溶血素4-羟苯丙酮酸二加氧酶介导灿烂弧菌致病的分子机制及调控研究

Vibrio splendidus is an important opportunistic pathogen in mariculture, leading to great loss to the aquaculture of fish, shellfish, crustacea and echinoderms. However, knowledge on the virulence factors and their pathogenic mechanism is largely limited, thus identification of virulence factors and deeply systematic investigation on the pathogenic mechanism is urgently recommended. In our previous work, a sole hemolysin gene named Vshppd encoding 4-HPPD protein with hemolytic activity was cloned and characterized from a V. splendidus isolated from diseased sea cucumber Apostichopus japonicus. The expression of Vshppd was regulated by ion, and the insertional inactivation of Vshppd led to the attenuated virulence. Based on this background, we intend to illustrate the molecular mechanism of ion regulation on both the expression and activity of Vshppd, by the construction of in-frame gene knockout and gene over-expression of Vshppd in V. splendidus, combined with the analyses of real-time reverse transcriptase PCR, western blot, HPLC, spectrometry and mass spectrum. To thoroughly interpret the entire regulatory network focused on Vshppd for better understanding of the pathogenic mechanism mediated by Vshppd, the differentially expressed proteins in V. splendidus strains of Vshppd gene knockout and gene over-expression were analyzed and identified by proteomics analysis. Overall, the present results will not only throw new insight on the pathogenic mechanism of V. splendidus, and but also supply solid data for the control of disease caused by V. splendidus.

灿烂弧菌是海水养殖动物的重要条件致病菌，能造成鱼类、双壳类、甲壳类和棘皮动物等养殖严重病害。然而，灿烂弧菌致病因子及致病机制的研究几近空白，亟待对该菌的致病因子和致病机制进行系统深入的探讨。申请人前期工作从源于刺参的灿烂弧菌中克隆了编码溶血素4-羟苯丙酮酸二加氧酶的Vshppd基因，发现Vshppd的表达受铁离子的调控，且该基因插入失活突变菌株的致病力明显减弱。基于此，本项目拟通过基因突变和过表达等技术，结合实时荧光定量、western blot、蛋白与DNA互作、HPLC、光谱和质谱等分析方法，阐明铁离子对Vshppd表达和蛋白活性双重调控的分子机制，借助蛋白质组学分析Vshppd基因敲除和过表达菌中的差异表达蛋白，解析与Vshppd基因表达相关的调控网络，阐明灿烂弧菌中Vshppd介导的致病机制，研究有望为认识灿烂弧菌的致病机制和控制灿烂弧菌感染疾病的发生奠定基础。

灿烂弧菌是海水中普遍存在的病原性弧菌，其易感宿主包括鱼、贝和参等经济养殖品种。溶血素是病原性弧菌中普遍存在的重要致病因子，而4-羟苯丙酮酸二加氧酶4-HPPD是灿烂弧菌LGP32基因组中注释的溶血素基因，因此，我们在本项目中进行了Vshppd基因致病相关功能及其介导的致病通路研究。首先克隆了Vshppd基因，并通过体外重组表达获得了重组蛋白，证明了其催化酪氨酸代谢产物羟苯丙酮酸4-HPP生成尿黑酸HGA，进而聚合成HGA-melanin。表征了HGA-melanin还原Fe3+、抗菌和抗紫外线等活性。进一步构建了Vshppd过表达菌株，Hppd的过表达介导刺参体腔细胞表面Fe3+为Fe2+，引起细胞氧化还原代谢相关基因表达变化，导致细胞氧化还原力的失衡，从而导致细胞裂解。本项目继而对HGA-melanin还原Fe3+的功能进行了深入探讨，提出了“HGA-melanin-Feo”的二价铁离子吸收通路。有鉴于铁吸收和铁调控在致病中的重要作用，本项目进一步阐明了灿烂弧菌中由“铁载体-IutA”介导的铁离子吸收通路，且两条铁吸收通路在不同的环境条件下协同调控灿烂弧菌的铁吸收和致病过程。为了进一步探究Vshppd介导的致病机制，我们利用自杀性质粒pDM4进行了Vshppd基因的框内缺失突变，获得了∆hppd菌株。Vshppd基因的缺失导致灿烂弧菌的致病力下降，LD50升高了三个数量级。突变株(MTVs)和野生型菌株（WTVs）的蛋白组分析表明，MTVs中与氨基酸代谢相关基因的表达下调，且与鞭毛组装和生物膜形成等通路相关基因的表达下调，因此，提出了“Vshppd通过上调三羧酸循环中关键氨基酸的代谢，参与鞭毛组装与生物膜合成从而介导灿烂弧菌致病力”这一致病通路。本项目在揭示灿烂弧菌铁吸收和铁调控基础上，提出基于“特洛伊木马”策略的硝酸稼化合物和基于铁吸收抑制的拮抗菌-弧菌V33的灿烂弧菌抑制策略，分别可达到不低于43%和60%的抑制效率。本项目的研究阐明了Vshppd在灿烂弧菌致刺参“腐皮综合症”发生中的功能及其介导的致病相关通路，研究结果不仅丰富了灿烂弧菌致病的分子机制内容，而且为发展灿烂弧菌的绿色防治提供了新颖的靶位点，有望为刺参养殖业的生态可持续发展保驾护航。