| Acute hepatopancreatic necrosis (AHPND) is a bacterial disease caused by Vibrio bacteria, severely affecting the Pacific white shrimp farming industry. Since AHPND was first found in China and Vietnam in 2010, it has subsequently been found in Malaysia (2011), Thailand (2012), Mexico (2013), the Philippines (2014), South Korea (2016), Bangladesh (2017), the United States (2017) and Japan (2020). AHPND causes enormous economic losses of more than 7 billion US$ of significant economic losses annually in the global shrimp farming industry. It has severely restricted the development of the global shrimp farming industry. Many studies have shown that the pathogen of AHPND is Vibrio, including V. parahaemolyticus, V. owensii, V. campbellii, V. harveyi, V. punensis, and V. anguillarum. The virulence of these pathogenic strains were derived from a 70 kb virulent plasmid (pVA1-type plasmid). The pVA1-type plasmid carries pirAB genes, which encode a 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). This trb-T4SS is capable of mediating conjugative transfer of pVA1-type virulent plasmid at high cell density for interspecific horizontal transfer.
The quorum sensing (QS) system, also known as the density-sensing system. In the surrounding environment, QS system allows bacteria to regulate the expression of multiple genes by sensing changes in the concentration of Autoinducer (AI) signaling molecules. At high cell density, high concentrations of AI signaling molecules bind to receptor proteins to inhibit cascade phosphorylation pathways and high cell density central regulator OpaR can be normally expressed. OpaR is involved in the regulation of various biological processes, such as biofilm-forming ability, motility, and the expression of Type Ⅲ secretion system (T3SS) and Type Ⅵ secretion system (T6SS). It has been reported that high concentrations of signaling molecules in the QS system regulate the expression of key genes of T4SS to increase the conjugative transfer efficiency of drug-resistant plasmids. However, the regulation of T4SS by OpaR in AHPND pathogen has not yet been reported.
In this study, OpaR, the high cell density regulator of the QS system in V. parahaemolyticus, was selected as the research object to explore the relationship between QS system and T4SS expression, as well as the conjugative transfer of virulent plasmid. And 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 technology. The opaR gene mutant strain Vp2S01::catΔopaR was constructed successfully. The effects of OpaR on Vp2S01::cat was explored by growth curve and motility, etc.
Vp2S01::cat and Vp2S01::catΔopaR were cultured continuously for about 24 h in a shaking bed at 28℃ and 180 rpm, and OD600 was measured every 2 h to compare the growth difference. The results showed that the growth curve of Vp2S01::cat and Vp2S01::catΔopaR had no significant difference. The biofilm-forming ability of strains were detected by crystal violet staining assay. The results showed that the biofilm-forming ability of Vp2S01::catΔopaR was significantly decreased. Growth zone diameter was recorded in 0.3% swimming LB semi-solid medium at 28℃ for 8 h to analyze the difference of swimming ability. We found that the swimming capacity of Vp2S01::catΔopaR was significantly increased by 2.67 times compared with Vp2S01::cat. The growth zone diameter was recorded in the 1.5% swarming LB solid medium at 28℃ for 12 h to analyze the differences of swarming ability. The results showed that there was no significant difference in swarming ability between Vp2S01::cat and Vp2S01::catΔopaR, but the colony of Vp2S01::catΔopaR has more missing. Using VcLMB29 as the receptor strain, the conjugative transfer efficiency of Vp2S01::cat and Vp2S01::catΔopaR at different time points was compared. It was found that the conjugative transfer efficiency of Vp2S01::catΔopaR increased after 12 h and 24 h. The 24-h conjugative transfer efficiency is increased by a factor of about 265.43. Thus, the RNA of Vp2S01::cat and Vp2S01::catΔopaR samples at 24 h of conjugative transfer were extracted and reverse-transcribed into cDNA as templates for quantitative fluorescence PCR (qPCR). GyrB was used as the reference gene, and the primers aphA-qRT, opaR-qRT and T4SS-qRT were used for qPCR detection. The relative expression rate of genes was calculated by 2-△△CT. The results showed that expression level of T4SS gene in Vp2S01::catΔopaR experimental group was significantly increased by 1.13-3.21 times, and the relative expression levels of conjugative-transfer key genes traF, trbE and traG were significantly increased by 1.96 times, 1.92 times and 3.21 times, respectively.
In summary, the opaR gene does not affect the growth characteristics and swarming motility, however it does affect the biofilm-forming ability and swimming motility of Vp2S01::cat. The high cell density regulator OpaR may affect the virulent plasmid’s conjugative transfer efficiency by regulating the expression levels of T4SS gene. This study provides fundamental data for analyzing the mechanism by which the QS system regulates the expression of T4SS and conjugative transfer of pVA1-type virulent plasmid in AHPND pathogenic bacteria, which can provide technical support for the control of virulent plasmid horizontal transfer in AHPND pathogenic bacteria. |
