Pathogenicity And Virulence Factors Of Nocardia Seriolae Isolated From Snakehead Fish

Nocardia seriolae is a Gram-positive aerobic bacterium that causes chronic infectious disease in fish with white nodules,which is the main pathogen of nocardiosis.Marine fish and freshwater fish can be infected,of which the fishes of Perciformes are more susceptible with serious infection.Guangdong province is the largest snakeheads production base in China.In recent years,with the development of high-density intensive culture of hybrid snakeheads,the disease caused by N.seriolae has occurred frequently,which has caused great economic losses to the aquaculture industry.In previous study,N.seriolae,NK201610020 strain,was isolated from a hybrid snakehead in a farm from Fashan.Pathogenicity and histopathology of the pathogen were investigated by experimental infections,LD₅₀ determination,histopathological analysis and cell infection.In order to investigate the pathogenic mechanism of N.seriolae and to further understand the biological information of N.seriolae at the genetic level,whole-genome sequencing was performed to obtain the full sequence of the genome of the test strain.Searches were performed against the virulence factor database full dataset.The phoP and phoR genes,which coding the PhoP-PhoR two-component system（TCS）of N.seriolae,were selected for bioinformatics analysis,gene cloning and protein purification.Pathogenicity and histopathological analysis of N.seriolae showed that except for the 1.5×10³ CFU/m L group and the control group,the mortality of other five experimental groups（1.5×10⁴CFU/m L to 1.5×10⁸CFU/m L）reached over 90%.The LD₅₀ was calculated by the Reed-Muench method as 1.079×10³ CFU/m L.When dissecting the infected fish,we found that there were a large number of well-defined,white nodules on the surface of the tissues.Histopathological observation showed that necrosis was obvious in liver,kidney and spleen,which were well-defined granulomatous structure with aggregation of epithelioid cells.The CCK-8 assay was performed to observe the mice macrophage Raw 264.7 infected with N.seriolae.We found that N.seriolae impaired the macrophages,and the longer the infection time,the more obvious the damage to macrophages.The whole genome sequencing of NK201610020 strain revealed that the strain consisted of a circular chromosome,with a size of 8 294 329 bp,and the overall GC content of the genome was 68.10%.Comparative genomics analysis showed that the gene sequences of N.seriolae from the same host were highly similar,with a homology as high as 99.93%,and there was no obvious difference in evolution and geographical differences.The homology of the strain comparing other hosts from freshwater fish and marine fish was 99.2%-99.8%,and there were certain differences in genomic function of survival and metabolism between them..By analyzing the virulence genes of N.seriolae,there are 171 coding sequences in the genome of the bacteria as potential virulence factors related to cell wall synthesis,nutrient metabolism,bacterial persistence,and signal regulation.The two-component system（TCS）of PhoP-PhoR were selected and to study the amino acid homology among different bacteria species.The results showed that the amino acid sequences of Phop and PhoR were 99%similar to N.seriolae UTF1 strain from yellowtail in Japan,78%～88%similar to other species of Nocardia spp.and63%～78%similar to genus of Mycobacterium.The phoP and phoR genes were cloned and expressed by prokaryotic expression.The recombinant plasmids of p ET32a-phoP and p ET32a-phoR were constructed and then transformed into Escherichia coli BL21（DE3）for protein expressions.The predicted molecular weights of the recombinant proteins were about 50 k Da and 70 k Da,respectively,which laid the foundation for further study of the functions of the PhoP-PhoR two-component system.In conclusion,N.seriolae NK201610020 has strong virulence,can cause granuloma nodules in tissues such as liver,spleen and kidney,and can impair mice macrophage.N.seriolae has a conserved genome sequence,and carries a variety of virulence factors related to cell wall synthesis,nutrient metabolism,bacterial persistence,and signal regulation.PhoP and PhoR proteins were purified and obtained,it provided a basis for further study on the functions of the PhoP-PhoR two-component system,and to illuminate pathogenic mechanism of nocardiosis.