The Functional Study Of Secreted Protein NsICL From Nocardia Seriolae

Nocardia seriolae is the main pathogen of fish nocardiosis because of its wide distribution and high pathogenicity.The incidence of fish nocardiosis has been increasing year by year,and the lacks of effective prevention and control measures,which caused huge economic losses to the aquaculture industry.Besides,the virulence factors and pathogenic mechanisms of N.seriolae are still unclear.Through the whole genome analysis of N.seriolae ZJ0503,an open reading frame(ORF)0653 encoding an isocitrate lyase(ICL)was discovered and named as NsICL.Isocitrate lyase is a key enzyme of the glyoxylic cycle(tricarboxylic acid cycle branch)with functions of catalyzing the cleavage of isocitrate into succinic acid and glyoxylic acid by passing the two basic steps of decarbonation in the tricarboxylic acid cycle,and changing the flow direction of the carbon source.The glyoxylic acid cycle is a metabolic branch that widely exists in prokaryotes,lower eukaryotes and plants,excluding vertebrates.Recent studies have shown that the glyoxylic cycle is highly related to the virulence of bacterial and fungal pathogens,thus NsICL is a potential virulence factor of N.seriolae.In this study,the NsICL gene was cloned,and bioinformatics analysis was performed according to its sequence.Further more,the mass spectrometry result of the collected extracellular products of N.seriolae verified that NsICL is a secreted protein.To explore the influence of deletion of NsICL gene on the physiological characteristics and the virulence of N.seriolae,a NsICL gene deletion strain(NS-(35)ICL)and a complement strain(NS-cICL)were constructed via homologous recombination technique.No differences of hyphae morphology and growth curves of the liquid medium between NS-(35)ICL,NS-cICL and the wild strain were discovered.In order to explore the effect of the NsICL gene on the intracellular viability of N.seriolae at cellular level,three strains were co-incubated with the primary macrophages of hybrid snakehead respectively.There was no significant difference in the ability of the three strains to induce the respiratory burst of macrophages,but the survival ability of NS-(35)ICL in macrophages was significantly declined.Moreover,the half-lethal concentration of NS-(35)ICL on the hybrid snakehead(2.60×10~5 CFU/m L)was significantly lower than those of NS-cICL(4.26×10~4 CFU/m L)and wild strains(4.32×10~4 CFU/m L),indicating that the virulence of N.seriolae was reduced by the deletion of NsICL.To sum up,the NsICL protein is a the secreted protein of N.seriolae,and thedeletion of NsICL gene does not affect its mycelial morphology and growth curve.The intracellular viability is significantly dropped,and the half-lethal concentration of the NS-(35)ICL was one order of magnitude higher than those of the NS-cICL and the wild strain.The NsICL gene deletion verified that ICL play an important role on the pathogenicity of N.seriolae.In this study,the secreted protein NsICL of N.seriolae was studied,and the role of NsICL in the pathogenicity was preliminarily elucidated,which provided a theoretical basis for the prevention and treatment of fish nocardiosis.