维氏气单胞菌不同毒力菌株比较蛋白质组学和基因组学分析及相关基因功能的初步研究
发布时间：2019-06-17 09:57
【摘要】：近年来,日益增多的病例表明维氏气单胞菌(Aeromonas veronii)已成为一种重要的人、兽及水生生物共患病原菌,并在食品安全上表现出重要意义,一些国家已把A.veronii及其同属菌作为水体质量和食品安全的检疫对象。目前,国内外有关A.veronii病例的报道逐年增多,其流行呈明显上升趋势。相关研究表明A.veronii的毒力在逐渐增强,但目前对A.veronii致病机制的研究相对较少,国内外有关A.veronii毒力因子的研究也并不多且主要是针对常见毒力因子的研究。而对A.veronii新毒力因子的挖掘和研究将有助于我们进一步了解其致病机制。因此,本研究基于串联质谱标签技术(Tandem Mass Tag,TMT)首次对A.veronii不同毒力菌株的蛋白质组进行比较分析,筛选出了不同毒力菌株间的部分差异表达蛋白并预测了其相互间可能存在的关系;并首次对鱼源致病性A.veronii强毒株TH0426的全基因组(完成图)进行了测序和分析,同时完成了对弱毒株AV161和无毒株CL8155基因组框架图的测定,在基因组水平上对其比较分析,初步揭示了A.veronii强、弱及无毒株在基因组水平上的差异;此外,通过比较基因组学分析了目前已公布的所有39株不同来源的A.veronii的遗传进化关系,初步了解了A.veronii的遗传进化规律;最后,利用基因敲除技术对强毒株TH0426中两个高表达蛋白(Porin蛋白和LamB蛋白)编码基因aha和lamB的功能进行了初步分析,主要研究结果如下:(1)TMT比较蛋白质组分析结果显示,A.veronii强、弱及无毒株间的蛋白表达差异明显,相互间存在多种上调和下调表达蛋白,其中强毒株TH0426和弱毒株AV161之间共鉴定出78个差异表达蛋白,强毒株TH0426和无毒株CL8155之间有90个差异表达蛋白,而弱毒株AV161和无毒株CL8155之间共鉴定出92个差异表达蛋白;分析发现强毒株TH0426中存在16个蛋白其表达量明显高于弱毒株和无毒株,经进一步GO和KEGG功能富集分析发现,这些蛋白主要参与两条代谢通路即细菌趋化过程和不同环境下的微生物代谢过程,表明TH0426对不同环境的适应能力可能更强;此外,分析还发现赖氨酸脱羧酶CadA,核酸内切酶L-PSP,麦芽糖孔蛋白LamB,普鲁兰酶和气溶素Aer这5个蛋白表达量的高低与菌株毒力强弱呈现出正相关的趋势,推测这五个蛋白可能与A.veronii的毒力密切相关;实时荧光定量PCR和质谱多反应监测(Multiple Reaction Monitoring,MRM)技术对部分差异表达蛋白的验证结果与TMT鉴定结果一致,表明A.veronii不同毒力菌株间差异表蛋白的鉴定结果准确可靠。(2)对A.veronii强毒株TH0426全基因组的测序和分析发现,基因组中存在多种毒力因子如菌毛、鞭毛、毒素、铁离子摄取系统以及各型分泌系统(II型、III型和VI型)等,而且TH0426基因组中还存在2个编码普鲁兰酶的基因(AMS64_19745和AMS64_20505)和2个编码几丁质酶的基因(A MS64_00725和AMS64_05940),与第一章差异表达蛋白的筛选结果一致,这两种酶在TH0426中的表达量明显高于AV161和CL8155;对A.veronii强、弱及无毒株的毒力因子比较分析发现,强毒株TH0426基因组中含有两套完整的III型分泌系统,其中III型分泌系统API2和铁离子转运系统是TH0426所特有的,此外TH0426还具有TA系统、前噬菌体序列、CRISPR-Cas系统以及重组系统等特有功能基因簇,这些功能基因簇对TH0426在抵抗感染、适应不同环境、遗传进化以及致病性等方面可能具有重要的作用。(3)对目前已公布的所有39株不同来源的A.veronii的基因组比较分析发现,39株A.veronii存在1993个共有的核心基因,这些共有核心基因可能是维持A.veronii基本特征所必需的基因;基于这些核心基因构建的39株A.veronii系统进化树显示菌株AMC34与另外38株A.veronii之间亲缘关系较远,而其余38株A.veronii之间亲缘关系很近,而且基于39株A.veronii全基因组之间的平均核苷酸同源性(Average Nucleotide Identity,ANI)分析结果与此一致,这表明菌株AMC34很可能不属于A.veronii;此外,基于对39株A.veronii共有核心基因和ANI值的分析可以看出A.veronii亲缘关系的远近与其分离地点没有明显的相关性,但在一定程度上与其来源有关,来源相同或相近的菌株其亲缘关系较近,这表明宿主环境对A.veronii进化的影响更为明显。(4)成功构建A.veronii强毒株TH0426的aha和lamB基因缺失突变株,其中缺失株△aha生物被膜形成能力显著下降3.7倍,对EPC细胞的黏附和侵袭能力也下降2.3倍,缺失株△aha对斑马鱼和小鼠的致病性明显减弱,其中对斑马鱼的LD50升高了80.4倍,对小鼠的LD50升高了10.1倍,毒力显著下降;与缺失株△aha相似,缺失株△lamB生物被膜形成能力显著下降5.6倍,比缺失株△aha下降更为明显,对EPC细胞的黏附和侵袭能力下降了有1.8倍,缺失株△lamB对斑马鱼和小鼠的致病性也明显减弱,对斑马鱼的LD50升高了13.7倍,对小鼠的LD50升高了5.6倍,毒力下降明显;而且aha和lamB基因的缺失,影响了TH0426鞭毛的稳定性,缺失株的游动能力完全丧失;试验结果表明aha和lamB基因的缺失使得TH0426的黏附能力和毒力显著下降。综上所述,本研究从蛋白质组水平和基因组水平上对A.veronii强、弱及无毒株之间的差异进行了初步探讨,分析发现存在于强毒株中的一些高表达蛋白以及基因组中的特有功能基因簇如III型分泌系统API2和铁离子摄取系统等可能是A.veronii TH0426毒力强于AV161和CL8155的原因;遗传进化分析显示A.veronii亲缘关系的远近与其分离地点没有明显的相关性,但宿主环境对A.veronii进化的影响更为明显;强毒株TH0426中高表达蛋白Porin和LamB的编码基因aha和lamB的缺失使得TH0426的黏附能力和毒力显著下降,强毒株中的这些高表达蛋白可能与TH0426的致病性密切相关。上述研究结果将会为进一步阐明A.veronii的致病机制提供一些依据。
[Abstract]:In recent years, an increasing number of cases have shown that Aeromonas veronii has become an important pathogen of human, animal and aquatic organisms, and is of great significance in food safety. Some countries have taken A. veronii and the same as the quarantine object of water quality and food safety. At present, the report of the case of A. veronii at home and abroad has increased year by year, and the prevalence of the case has increased significantly. The related studies show that the virulence of A. veronii is gradually enhanced, but there are relatively few studies on the pathogenesis of A. veronii, and there are few studies on the virulence factors of A. veronii at home and abroad, and mainly for the study of the common virulence factors. The research on the new virulence factors of A. veronii will help us to understand the pathogenesis of A. veronii. In this study, the protein groups of the different virulence strains of A. veronii were compared and analyzed for the first time on the basis of Tandem Mass Tag (TMT). The whole genome (complete graph) of the highly pathogenic A. veronii virulent strain TH0426 of the fish source was sequenced and analyzed for the first time. In addition, the genetic evolution of A. veronii from all the 39 different sources, which has been published, is analyzed by comparative genomics, and the genetic evolution of A. veronii is first known; and finally, The functions of two high-expression proteins (Porin protein and Lamb B protein) in the strong strain TH0426 were preliminarily analyzed by using the gene knock-out technique, and the main results are as follows: (1) The results of the analysis of the TMT comparison protein group show that the A. veronii is strong, There were a variety of up-and down-regulated expression proteins among the weak and non-virulent strains, of which 78 differential expression proteins were identified between the strong strain TH0426 and the weak strain AV161, and 90 differential expression proteins were found between the strong strain TH0426 and the non-strain CL8155. A total of 92 differentially expressed proteins were identified between the attenuated strain AV161 and the non-strain CL8155. The analysis found that the expression of the 16 proteins in the strong strain TH0426 was significantly higher than that of the weak and non-virulent strains, and was found by the enrichment and analysis of the further GO and KEGG function. These proteins are mainly involved in two metabolic pathways, namely the bacterial chemotaxis process and the microbial metabolic processes in different environments, indicating that TH0426 may be more adaptive to different environments; in addition, the analysis also found that the lysine deaminase CadiA, the endonuclease L-PSP, the maltose pore protein Lamb, The expression of pullulanase and Aer in Aer showed a positive correlation with the virulence of the strain, and it was suggested that the five proteins might be closely related to the virulence of A.veronii, and the real-time fluorescence quantitative PCR and the multiple reaction monitoring (MS). The results of the validation of the partial differential expression protein (MMRM) were consistent with the results of the TMT identification, indicating that the results of the identification of the differentially expressed protein in A. veronii were accurate and reliable. (2) The sequencing and analysis of the whole genome of A. veronii virulent strain TH0426 found that there are multiple virulence factors such as pili, flagella, toxin, iron ion uptake system and various secretion systems (type II, type III and VI) in the genome. In addition, two genes encoding the pullulanase (AMS64 _ 19745 and AMS64 _ 20505) and two genes encoding the chitinase (A MS64 _ 00725 and AMS64 _ 05940) were also present in the genome of the TH0426, and the expression levels of the two enzymes in the TH0426 were significantly higher than that of the AV161 and CL8155; and A. veronii was strong, The comparative analysis of the virulence factors of weak and non-virulent strains found that the genome of the strong strain TH0426 contains two sets of complete III-type secretion systems, in which the type III secretion system API2 and the iron ion transport system are specific to the TH0426, and the TH0426 also has a TA system, a pre-bacteriophage sequence, The special functional gene cluster, such as the CRISPR-Cas system and the recombination system, can play an important role in resisting infection, adapting to different environment, genetic evolution and pathogenicity. (3) A comparative analysis of the genome of A. veronii, which is currently published, has found that 39 A. veronii has a total of 1993 core genes, which may be the genes necessary to maintain the essential characteristics of A. veronii; A 39-strain A. veronii system based on these core genes showed a relatively distant relationship between the bacterial strain AMC34 and the other 38 strains of A. veronii, while the remaining 38 strains A. veronii were very close to each other and were based on the average core-to-acid homology between the 39 strains of A. veronii (average Nucleotide Identity, The results of the ANI analysis are consistent with this, indicating that the strain AMC34 is likely not to belong to A. veronii; in addition, it can be seen from the analysis of the total core gene and the ANI value of the 39 A. veronii, that the near and near relationship of the A. veronii is not significantly associated with its separation site, but is to some extent relevant to its origin, The genetic relationship of the same or similar strains is closer, which indicates that the host environment has more effect on the evolution of A. veronii. (4) The Ha and the lamB gene deletion mutant of the A. veronii virulent strain TH0426 was successfully constructed, in which the capacity of the missing strains of the saha was significantly reduced by 3.7 times, and the adhesion and the invasion ability of the EPC cells were also decreased by 2.3 times, and the pathogenicity of the deleted strain Sahaa to the zebrafish and the mice was significantly reduced, the LD50 of the zebrafish is increased by 80.4 times, the LD50 of the mouse is increased by 10.1 times, and the virulence is obviously reduced; and the loss of the laminB of the deleted strain is significantly reduced by 5.6 times compared with that of the missing strain Sahia, The adhesion and invasion ability of the EPC cells decreased by 1.8 times, and the pathogenicity of the deleted strains of lamB to the zebrafish and the mice is also obviously reduced, the LD50 of the zebrafish is increased by 13.7 times, the LD50 of the mouse is increased by 5.6 times, and the virulence is obviously reduced; and the loss of the aa and the lamB gene is reduced, The stability of the flagellum of the TH0426 was affected, and the swimming ability of the deleted strain was completely lost; the results of the test showed that the deletion of the aa and the lamB gene resulted in a significant decrease in the adhesion and the virulence of the TH0426. To sum up, the differences between A. veronii, weak and non-virulent strains were discussed in this study from the level of the protein and the genomic level. It is found that some of the high-expression proteins present in the virulent strain and the specific functional gene clusters in the genome such as the III-type secretion system API2 and the iron ion-uptake system may be A. veronii TH0426 is more virulent than AV161 and CL8155; The genetic evolution analysis shows that the relationship of A. veronii has no significant correlation with its separation site, but the host environment has more effect on the evolution of A. veronii; the deletion of the encoding genes aa and lamB of the high-expression protein Porin and Lamb B in the strong strain TH0426 makes the adhesion and virulence of the TH0426 to be significantly reduced, These high-expression proteins in the virulent strain may be closely related to the pathogenicity of TH0426. The results of this study will provide some evidence for further elucidating the pathogenesis of A.veronii.
【学位授予单位】：吉林农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S941.4
，
本文编号：2500905
[Abstract]:In recent years, an increasing number of cases have shown that Aeromonas veronii has become an important pathogen of human, animal and aquatic organisms, and is of great significance in food safety. Some countries have taken A. veronii and the same as the quarantine object of water quality and food safety. At present, the report of the case of A. veronii at home and abroad has increased year by year, and the prevalence of the case has increased significantly. The related studies show that the virulence of A. veronii is gradually enhanced, but there are relatively few studies on the pathogenesis of A. veronii, and there are few studies on the virulence factors of A. veronii at home and abroad, and mainly for the study of the common virulence factors. The research on the new virulence factors of A. veronii will help us to understand the pathogenesis of A. veronii. In this study, the protein groups of the different virulence strains of A. veronii were compared and analyzed for the first time on the basis of Tandem Mass Tag (TMT). The whole genome (complete graph) of the highly pathogenic A. veronii virulent strain TH0426 of the fish source was sequenced and analyzed for the first time. In addition, the genetic evolution of A. veronii from all the 39 different sources, which has been published, is analyzed by comparative genomics, and the genetic evolution of A. veronii is first known; and finally, The functions of two high-expression proteins (Porin protein and Lamb B protein) in the strong strain TH0426 were preliminarily analyzed by using the gene knock-out technique, and the main results are as follows: (1) The results of the analysis of the TMT comparison protein group show that the A. veronii is strong, There were a variety of up-and down-regulated expression proteins among the weak and non-virulent strains, of which 78 differential expression proteins were identified between the strong strain TH0426 and the weak strain AV161, and 90 differential expression proteins were found between the strong strain TH0426 and the non-strain CL8155. A total of 92 differentially expressed proteins were identified between the attenuated strain AV161 and the non-strain CL8155. The analysis found that the expression of the 16 proteins in the strong strain TH0426 was significantly higher than that of the weak and non-virulent strains, and was found by the enrichment and analysis of the further GO and KEGG function. These proteins are mainly involved in two metabolic pathways, namely the bacterial chemotaxis process and the microbial metabolic processes in different environments, indicating that TH0426 may be more adaptive to different environments; in addition, the analysis also found that the lysine deaminase CadiA, the endonuclease L-PSP, the maltose pore protein Lamb, The expression of pullulanase and Aer in Aer showed a positive correlation with the virulence of the strain, and it was suggested that the five proteins might be closely related to the virulence of A.veronii, and the real-time fluorescence quantitative PCR and the multiple reaction monitoring (MS). The results of the validation of the partial differential expression protein (MMRM) were consistent with the results of the TMT identification, indicating that the results of the identification of the differentially expressed protein in A. veronii were accurate and reliable. (2) The sequencing and analysis of the whole genome of A. veronii virulent strain TH0426 found that there are multiple virulence factors such as pili, flagella, toxin, iron ion uptake system and various secretion systems (type II, type III and VI) in the genome. In addition, two genes encoding the pullulanase (AMS64 _ 19745 and AMS64 _ 20505) and two genes encoding the chitinase (A MS64 _ 00725 and AMS64 _ 05940) were also present in the genome of the TH0426, and the expression levels of the two enzymes in the TH0426 were significantly higher than that of the AV161 and CL8155; and A. veronii was strong, The comparative analysis of the virulence factors of weak and non-virulent strains found that the genome of the strong strain TH0426 contains two sets of complete III-type secretion systems, in which the type III secretion system API2 and the iron ion transport system are specific to the TH0426, and the TH0426 also has a TA system, a pre-bacteriophage sequence, The special functional gene cluster, such as the CRISPR-Cas system and the recombination system, can play an important role in resisting infection, adapting to different environment, genetic evolution and pathogenicity. (3) A comparative analysis of the genome of A. veronii, which is currently published, has found that 39 A. veronii has a total of 1993 core genes, which may be the genes necessary to maintain the essential characteristics of A. veronii; A 39-strain A. veronii system based on these core genes showed a relatively distant relationship between the bacterial strain AMC34 and the other 38 strains of A. veronii, while the remaining 38 strains A. veronii were very close to each other and were based on the average core-to-acid homology between the 39 strains of A. veronii (average Nucleotide Identity, The results of the ANI analysis are consistent with this, indicating that the strain AMC34 is likely not to belong to A. veronii; in addition, it can be seen from the analysis of the total core gene and the ANI value of the 39 A. veronii, that the near and near relationship of the A. veronii is not significantly associated with its separation site, but is to some extent relevant to its origin, The genetic relationship of the same or similar strains is closer, which indicates that the host environment has more effect on the evolution of A. veronii. (4) The Ha and the lamB gene deletion mutant of the A. veronii virulent strain TH0426 was successfully constructed, in which the capacity of the missing strains of the saha was significantly reduced by 3.7 times, and the adhesion and the invasion ability of the EPC cells were also decreased by 2.3 times, and the pathogenicity of the deleted strain Sahaa to the zebrafish and the mice was significantly reduced, the LD50 of the zebrafish is increased by 80.4 times, the LD50 of the mouse is increased by 10.1 times, and the virulence is obviously reduced; and the loss of the laminB of the deleted strain is significantly reduced by 5.6 times compared with that of the missing strain Sahia, The adhesion and invasion ability of the EPC cells decreased by 1.8 times, and the pathogenicity of the deleted strains of lamB to the zebrafish and the mice is also obviously reduced, the LD50 of the zebrafish is increased by 13.7 times, the LD50 of the mouse is increased by 5.6 times, and the virulence is obviously reduced; and the loss of the aa and the lamB gene is reduced, The stability of the flagellum of the TH0426 was affected, and the swimming ability of the deleted strain was completely lost; the results of the test showed that the deletion of the aa and the lamB gene resulted in a significant decrease in the adhesion and the virulence of the TH0426. To sum up, the differences between A. veronii, weak and non-virulent strains were discussed in this study from the level of the protein and the genomic level. It is found that some of the high-expression proteins present in the virulent strain and the specific functional gene clusters in the genome such as the III-type secretion system API2 and the iron ion-uptake system may be A. veronii TH0426 is more virulent than AV161 and CL8155; The genetic evolution analysis shows that the relationship of A. veronii has no significant correlation with its separation site, but the host environment has more effect on the evolution of A. veronii; the deletion of the encoding genes aa and lamB of the high-expression protein Porin and Lamb B in the strong strain TH0426 makes the adhesion and virulence of the TH0426 to be significantly reduced, These high-expression proteins in the virulent strain may be closely related to the pathogenicity of TH0426. The results of this study will provide some evidence for further elucidating the pathogenesis of A.veronii.
【学位授予单位】：吉林农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S941.4
，
本文编号：2500905
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