Novel insights into host-pathogen interactions of large yellow croakers(Larimichthys crocea)and pathogenic bacterium Pseudomonas plecoglossicida using time-resolved dual RNA-seq of infected spleens

Host-pathogen interactions are highly complex,involving large dynamic changes in gene expression during infection. These interactions are fundamental to understanding anti-infection immunity of hosts, as well as the pathogenesis of pathogens. For bacterial pathogens interacting with animal hosts, timeresolved dual RNA-seq of infected tissue is difficult to perform due to low pathogen load in infected tissue. In this study, an acute infection model of Larimichthys crocea infected by Pseudomonas plecoglossicida was established. The spleens of infected fish exhibited typical symptoms, with a maximum bacterial load at two days post-injection(dpi). Time-resolved dual RNA-seq of infected spleens was successfully applied to study hostpathogen interactions between L. crocea and P.plecoglossicida. The spleens of infected L. crocea were subjected to dual RNA-seq, and transcriptome data were compared with those of noninfected spleens or in vitro cultured bacteria. Results showed that pathogen-host interactions were highly dynamically regulated, with corresponding fluctuations in host and pathogen transcriptomes during infection. The expression levels of many immunogenes involved in cytokine-cytokine receptor,Toll-like receptor signaling, and other immunerelated pathways were significantly up-regulated during the infection period. Furthermore, metabolic processes and the use of oxygen in L. crocea were strongly affected by P. plecoglossicida infection. The WGCNA results showed that the metabolic process was strongly related to the entire immune process.For P. plecoglossicida, the expression levels of motility-related genes and flagellum assemblyrelated genes were significantly up-regulated. The results of this study may help to elucidate the interactions between L. crocea and P.plecoglossicida.

Dual RNA-seq provides novel insight into the roles of dks A from Pseudomonas plecoglossicida in pathogenhost interactions with large yellow croakers(Larimichthys crocea)

Pseudomonas plecoglossicida is a rod-shaped,gram-negative bacterium with flagella.It causes visceral white spot disease and high mortality in Larimichthys crocea during culture,resulting in serious economic loss.Analysis of transcriptome and quantitative real-time polymerase chain reaction(PCR)data showed that dks A gene expression was significantly up-regulated after 48 h of infection with Epinephelus coioides(log2 FC=3.12,P<0.001).RNAi of five sh RNAs significantly reduced the expression of dks A in P.plecoglossicida,and the optimal silencing efficiency was 96.23%.Compared with wild-type strains,the symptoms of visceral white spot disease in L.crocea infected with RNAi strains were reduced,with time of death delayed by 48 h and mortality reduced by 25%.The dks A silencing led to a substantial down-regulation in cellular component-,flagellum-,and ribosome assembly-related genes in P.plecoglossicida,and the significant up-regulation of fli C may be a way in which virulence is maintained in P.plecoglossicida.The GO and KEGG results showed that RNAi strain infection in L.crocea led to the down-regulation of inflammatory factor genes in immune-related pathways,which were associated with multiple immune response processes.Results also showed that dks A was a virulence gene in P.plecoglossicida.Compared with the wild-type strains,RNAi strain infection induced a weaker immune response in L.crocea.

Transcriptomic and metabolomic insights into the role of the flgK gene in the pathogenicity of Pseudomonas plecoglossicida to orange-spotted grouper(Epinephelus coioides)

Pseudomonas plecoglossicida is the pathogen responsible for visceral white spot disease in large yellow croaker(Larimichthys crocea)and orangespotted grouper(Epinephelus coioides).Previously,RNA sequencing showed that P.plecoglossicida flgK gene expression was significantly up-regulated in orange-spotted grouper spleens during infection.To explore the role of flgK in P.plecoglossicida pathogenicity,RNA interference(RNAi)was performed to silence the P.plecoglossicida flgK gene,and the mutant(flgK-RNAi strain)with the best silencing efficiency(89.40%)was chosen for further study.Results showed that flgK gene silencing significantly attenuated P.plecoglossicida motility,adhesion,and biofilm formation.Compared to those fish infected with the wild-type strain of P.plecoglossicida,orange-spotted grouper infected with the flgK-RNAi strain showed a 55%increase in the survival rate and a one-day delay in time of first death,with fewer pathogens in the spleen and fewer white spots on the spleen surface.RNAi of flgK significantly affected the transcriptome and metabolome of the spleen in infected orange-spotted grouper.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis showed that the C-type lectin receptor signaling pathway was the most significantly changed immune-related pathway and the mitogen-activated protein kinase(MAPK)signaling pathway was related to multiple immunerelated pathways.Furthermore,arginine biosynthesis and glycerophospholipid metabolism were the most significantly changed metabolism-related pathways.These findings suggest that flgK is a virulence gene of P.plecoglossicida.Furthermore,flgK appears to be involved in the regulation of motility,adhesion,and biofilm formation in P.plecoglossicida,as well as in the regulation of inflammatory and immune responses of orange-spotted grouper to P.plecoglossicida infection.