Vibrio parahaemolyticus becomes lethal to post-larvae shrimp via acquiring novel virulence factors

ABSTRACT Translucent post-larvae disease (TPD), caused by Vibrio parahaemolyticus ( Vp TPD ), has become an emerging shrimp disease, affecting more than 70%–80% of coastal shrimp nurseries in China in spring 2020. Here, we investigated the key virulence factors of Vp TPD by analyzing protein fragments, related genomic information, as well as experimental challenge tests. After investigating the toxic effects of purified protein fragments with different molecular weights (MWs) from Vp TPD, we found that only the protein fragments with MW >100 kDa showed similar lethality to live Vp TPD in the experimental challenge test using post-larvae shrimp. Meanwhile, similar histopathological changes exhibiting in the hepatopancreas and midgut of the diseased individuals were observed in the post-larvae shrimp challenged with either bacterial protein fragments (MW >100 kDa) or live Vp TPD . Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analyses, two novel proteins, Vibrio high virulent protein (VHVP)-1 and VHVP-2, were identified as the candidates of key virulence factors to cause TPD. Moreover, VHVP-1 and VHVP-2 were found to be encoded by two genes ( vhvp-1 and vhvp-2 ) tandemly located on a 187,791-bp plasmid and were predicted to depend on the same promoter following a comparative genomic analysis. Further epidemiological investigation and challenge test indicated that the V. parahaemolyticus isolate carrying only the vhvp-1 gene and lacking vhvp-2 gene could not cause mortality of experimental Penaeus vannamei post-larvae. The mutant (Δvhvp-2) by deleting vhvp-2 gene could only cause 4.92% of accumulative mortality of post-larvae that is similar to the non- Vp TPD Vibrio strain. Additionally, the complemented strains, Δvhvp-2/pBT3-vhvp-2 and Δvhvp-2/pwtCas9-vhvp-2, showed similar virulence to the wild-type Vp TPD . The results demonstrated that V. parahaemolyticus becomes lethal to post-larval shrimp by acquiring the VHVP-2 virulence factor. This study sheds light on further investigations of the pathogenic mechanism of Vp TPD and the development of strategies for early diagnosis of TPD in shrimp hatcheries. IMPORTANCE As a severe emerging shrimp disease, TPD has heavily impacted the shrimp aquaculture industry and resulted in serious economic losses in China since spring 2020. This study aimed to identify the key virulent factors and related genes of the Vp TPD , for a better understanding of its pathogenicity of the novel highly lethal infectious pathogen, as well as its molecular epidemiological characteristics in China. The present study revealed that a novel protein, Vibrio high virulent protein-2 (MW >100 kDa), is responsible to the lethal virulence of V. parahaemolyticus to shrimp post-larvae. The results are essential for effectively diagnosing and monitoring novel pathogenic bacteria, like Vp TPD , in aquaculture shrimps and would be beneficial to the fisheries department in early warning of Vp TPD emergence and developing prevention strategies to reduce economic losses due to severe outbreaks of TPD. Elucidation of the key virulence genes and genomics of Vp TPD could also provide valuable information on the evolution and ecology of this emerging pathogen in aquaculture environments.