摘 要

杀鲑气单胞菌（Aeromonas salmonicida, A. salmonicida），作为最主要的一种可以引起鱼体疖疮病的细菌，正影响着全世界范围内的鲑科鱼类的养殖，尤其是大西洋鲑（Salmo salar）的养殖。为了增加我们对这个病原菌的了解，同时从大西洋鲑体内筛选出可以用来抵抗该病菌侵袭的潜在生物指示因子，我们采用代谢组学、蛋白质组学、磷酸化蛋白质学和分子生物学的方法进行实验分析和筛选。由于该细菌主要发病的器官是鱼体的重要免疫器官-肾脏组织。因此，我们将肾脏作为本实验研究的主体。另外，我们从实验分析的组学数据中筛选出醛脱氢酶家族的7A1蛋白（ALDH7A1），并将其进行蛋白重组表达纯化。发现其在大西洋鲑感染杀鲑气单胞菌的早期，有着明显的抗杀鲑气单胞菌的能力并且可以显著降低鱼体的死亡率。所有得到的主要结果如下所示：

1 代谢组学对感染杀鲑气单胞菌的大西洋鲑的肾脏组织进行分析

本部分实验中，我们首次通过¹H核磁共振（NMR）光谱分析法来分析感染杀鲑气单胞菌的大西洋鲑的肾脏组织中代谢物的变化情况。通过NOESYPR1D光谱、主成分分析（PCA）和正交偏最小二乘判别分析模型（OPLS-DA）两种多元化分析（刘鹏飞，2015），对7天和14天的高浓度感染实验组与对照组中的实验大西洋鲑的肾脏组织进行比较分析。本部分实验最主要的目的就是筛选出可以抵抗杀鲑气单胞菌感染鱼体的重要代谢物，同时进一步了解该病菌在大西洋鲑体内的致病机理。经分析，我们发现分布在TCA循环、糖酵解/糖异生、色氨酸代谢和尿素循环等四大代谢通路中的延胡索酸盐、丙氨酸、缬氨酸、甘氨酸、天冬氨酸盐、胆碱、磷酸胆碱和甜菜碱等9种差异代谢物，可能是由于杀鲑气单胞菌的刺激和影响，而在鱼体内出现明显增高的表达趋势。

2 蛋白质组学对感染杀鲑气单胞菌的大西洋鲑的肾脏组织进行分析

本部分实验通过高通量蛋白质组学（iTRAQ标记法），对在7天和14天两个高、低浓度不同的感染杀鲑气单胞菌的实验组中的大西洋鲑的肾脏组织进行分析和鉴定。我们通过LC/MS/MS质谱仪分析后共筛选出4009个总蛋白。随后，我们将140个差异极显著的蛋白（差异倍数≥1.5和P<0.01）进行蛋白网络互作分析，共筛选出39个蛋白可以作为大西洋鲑抵抗杀鲑气单胞菌侵袭的潜在生物指示因子。然后，我们从中筛选了9个重要的差异蛋白进行实时定量PCR来检验这些基因的转录水平表达情况，发现与蛋白水平变化基本相同。本实验是第一次通过iTRAQ技术对感染杀鲑气单胞菌的大西洋鲑的肾脏组织进行分析鉴定，最终我们推测甘油醛-3-磷酸脱氢酶、醛脱氢酶家族7A1、醛脱氢酶家族9A1、L-乳氨酸脱氢酶B链、羟烷基-辅酶A脱氢酶、β-血红蛋白亚基、α-4-血红蛋白亚基、T-复合蛋白1-eta亚基、磷酸丙糖异构酶-A和补体因子B等10个蛋白可作为潜在的生物指示因子进行深入研究。

3 ALDH7A1蛋白可以保护大西洋鲑抵抗杀鲑气单胞菌

醛脱氢酶家族（ALDHs）是一个具有解毒功能的超级蛋白家族，同时该家族蛋白在维持上皮细胞内稳态，并保护细胞免受醛类毒素的影响和抗药性上有着重要的作用。然而，对于这些解毒蛋白如何行使功能，我们至今还无从得知。在本部分的研究中，我们表达并纯化了ALDH7A1蛋白来研究其在感染杀鲑气单胞菌的大西洋鲑时的作用。我们首先通过SDS-PAGE和western blot的方法来验证重组表达的ALDH7A1（rALDH7A1）蛋白。该蛋白分子量大概为58.9 kDa，等电点为7.09，且该重组蛋白并无信号肽分子，而氨基酸序列中只有一个低复杂区域（¹⁴¹ yvegvgevqeyvdv ¹⁵³）。另外，ELISA的结果表明，rALDH7A1可以与杀鲑气单胞菌结合。且ALDH7A1的基因在鱼的肾脏、肝脏、肠、和血液中表达最高（依次降低），而在鳃和脾中表达最低。随后再通过实时荧光定量PCR检验大西洋鲑的肾脏组织后，我们发现注射rALDH7A1蛋白的感染杀鲑气单胞菌的实验组中，在感染早期，杀鲑气单胞菌的拷贝数明显低于同浓度的感染实验组。与此同时，我们测定了在肾脏和脾脏组织中，NF-kB、P-38 MAPK、caspase-3和TNF-α等4个基因的表达情况。结果表明，主要在72 h的rALDH7A1+高/低浓度感染组的肾脏和肝脏中，这4个基因受到rALDH7A1蛋白的作用表达量明显高于其他实验组，而在脾脏组织中则是在12 h时，在rALDH7A1+高/低浓度感染组中出现与72 h相同的变化趋势。再通过体内中和实验，我们发现rALDH7A1+高/低浓度感染组中的鱼与BSA和PBS对照组相比，鱼的死亡率会降低并出现明显延后致死的情况。

4 磷酸化蛋白质组学对感染杀鲑气单胞菌的大西洋鲑的肾脏组织进行分析

本部分实验通过磷酸化蛋白质组对感染细菌时显著差异表达的蛋白的磷酸化位点进行分析。这是首次对感染杀鲑气单胞菌的大西洋鲑进行磷酸化位点的鉴定。经分析，共有3112个蛋白含5635个磷酸化位点被鉴定到，并以1.5倍差异倍数为基础，共鉴定到1502个上调蛋白和77个下调蛋白。再结合之前得到的蛋白质组学的数据以及基序（motif）分析，我们推测5个在免疫过程和细胞通路中调控细胞凋亡和细胞骨架的激酶（VRK3, GAK, HCK, PKCδ和RSK6）可以作为研究大西洋鲑抵抗杀鲑气单胞菌侵袭的潜在生物因子。另外，通过蛋白网络互作图分析，我们发现fga在整个蛋白网络的最中心部位，同样可能在感染杀鲑气单胞菌的大西洋鲑体内起着重要的调控作用。

Abstract

Aeromonas salmonicida (A. salmonicida) is a major etiologic agent which induces furunculosis and is globally harmful in salmonid cultures, especially in Atlantic salmon (Salmo salar) farming. In order to improve knowledge of its poorly understood pathogenesis and select potential biomarkers from Atlantic salmon against this bacteria, methods including metabolomic, proteomic, phosphoproteomic and molecular biology were utilized in our work. As symptoms mainly existed in kidney, which was also the important organ provoking immune responsiveness in fish. Therefore, we set kidney of fish as the main target organ in our research. Otherwise, we purified recombinant protein ALDH7A1 which was obtained from omics data. And it could act in host immune defense against bacterial infection and decrease the mortality rate of Atlantic salmon at early stages of infection with A. salmonicida. All main results are as follows:

1. Metabolic profiling in kidneys of Atlantic salmon infected with A. salmonicida

Here, we used ¹H nuclear magnetic resonance (NMR) to comprehensively analyze the metabolic changes in the kidney of Atlantic salmon infected with A. salmonicida. Through the NOESYPR1D spectrum combined with multivariate pattern recognition analysis, including principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) models, significant metabolic changes were observed in post-infection groups compared to control groups at seven and 14 days (Liu et al. 2016). Hence, the main objective of this study was to estimate the significant metabolites with resistance to furunculosis and further understand the mechanism of A. salmonicida in Atlantic salmon. Notably, substantial alterations of kidney metabolites were observed, such as with fumarate, alanine, valine, glycine, aspartate, choline, glycerophosphocholine and betaine, and summarized by metabolic pathways including the citrate cycle, glycolysis/gluconeogenesis, tryptophan metabolism, and urea cycle, respectively. After analyzing the alteration trend of these metabolites, we inferred that A. salmonicida caused absorption inhibition of amino acids and disturbed protein metabolism as well as cell metabolism in favor of its replication.

2. Proteomic analyses in kidneys of Atlantic salmon infected with A. salmonicida

We utilized high-throughput proteomics to display differentially expressed proteins in the kidney of Atlantic salmon challenged with high and low infection dose of A. salmonicida at 7 and 14 days. In this study, 4009 distinct proteins were identified in three two-dimensional LC/MS/MS analyses. Then we chose 140 proteins (fold change ratio ≥ 1.5 and P < 0.01) combined with protein–protein interaction analysis to ultimately obtain 39 proteins in network which could be considered as potential biomarkers for Atlantic salmon immune responses. Nine significant differentially expressed proteins were consistent with those at the proteomic level used to validate genes at the transcriptomic level by Real-Time quantative PCR (RT-qPCR). Collectively, these data was first reported using an iTRAQ approach and expression of ten proteins including glyceraldehyde-3-phosphate dehydrogenase, ALDH7A1, ALDH9A1, L-lactate dehydrogenase B chain, HADH, hemoglobin subunit beta, hemoglobin subunit alpha-4, CCT7, triosephosphate isomerase A and complement factor B-like increased significantly in infected groups.

3. ALDH7A1 is a protein that protects Atlantic salmon against A. salmonicida

Aldehyde dehydrogenases (ALDHs) belong to a super-family of detoxifying proteins and perform a significant role in developing epithelial homeostasis, protecting cells from toxic aldehydes and drug resistance. However, the activity and function of these detoxifying proteins remain unknown. In our research, we had expressed and purified recombinant protein ALDH7A1 to study its function in Atlantic salmon infected by Aeromonas salmonicida. The rALDH7A1 protein was verified by SDS-PAGE and western blot. The molecular mass of the deduced amino acid sequence of rALDH7A1 is 58.9 kDa with an estimated pI of 7.09. And only a low complexity region (¹⁴¹ yvegvgevqeyvdv ¹⁵³) without signal peptide was existed in rALDH7A1. Results of ELISA indicated that rALDH7A1 exhibited apparent binding activities with A. salmonicida. And ALDH7A1 expression was high in fish kidney, liver intestine and blood (decrease successively), and low in gill and spleen. The quantification of bacteria copy numbers were obtained by a Real-Time quantative PCR (RT-qPCR) assay in kidneys to confirm that fish in this experiment were authentically infected and bacterial loads in rALDH7A1-adminsitered fish were significantly reduced at an early stage of infection. Meanwhile, the mRNA expression of NF-kB, P-38 MAPK, caspase-3 and TNF-α in tissues of kidneys, livers and spleens were quantified. Of the genes that were stimulated by rALDH7A1 that were mainly up-regulated in rALDH7A1+high-dose infection group at 72 h of fish kidneys and livers at 72 h, and the same variation trend was also found in fish spleens at 12 h. Consistent with these observations, neutralization experiments in vivo indicated that rALDH7A1 could obviously reduce the death rate compared to the BSA and control group.

4. Phosphoproteomic analyses in kidneys of Atlantic salmon infected with A. salmonicida

Phosphoproteomic analyses were conducted to further clarify the dynamics changes of protein phosphorylation pattern triggered by bacterial infection. To our knowledge, this is the first study to characterize phosphorylation events in proteins from A. salmonicida-infected Atlantic salmon. Overall, we identified over 5635 phosphorylation sites in 3112 proteins, and 1502 up-regulated and 77 down-regulated proteins, quantified as a 1.5-fold or greater change, respectively. Based on the combined data from proteomic and motif analyses, we hypothesize that five prospective novel kinases (VRK3, GAK, HCK, PKCδ and RSK6) with common functions in inflammatory processes and cellular pathways to regulate apoptosis and the cytoskeleton could serve as potential biomarkers against bacterial propagation in fish. Therefore, further research could be processed to prove defensive mechanism of these proteins on their sites. Data from STRING-based functional network analyses indicate that fga is the most central protein which could act as an important regulatory factor in kidneys of Atlantic salmon infected with A. salmonicida.