Transcriptional Response To Aeromonas Hydrophila And Functional Analysis Of The Key Genes TLR25 And IRF1 In Schizothorax Prenanti

Schizothorax prenanti(S.prenanti),a regional special economic cold-water fish with high nutritional,cultural and economic values,is very popular in the local aquatic product market.Intensive farming is the main aquaculture pattern for S.prenanti,but the highdensity environment is easy to induce various diseases caused by pathogenic microorganism,such as Aeromonas hydrophila(Ah)-induced bacterial septicemia.Ah is an opportunistic pathogen that wildly distribute in the nature,especially in water environment,and give rise to great harm to aquaculture since it is primary pathogen for fish farming.Ah belongs to Gram-negative bacterium,and its Lipopolysaccharide(LPS)distributed in the outermost layer of bacterial cell wall is one of the crucial pathogen-associated molecular patterns(PAMPs)for activating organism immune response.The recognition and elimination of invasive LPS by fishes and mammals exists a major difference,but the mechanism is still poorly understood.To illuminate the mechanism of immune response against Ah and LPS in S.pregnant,this study carried out a comprehensive understanding of including transcriptional response mechanism after Ah and LPS stimulations in S.pregnant,screening of the crucial functional genes and immune function of the candidate genes toll-like receptor 25(TLR25)and interferon regulatory factor 1(IRF1)by transcriptome analysis and molecular biology approaches.The experiments and results as followed:Part 1: Transcriptional response to Ah in S.prenanti.This study firstly observed pathological changes by HE stained section,which showed the increased lymphocytes in the spleen of S.prenanti after infection with Ah.Then,RNA-seq was used to analyze the gene expression profiles,especially for the expression profiles of immune-related genes,in the spleen of S.prenanti after infection with Ah.A total of 6,213 different expression genes(DEGs)were obtained,including 3,066 up-regulated DEGs and 3,147 down-regulated DEGs.These DEGs underwent KEGG and GO enrichment analysis,so that the immunerelated DEGs(IRDs)can be identified by the enrichment in immune-related pathways of KEGG database and subtypes of GO database.Then,the interesting IRDs,including TLRs,complements,cytokines and other signaling molecules,were screened to build heat map,and the reliability of the transcriptome data was validated by q PCR.According to the expression levels of the IRDs and the known research,the potential signaling pathway initiated by TLRs was predicted.In this pathway,TLR25 and TLR5 mediate the NF-κB and AP-1 signaling pathways.Meanwhile,the type I IFNs induced by IRF1 and IRF3/7may play an important role in the anti-bacterial immunity of S.prenanti.This study preliminarily reveals the transcriptional response mechanism of IRDs after Ah infection in S.prenanti,which provides a theoretical foundation to further research the molecular mechanism of anti-bacterial immunity in fishes.Part 2: Transcriptional response to LPS form Ah in S.prenanti.This study firstly observed pathological changes by HE stained section,which showed the aggregated erythrocytes in the blood sinus of spleen,and the increased lymphocytes and the decreased granulocytes in head kidney in S.prenanti after stimulation with Ecoli.LPS.Anti-LPS immunohistochemical analysis indicated that the immune cells of spleen and head kidney could interact with LPS.Then,RNA-seq was used to analyze the gene expression features,especially for the expression of immune-related genes,in the spleen of S.prenanti after stimulations with two sources of LPS extracted from Ah and Escherichia coli(Ah.LPS and Ecoli.LPS),respectively.After Ah.LPS and Ecoli.LPS stimulations,921 and 975 DEGs were acquired,respectively,of which Ah.LPS and Ecoli.LPS stimulation shared 470 DEGs.These DEGs underwent KEGG and GO enrichment analysis to screen IRGs,showing 88 shared IRGs after Ah.LPS and Ecoli.LPS stimulation.Among the shared IRGs,four pattern-recognition genes(TLR5,TLR25,PTX3 and C1q)were induced with high expression foldchange,which may associate with LPS recognition and the signal transduction.Meanwhile,inflammatory signals were highlighted by up-regulating the expression of inflammatory cytokines(IL-1β,IL-10 and IL-8).Moreover,some non-shared IRGs(including TLR2 and TLR4)were identified,suggesting that different sources of LPS own different potentials for the induction of immune gene expression.Moreover,the expression change of the potential functional genes for LPS recognition and the signal transduction was analyzed in S.prenanti head kidney primary macrophages and granulocytes after LPS stimulation.Finally,according to the expression levels of the IRDs and known research,TLR5,TLR25,PTX3 and C1 q may be the critical molecules of LPS recognition and signal activation in S.prenanti.Amongst,TLR25/TLR5 may mediate the activation of inflammatory signal,while PTX3/C1 q may modulate the activation of complement pathway.This study preliminarily reveals the transcriptional response mechanism of IRDs after LPS stimulation in S.prenanti,which provides a theoretical foundation to further research the molecular mechanism of anti-LPS immunity in fishes.Part 3: Transcriptome-based evaluation and validation of the reference gene for quantification real-time PCR.Quantification real-time PCR(qRT-PCR)is a common method in analysis of gene expression,but selection of stable reference genes for the normalization analysis must be very cautious.In this study,we selected eight candidate reference genes(18S,Actin,EF-1α,40 S,B2M,TUBA,UBCE and GAPDH)basing on transcriptome analysis and the traditional housekeeping genes,and analyzed the stability of the reference genes in spleen,head kidney and head kidney leukocytes(HKLs)after pathogen stimulations in S.prenanti.Three common programs(ge Norm,Norm Finder and Bestkeeper)were used to evaluate the stability of the candidate reference genes.Two reference genes,Actin and EF-1α presented higher stability,while 18 S and GAPDH were the genes with low stability.Toll-like receptor 22a(TLR22a)was used to validate the stability of the candidate reference genes(Actin and EF-1α)across different experiment treatments,the results showed that Actin and EF-1α are quite suitable reference genes for quantification real-time PCR under the condition of the pathogen stimulations in S.prenanti(stability: Actin > EF-1α).This study acquired the suitable reference gene for the qRT-PCR assay under specific experiment condition in S.prenanti,which laid a foundation for accurate analysis of gene expression in S.prenanti.Part 4: Function analysis of the crucial genes TLR25 and IRF1 for anti-bacterial immune responses in S.prenanti.Based on the transcriptome data,this study screened 59 IRGs,including the potential key genes TLR25 and IRF1,that highly expressed after Ah and LPS stimulations in S.prenanti.Next,the coding sequence(CDS)of TLR25 was cloned from S.prenanti(named sp TLR25),and sp TLR25 is 2,454 bp in length and codes a protein of 817 aa.The sp TLR25 protein contains a signal peptide,twenty leucine-rich repeat(LRR)domains,a LRR C terminal(LRRCT)motif,a transmembrane region and a Toll/IL-1 receptor(TIR)domain.Phylogenetic analysis indicates that sp TLR25 has the closest relationship with Cyprinus carpio TLR25-2.The 3D structure of sp TLR25 exhibits5 α-helices and 3 β-sheets in the TIR domain,and 8 α-helices and 6 β-sheets in the LRR domains.Tissue expression distribution showed that sp TLR25 is mainly expressed in immune-related tissues and peripheral blood leukocytes(PBLs).Furthermore,the expression levels of sp TLR25 were upregulated in spleen,head kidney and liver when S.prenanti was challenged with LPS or Ah,and the upregulation was also detected in HKLs after LPS and Poly(I:C)stimulations.Dual-luciferase reporter system analysis indicates that sp TLR25 is able to activate the downstream NF-κB signaling pathway,and the TIR and LRR domains of sp TLR25 play important roles in the signal activation.Interaction between sp TLR25 and the downstream adapter molecules(sp My D88,sp TIRF and sp TIRAP)was analyzed via dual-luciferase reporter system and GST-pulldown analysis,which reveals that sp TLR25 directly interacts with sp My D88,suggesting that sp TLR25 activates the downstream NF-κB signaling pathway via My D88-dependent pathway.Subcellular localization showed that sp TLR25 was located to intracellular region.Finally,the special recognition of sp TLR25 to PAMPs was screened via dual-luciferase reporter system,suggesting that sp TLR25,instead of recognizing PAMPs,may be an “equilibrium molecular” during the signal transduction.In this study,the coding sequence(CDS)of IRF1 was also cloned from S.prenanti(named sp IRF1),and sp IRF1 is 867 bp in length and codes a protein of 288 aa.Multiple sequence alignment showed that sp IRF1 is composed of a conservative N-terminal DNA binding domain(DBD),a transactivation domain(TD)and a C-terminal IRF association domain 2(IAD2).Luciferase reporter assay found that IRF1 could activate type I IFNs signaling pathway.In Ah-and LPS-stimulated S.prenanti,the expression levels of sp IRF1 and sp IFN were increased in different degrees,of which Ah-induced the expression levels of sp IRF1 and sp IFN were highest and showed the potential relevance between them.Further,overexpression of sp TLR25/sp IRF1 in EPC cells showed that sp IRF1 could activate type I IFNs signaling pathway and sp TLR25 synergistically regulated sp IRF1-induced activation of type I IFNs.This study preliminarily reveals that TLR25-mediated NF-κB signaling pathway and IRF1-induced type I IFNs signaling pathway may play critical roles in anti-bacterial and anti-LPS immune responses in S.prenanti,which lays a foundation for investigating the immune mechanism of fish TLR25 and IRF1.In conclusion,this study revealed the transcriptional response mechanism of immunerelated genes to Ah and LPS via RNA-seq and identified various potential effector genes in S.prenanti.Combination with the RNA-seq results,the stable reference gene for qRT-PCR analysis was screened and evaluated in S.prenanti under the condition of immune stimulations,and Actin was identified as the optimal reference gene.Next,TLR25 and IRF1 were identified as the key effector genes for anti-bacteria immune response in S.prenanti.Function analysis of TLR25 was carried out via a series of molecular biology methods,which uncovers that TLR25 responds to various pathogens,activates NF-κB signal cascade via My D88-dependent manner,and synergistically regulates IRF1-induced type I IFNs signaling pathway.This study provided a theoretical foundation to the immunological research and the control and prevention of disease in fishes.