以及致病具有重要意义,目前还没有相关研究,因此本课题的目的是挖掘K.pneumoniae临床耐药菌株中的基因组岛,探讨K.pneumoniae临床耐药菌株播散的相关分子,为临床K.pneumoniae临床耐药菌株播散的检测以及控制提供一定的线索。由于基因组岛大小不等,变异较大,因此很难找到一个简单高效的筛选基因组岛的方法。有人曾采用基因芯片的方法对基因组岛进行筛选,但此方法费用较为昂贵,难以用于临床菌株基因组岛的普筛。由于基因组岛的一个典型特征就是位于tRNA基因位点后面,欧z延畹热瞬捎蒙镄畔⒀Х椒ǘ苑窝卓死撞甑膖RNA基因位点进行分析,确定了一些可能为基因组岛插入热点的tRNA基因位点。同时进一步对这些位点的上下游保守序列进行分析,基于该位点上下游的保守序列设计特异性的引物对该位点进行筛选,根据PCR结果来确定是否有基因组岛插入。 (1)K.pneumoniae临床耐药菌株中基因组岛的筛选针对生物信息学分析确定的基因组岛可能的插入热点进行PCR筛选,我们发现arg6 tRNA、asn34 tRNA、phe55 tRNA以及met56 tRNA这四个基因位点确实是基因组岛的插入热点。我们发现51株临床菌株在这四个位点上有83个位点上可能有基因组岛的插入,其中arg6 tRNA基因位点16个,asn34 tRNA基因位点9个,met56 tRNA基因位点7个,而phe55 tRNA基因位点则全部没有得到空位点长度的PCR产物。51株K.pneumoniae临床菌株phe55 tRNA位点处有44株临床菌株得到了3.7 kb的PCR扩增产物,我们将这个片段命名为KpGI-1,1株临床菌株(HS04160)得到了6.4 kb的PCR扩增产物,我们将这个片段命名为KpGI-2,6株临床菌株没有得到PCR扩增产物,怀疑有大的基因组岛插入。在K.pneumoniae MGH78578基因组中,在这个位点有一个12.6 kb的基因组岛插入,我们将它命名为KpGI-3。基因组岛KpGI-3中携带了1个整合酶基因和7个鞭毛编码相关基因。通过PCR筛选,我们发现菌株HS04053扩增得到了KpGI-3中7个鞭毛编码相关基因,但是没有整合酶基因,而是一个更长的基因片段取代了这个整合酶基因。因此我们可以判断在HS04053携带有一个变异了的KpGI-3基因组岛。 (2)KpGI-1和KpGI-2确定为新的基因组岛我们将3.7 kb的PCR产物KpGI-1和6.4 kb的PCR产物KpGI-2进行测序,我们对KpGt-1和KpGI-2的基因序列分析发现,KpGI-1的GC含量为46.75%,KpGI-2的GC含量为38.03%,与K.pneumoniae MGH78578基因组57.5%的GC含量存在明显不同。同时KpGI-1和KpGI-2基因序列中,存在着163 bp的重复序列(DR),这个163 bp的DR序列与K.pneumoniae MGH78578中的KpGI-3中的163 bp的DR序列完全一致。KpGI-1的orf2中携带有一个与转座酶一致的保守的结构域(pfam01527),KpGI-2的orf1中携带有一个不完整的整合酶基因(CAD06800)。KpGI-1和KpGI-2的这些特点完全符合基因组岛的特征,因此可以将KpGI-1和KpGI-2定义为两个新的基因组岛。通过对新基因组岛KpGI-1和KpGI-2的DNA序列进行分析,KpGI-1中的orfs与目前已知的蛋白同源性均较低,但是有的基因中含有与已知基因一致的保守的结构域。KpGI-1中的orf3含有的结构域与乙酰基转移酶的结构域(pfam00583)一致,KpGI-1中的orf4含有一个未知的结构域。与KpGI-1相似,KpGI-2中的大部分orfs与目前已知的蛋白同源性均较低,KpGI-2中的orf4含有结构域DEXDc(DEAD-like helicases superfamily)(CDD accession no.cd00046)和HELICc(Helicase superfamily c-terminal domain)(pfam00271)。KpGI-2中的ORF5与Salmonella enterica Weltevreden HI_N05-537菌株中的Fic蛋白具有高度同源性,并且ORF5同时含有Fic(filamentation induced by cAMP,Fic)蛋白家族的结构域(pfam02661)。 (3)基因组岛KpGI-2来源分析 fic基因最初在E.coli菌株中发现,被认为参与了cAMP诱导下细菌生长的调节。我们发现fic基因不仅在E.coli菌属中高度保守,同时在K.pneumoniae菌属和S.enteria菌属中也高度保守。与E.coli菌属和S.enteria菌属不同的是,在临床菌株来源的已测序菌株K.pneumoniae MGH78578基因组中含有两个fic基因(kpn_03747和kpn_03553)。kpn_03747在K.pneumoniae菌属中高度保守且与E.coli菌属和S.enteria菌属中的fic基因具有高度相似性,而另一个fic基因kpn_03553则与保守的fic基因kpn_03747相似性较低。在51株K.pneumoniae临床菌株中,所有的菌株都含有基因kpn_03747,且与邻近的基因也都具有高度的连锁性。而51株K.pneumoniae临床菌株中有3株失去了基因kpn_03553,其中一株为HS04160,失去了基因kpn_03553但携带有另一个fic基因,该fic基因位于基因组岛KpGI-2中。同时我们进一步分析发现KpGI-2中的fic基因与Salmonella Weltevreden HI N05-537中的fic基因Sew_A3907具有高度的相似性,而我们分析发现Sew_A3907也位于SalmonellaWeltevreden HI_N05-537菌株中phe tRNA基因后的一个14.6 kb的基因组岛上。由此我们推测HS04160中的基因组岛KpGI-2可能由S.enteria菌株中的基因Sew_A3907水平转移而来。 (4)KpGI-2基因组岛的功能研究在E.coli K-12菌株中,cAMP可以诱导携带有fic基因的菌株发生丝化,并且使其生长也受到抑制,而fic基因突变株和cAMP受体(CRP)突变株在cAMP诱导后则没有细菌丝化现象以及生长抑制现象出现。因此,fic基因参与了cAMP对细菌丝化和生长的调节过程。我们将KpGI-2中携带的基因分别进行克隆,观察这些基因以及KpGI-2在cAMP作用下对细菌形态和生长的影响,我们发现这些基因在cAMP作用下对细菌丝化的诱导为非特异性的,但是对细菌生长却具有不同的作用。基因orf2+orf3在cAMP作用下明显抑制了细菌的生长,而orf4和orf5则与之相反,在cAMP作用下明显促进了细胞的生长,而整个基因组岛KpGI-2在cAMP的作用下对细胞的生长的影响与含有空质粒的对照菌株相比没有明显差异。由于Fic蛋白被认为是毒素/抗毒素系统中Doc/PhD的前体,Doc蛋白有杀细胞作用,而PhD蛋白可以拮抗Doc蛋白的杀细胞作用,因此我们推测KpGI-2可能也是毒素/抗毒素系统的一种,携带的基因对细胞的生长具有相反的作用。
[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.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R378

【引证文献】