opaR基因缺失对AHPND致病菌生物学特性及毒力质粒接合转移的影响

Effects of opaR mutation on the biological characteristics and conjugative transfer of the virulence plasmid of acute hepatopancreatic necrosis disease-causing Vibrio parahaemolyticus

中文关键词: 副溶血弧菌  opaR基因  生物学特性  Ⅳ型分泌系统  水平转移

英文关键词: Vibrio parahaemolyticus  opaR gene  Biological characteristics  Type Ⅳ secretion system  Horizontal transfer

中文摘要:

      急性肝胰腺坏死病(acute hepatopancreatic necrosis disease, AHPND)严重影响了我国乃至全球对虾养殖业的发展。近期研究发现，致病菌毒力质粒携带trb型Ⅳ型分泌系统(type Ⅳ secretion system, T4SS)可在高菌体密度下介导毒力质粒发生接合转移，从而导致AHPND致病菌多样性。为探究群体感应与T4SS表达以及毒力质粒接合转移的关系，本研究以副溶血弧菌(Vibrio parahaemolyticus)群体感应系统的高密度调控子OpaR为研究对象，在致AHPND副溶血弧菌20130629002S01::cat(Vp2S01::cat)菌株基础上，利用同源重组技术构建opaR基因缺失株Vp2S01::catΔopaR。比较了出发菌株和缺失株在生长性能、运动性和生物被膜形成能力等方面的差异，分析了opaR基因对T4SS基因表达量以及质粒接合转移效率的影响。结果显示，opaR基因缺失不影响细菌的生长特性和群集运动，但泳动能力显著增加，生物被膜形成能力显著下降；接合转移实验显示，opaR基因缺失显著提高T4SS表达水平和接合转移效率。本研究为解析群体感应系统调控AHPND致病菌T4SS表达以及毒力质粒接合转移机制提供了基础数据，可为控制AHPND致病菌毒力质粒传播提供技术支撑。

英文摘要:

      Acute hepatopancreatic necrosis (AHPND) is a bacterial disease caused by Vibrio bacteria that severely affects the Pacific white shrimp farming industry. Since AHPND was first identified in China and Vietnam in 2010, it was subsequently identified in Malaysia (2011), Thailand (2012), Mexico (2013), the Philippines (2014), South Korea (2016), Bangladesh (2017), the United States (US) (2017), and Japan (2020). AHPND causes significant economic losses of more than 7 billion US$ annually to the global shrimp farming industry. It has severely restricted the development of the global shrimp farming industry. Many studies have shown that the pathogens of AHPND are Vibrio spp., including V. parahaemolyticus, V. owensii, V. campbellii, V. harveyi, V. punensis, and V. anguillarum. The virulence of these pathogenic strains was derived from a 70 kb virulent plasmid (pVA1-type plasmid). The pVA1-type plasmid carries pirAB genes encoding the binary toxin pirAB homologous to the insecticidal protein of Photobacterium phosphoreum. The pVA1-type plasmid has been confirmed to carry a novel trb type Ⅳ secretion system (T4SS). Trb-T4SS can mediate the conjugative transfer of the pVA1-type plasmid at high cell density for interspecific horizontal transfer. The quorum sensing (QS) system is also known as the density-sensing system. In the surrounding environment, the QS system allows bacteria to regulate the expression of multiple genes by sensing changes in the concentration of autoinducer signaling molecules. At high cell density, high concentrations of autoinducer signaling molecules bind to receptor proteins to inhibit phosphorylation cascades and high cell density master regulator OpaR is normally expressed. OpaR is involved in the regulation of various biological processes, such as biofilm-forming ability, motility, and the expression of the type Ⅲ and type Ⅳ secretion systems. Reportedly, high concentrations of signaling molecules in the QS system regulate the expression of key genes of the T4SS to increase the conjugative transfer efficiency of drug-resistant plasmids. However, the regulation of the T4SS by opaR in AHPND is not yet reported. In this study, OpaR, the high cell density master regulator of the QS system in V. parahaemolyticus, was selected to explore the relationship between the QS system and T4SS expression, as well as the conjugative transfer of the pVA1-type plasmid. The V. parahaemolyticus 20130629002S01::cat (Vp2S01::cat) strain, a pathogenic strain of AHPND, was used as the starting strain. The opaR gene was replaced with the erythromycin resistance gene (ermB) by homologous recombination and electroporation. The opaR mutant strain (Vp2S01::catΔopaR) was successfully constructed. The effects of OpaR on Vp2S01::cat were explored by performing growth curve and motility assays. Vp2S01::cat and Vp2S01::catΔopaR were cultured continuously for approximately 24 h in a shaking bed at 28 ℃ and 180 r/min. OD600 was measured every 2 h to compare the difference in growth. The results showed that there were no significant differences in the growth curves for Vp2S01::cat and Vp2S01::catΔopaR. Biofilm-forming ability was detected using a crystal violet staining assay. The results showed that the biofilm-forming ability of Vp2S01::catΔopaR was significantly reduced. Growth zone diameter was recorded in 0.3% (swimming) LB agar plates at 28 ℃ for 8 h to analyze the difference in swimming ability. We found that the swimming ability of Vp2S01::catΔopaR increased significantly by 2.67 times compared with that of Vp2S01::cat. Growth zone diameter was recorded in 1.5% (swarming) LB agar plates at 28 ℃ for 12 h to analyze the difference in swarming ability. The results showed that there was no significant difference in swarming ability between Vp2S01::cat and Vp2S01::catΔopaR. However, Vp2S01::catΔopaR had more missing colonies. Using VcLMB29 as the receptor strain, the conjugative transfer efficiency of Vp2S01::cat and Vp2S01::catΔopaR was compared at different time points. We found that the conjugative transfer efficiency of Vp2S01::catΔopaR increased after 12 and 24 h. The conjugative transfer efficiency after 24 h was increased by a factor of 265.43. RNA of Vp2S01::cat and Vp2S01::catΔopaR was extracted after 24 h of conjugative transfer and reverse-transcribed into cDNA for real-time quantitative fluorescence PCR (RT-qPCR). GyrB was used as the reference gene. AphA-qRT, opaR-qRT, and T4SS-qRT primers were used for RT-qPCR. The relative expression levels of genes were calculated using the 2–ΔΔCt method. The results showed that the relative expression levels of T4SS in the Vp2S01::catΔopaR experimental group were significantly increased by 1.13–3.21 times and that the relative expression levels of the key conjugative transfer genes traF, trbE, and traG were significantly increased by 1.96, 1.92, and 3.21 times, respectively. In conclusion, the opaR gene does not affect the growth and swarming motility but does affect the biofilm-forming and swimming ability of Vp2S01::cat. The high cell density master regulator OpaR may affect the conjugative transfer efficiency of the virulent plasmid by regulating the expression levels of T4SS. This study provides fundamental data for analyzing the mechanism by which the QS system regulates the expression of T4SS and the conjugative transfer of the pVA1-type plasmid in AHPND pathogenic bacteria, offering technical support for the control of the horizontal transfer of the virulent plasmid in AHPND-causing bacteria.