【摘要】：近年來,日益增多的病例表明維氏氣單胞菌(Aeromonas veronii)已成為一種重要的人、獸及水生生物共患病原菌,并在食品安全上表現(xiàn)出重要意義,一些國家已把A.veronii及其同屬菌作為水體質(zhì)量和食品安全的檢疫對象。目前,國內(nèi)外有關(guān)A.veronii病例的報道逐年增多,其流行呈明顯上升趨勢。相關(guān)研究表明A.veronii的毒力在逐漸增強(qiáng),但目前對A.veronii致病機(jī)制的研究相對較少,國內(nèi)外有關(guān)A.veronii毒力因子的研究也并不多且主要是針對常見毒力因子的研究。而對A.veronii新毒力因子的挖掘和研究將有助于我們進(jìn)一步了解其致病機(jī)制。因此,本研究基于串聯(lián)質(zhì)譜標(biāo)簽技術(shù)(Tandem Mass Tag,TMT)首次對A.veronii不同毒力菌株的蛋白質(zhì)組進(jìn)行比較分析,篩選出了不同毒力菌株間的部分差異表達(dá)蛋白并預(yù)測了其相互間可能存在的關(guān)系;并首次對魚源致病性A.veronii強(qiáng)毒株TH0426的全基因組(完成圖)進(jìn)行了測序和分析,同時完成了對弱毒株AV161和無毒株CL8155基因組框架圖的測定,在基因組水平上對其比較分析,初步揭示了A.veronii強(qiáng)、弱及無毒株在基因組水平上的差異;此外,通過比較基因組學(xué)分析了目前已公布的所有39株不同來源的A.veronii的遺傳進(jìn)化關(guān)系,初步了解了A.veronii的遺傳進(jìn)化規(guī)律;最后,利用基因敲除技術(shù)對強(qiáng)毒株TH0426中兩個高表達(dá)蛋白(Porin蛋白和LamB蛋白)編碼基因aha和lamB的功能進(jìn)行了初步分析,主要研究結(jié)果如下:(1)TMT比較蛋白質(zhì)組分析結(jié)果顯示,A.veronii強(qiáng)、弱及無毒株間的蛋白表達(dá)差異明顯,相互間存在多種上調(diào)和下調(diào)表達(dá)蛋白,其中強(qiáng)毒株TH0426和弱毒株AV161之間共鑒定出78個差異表達(dá)蛋白,強(qiáng)毒株TH0426和無毒株CL8155之間有90個差異表達(dá)蛋白,而弱毒株AV161和無毒株CL8155之間共鑒定出92個差異表達(dá)蛋白;分析發(fā)現(xiàn)強(qiáng)毒株TH0426中存在16個蛋白其表達(dá)量明顯高于弱毒株和無毒株,經(jīng)進(jìn)一步GO和KEGG功能富集分析發(fā)現(xiàn),這些蛋白主要參與兩條代謝通路即細(xì)菌趨化過程和不同環(huán)境下的微生物代謝過程,表明TH0426對不同環(huán)境的適應(yīng)能力可能更強(qiáng);此外,分析還發(fā)現(xiàn)賴氨酸脫羧酶CadA,核酸內(nèi)切酶L-PSP,麥芽糖孔蛋白LamB,普魯蘭酶和氣溶素Aer這5個蛋白表達(dá)量的高低與菌株毒力強(qiáng)弱呈現(xiàn)出正相關(guān)的趨勢,推測這五個蛋白可能與A.veronii的毒力密切相關(guān);實(shí)時熒光定量PCR和質(zhì)譜多反應(yīng)監(jiān)測(Multiple Reaction Monitoring,MRM)技術(shù)對部分差異表達(dá)蛋白的驗(yàn)證結(jié)果與TMT鑒定結(jié)果一致,表明A.veronii不同毒力菌株間差異表蛋白的鑒定結(jié)果準(zhǔn)確可靠。(2)對A.veronii強(qiáng)毒株TH0426全基因組的測序和分析發(fā)現(xiàn),基因組中存在多種毒力因子如菌毛、鞭毛、毒素、鐵離子攝取系統(tǒng)以及各型分泌系統(tǒng)(II型、III型和VI型)等,而且TH0426基因組中還存在2個編碼普魯蘭酶的基因(AMS64_19745和AMS64_20505)和2個編碼幾丁質(zhì)酶的基因(A MS64_00725和AMS64_05940),與第一章差異表達(dá)蛋白的篩選結(jié)果一致,這兩種酶在TH0426中的表達(dá)量明顯高于AV161和CL8155;對A.veronii強(qiáng)、弱及無毒株的毒力因子比較分析發(fā)現(xiàn),強(qiáng)毒株TH0426基因組中含有兩套完整的III型分泌系統(tǒng),其中III型分泌系統(tǒng)API2和鐵離子轉(zhuǎn)運(yùn)系統(tǒng)是TH0426所特有的,此外TH0426還具有TA系統(tǒng)、前噬菌體序列、CRISPR-Cas系統(tǒng)以及重組系統(tǒng)等特有功能基因簇,這些功能基因簇對TH0426在抵抗感染、適應(yīng)不同環(huán)境、遺傳進(jìn)化以及致病性等方面可能具有重要的作用。(3)對目前已公布的所有39株不同來源的A.veronii的基因組比較分析發(fā)現(xiàn),39株A.veronii存在1993個共有的核心基因,這些共有核心基因可能是維持A.veronii基本特征所必需的基因;基于這些核心基因構(gòu)建的39株A.veronii系統(tǒng)進(jìn)化樹顯示菌株AMC34與另外38株A.veronii之間親緣關(guān)系較遠(yuǎn),而其余38株A.veronii之間親緣關(guān)系很近,而且基于39株A.veronii全基因組之間的平均核苷酸同源性(Average Nucleotide Identity,ANI)分析結(jié)果與此一致,這表明菌株AMC34很可能不屬于A.veronii;此外,基于對39株A.veronii共有核心基因和ANI值的分析可以看出A.veronii親緣關(guān)系的遠(yuǎn)近與其分離地點(diǎn)沒有明顯的相關(guān)性,但在一定程度上與其來源有關(guān),來源相同或相近的菌株其親緣關(guān)系較近,這表明宿主環(huán)境對A.veronii進(jìn)化的影響更為明顯。(4)成功構(gòu)建A.veronii強(qiáng)毒株TH0426的aha和lamB基因缺失突變株,其中缺失株△aha生物被膜形成能力顯著下降3.7倍,對EPC細(xì)胞的黏附和侵襲能力也下降2.3倍,缺失株△aha對斑馬魚和小鼠的致病性明顯減弱,其中對斑馬魚的LD50升高了80.4倍,對小鼠的LD50升高了10.1倍,毒力顯著下降;與缺失株△aha相似,缺失株△lamB生物被膜形成能力顯著下降5.6倍,比缺失株△aha下降更為明顯,對EPC細(xì)胞的黏附和侵襲能力下降了有1.8倍,缺失株△lamB對斑馬魚和小鼠的致病性也明顯減弱,對斑馬魚的LD50升高了13.7倍,對小鼠的LD50升高了5.6倍,毒力下降明顯;而且aha和lamB基因的缺失,影響了TH0426鞭毛的穩(wěn)定性,缺失株的游動能力完全喪失;試驗(yàn)結(jié)果表明aha和lamB基因的缺失使得TH0426的黏附能力和毒力顯著下降。綜上所述,本研究從蛋白質(zhì)組水平和基因組水平上對A.veronii強(qiáng)、弱及無毒株之間的差異進(jìn)行了初步探討,分析發(fā)現(xiàn)存在于強(qiáng)毒株中的一些高表達(dá)蛋白以及基因組中的特有功能基因簇如III型分泌系統(tǒng)API2和鐵離子攝取系統(tǒng)等可能是A.veronii TH0426毒力強(qiáng)于AV161和CL8155的原因;遺傳進(jìn)化分析顯示A.veronii親緣關(guān)系的遠(yuǎn)近與其分離地點(diǎn)沒有明顯的相關(guān)性,但宿主環(huán)境對A.veronii進(jìn)化的影響更為明顯;強(qiáng)毒株TH0426中高表達(dá)蛋白Porin和LamB的編碼基因aha和lamB的缺失使得TH0426的黏附能力和毒力顯著下降,強(qiáng)毒株中的這些高表達(dá)蛋白可能與TH0426的致病性密切相關(guān)。上述研究結(jié)果將會為進(jìn)一步闡明A.veronii的致病機(jī)制提供一些依據(jù)。
[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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S941.4
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本文編號：2500905