Study On The Mechanism Of Eriocheir Sinensis Infecting 3T6 Cells In Mice

The genus Spiroplasma,belonging to the Class Mollicutes,comprises a group of small,wall-less,helical,motile and genome-reduced tiny bacteria.Spiroplasma eriocheiris was previously identified as a causative pathogen of Chinese mitten crab Eriocheir sinensis causing the tremor disease(TD),and was the first spiroplasma pathogen found in aquatic crustaceans.Phylogenetic analysis of the 16S rRNA gene sequences of S.eriocheiris showed that this spiroplamsa strain had a close,98%similarity with Spiroplasma mirum isolated from rabbit ticks.It was confirmed that S.mirum can infect suckling vertebrates and cause cataract.It can also proliferate in chicken embryos,resulting in embryo death.Like S.mirum,S.eriocheiris also had the ability to infect the newborn mice(adult mice are not infected)and cause cataract,and the same situation in chicken embryos,S.eriocheiris could be propagated in the yolk sacs of chicken embryos,and transmit to all the tissues and subsequently appeared in the brains of embryonated chickens.The histopathology of spiroplasma infection was evident,but the infection mechanism was almost blank.Based on the in vitro S.eriocheiris infection models we had established,the 3T6-Swiss albino cells,which had been proven to be the target cells of S.mirum,we employed an integrative methodology to combine data from the two global profiling technologies pathogenesis studies(global mRNA and microRNA expression profiling)to explore more with bioinformatics analysis.At the same time,we used a yeast two-hybrid(Y2H)assay to analysis the Adhesin-like Protein(ALP)of S.eriocheiris,an important protein involved in the interaction between pathogen and host,and plays an important and complex role in the process of bacterial entry into the host cell.All these can be used for further study on molecular mechanism of how S.eriocheiris can infect the suckling mouse(vertebrate)beyond its natural invertebrate hosts.1.mRNA microarray analysis in 3T6 cells infected with S.eriocheirisIn order to study the molecule mechanism of S.eriocheiris infection the 3T6 cells,the total RNA were extracted using RNeasy Kit before and after S.eriocheiris infection 6h of 3T6 cells respectively.The RNA quality and yields were analyzed using Agilent Bio-analyzer and Nanodrop.Only cases with RNA Integrity Numbers(RIN)>7 and 28S/18S>0.7 were used for further study.Gene expression analysis was carried out on the Agilent mouse GE 4x44K v2 microarray contains 39,430 probesets.Total RNA(1?g)was reverse transcribed with T7-oligo(dT)primer and labeled with Cy3-dCPT using Agilent Low Input Quick Amp Labeling kit according to the manufacturer's instructions.The methodology and criteria used for total RNA purification,cRNA sample processing and hybridization to hamster microarrays according to protocol.Total RNA of 6 samples(3 uninfected 3T6 samples and 3 infected 3T6 samples)were labeled and hybridized separately in different slides to generate technical replicates.From differential expressions analysis of the mRNA data we identified 619 non-redundant annotated transcripts(183 up-regulated and 436 down-regulated)among 39430 transcripts in 3T6 cells using Agilent mouse GE 4x44K v2 microarray after 6h S.eriocheiris infection compared to control group(p<0.05).We used DAVID online database to analyse the functional enrichment of significant differentially expressed genes,and the distribution in protein interaction network was studied based on STING online database.Combined with GO and KEGG pathway analysis,we found that the differentially expressed genes focused on Neurotrophin signaling pathway,Alzheimer's disease,Insulin signaling pathway,Focal adhesion,Axon guidance and other 14 pathways.2.microRNA microarray analysis in 3T6 cells infected with S.eriocheirisThe microRNA microarray analysis was performed by LC Sciences using their MRA-1002 miRmouse v20 microarrays based on Sanger miRBase Release 20.Multiple control probes are included in each chip.Among the control probes,PUC2PM-20B and PUC2MM-20B are the perfect match and single-based match detection probes,respectively,of a 20-mer RNA positive control sequence that is spiked into the RNA samples before labeling.Total RNA of 6 samples were the same as the mRNA microarray analysis above.One may assess assay stringency from the intensity ratio of PUC2PM-20B and PUC2MM-20B,which is normally larger than 20.One-way ANOVA test was used to compute the p value.Clustering and principal component analysis(PCA)were performing using Cluster 3.0 and TreeView.The expression profiles of 1777 mature miRNAs were assessed using miRNA microarrays(Sanger miRbase v20.0)in 3T6 cells in uninfected and S.eriocheiris-infected groups at 6h.In total,1982 miRNAs in 3T6 cells were identified.Among these,22 miRNAs were differential expressions(8 up-regulated and 14 down-regulated)after 6h S.eriocheiris infection compared to control group.We used TargetScan,miRanda and PicTar software to predict the potential 22 differentially expressed miRNA targets based on the 'seed' sequence,and 3781 miRNA targets were found.Among 22 miRNAs,5 miRNAs(2 up-regulated and 3 down-regulated)have a signal intensity of<500.The result suggests a unique miRNA expression pattern in 3T6 cells under S.eriocheiris infection status.3.RNA conjoint analysis and functional verificationThe homologous genes of differential expression miRNA targets and the differential expression genes of the mRNA microarray were collected and 177 common genes were used to do RNA conjoint analysis.The common genes of significant changed miRNA negative correlation target genes and significant changed mRNA was analyzed by KEGG pathway,and the analysis results showed that focal adhesion and MAPK signaling pathway were two important pathways to S.eriocheiris infection.To validate the results of microarray,eight mRNAs from the two important pathways were selected in quantitative real-time PCR and Western blotting to confirm the results:focal adhesion(?-Catenin,Parvin,Grb2 and ERK)and MAPK signaling pathway(FGFR,Grb2,ERK,MKK3,p38 and JNK),GAPDH as the housekeeping gene.The changed trends of ?-Catenin(up-regulated),Parvin(up-regulated),FGFR(down-regulated),ERK(down-regulated),MKK3(down-regulated)and p38(down-regulated)from microarray and qPCR analysis were same.The Grb2 was down-regulated in microarray analysis,but was not change significantly in qPCR.The results of Western blotting were in agreement with the genes expression trends in transcription level.Eight miRNAs(miR-143-3p,miR-214-5p,miR-322-3p,miR-328-5p,miR-351-5p,miR-466h-5p,miR-503-5p and miR-30c-1-3p)and the housekeeping gene U6 miRNA were assayed by qPCR to confirm the results of microarray.Five miRNAs from MAPK signaling pathway were all up-regulated both in microarray and qPCR analysis.We speculate that when the 3T6 cell was infected by the bacteria,the MAPK pathway were restrained,and the cells normal metabolism were destroyed,so the S.eriocheiris was more likely to infect the cell.4.The role of ALP in the process of 3T6 cell infected by S.eriocheirisIn this study,we use immunogold electron Microscopy and Western blotting to analyze the location of the ALP from S.eriocheiris.Transmission eletctron microscope(TEM)immune-gold labeling showed that native ALP localizes on the surface of the S.eriocheiris.After preparation of membrance protein,cytoplasmic protein,and total protein of S.eriocheiris,ALP aniserum(1:2000)was used to determine whether immune-accessible domains were present on the S.eriocheiris surface using western blotting.In agreement with TEM immune-gold labeling,the results showed that the ALP signal was detected in the lanes of total protein and membrane proteins but not in cytoplasmic protein,thus indicating that ALP is exposed on the surface of S.eriocheiris.We also found ALP to be involved in the bacterium's infection of mouse embryo fibroblasts(3T6-Swiss albino)using antibody neutralization experiment.The S.eriocheiris were incubated with anti-ALP for lh at 30?,and then bacterial adhesion and infection of 3T6 cells were monitored.The results showed that the quantities of S.eriocheiris infection within the 3T6 cell interior were significantly lower than that in the control group at 36h post-inoculation.In short,ALP as a S.eriocheiris surface protein,appears to be an effector protein involved in the direct interactions with eukaryotic proteins and was critical for S.eriocheiris infection.5.Screening and functional identification of ALP interaction proteinsWe used the Yeast Two-Hybrid System to screen the mouse protein that could interact with S.eriocheiris ALP.The results showed that ALP could interact with 137 gene fragments in mouse.The interactions between recombinant partial fibulin7(FBLN7;including two epidermal growth factor[EGF]domains)and ALP were confirmed by Far-western blotting and colocalization.We synthetized the domains of FBLN7[EGF domain:amino acids 136-172 and complement control protein(CCP)domain:81-134 amino acids],and demonstrated that only EGF domain of FBLN7 can interact with ALP.Because the EGF domain has high degree of similarity to EGF,it can activate the downstream EGFR signaling pathway,in key site amino acids.The EGFR pathway in 3T6 cells was restrained after rALP stimulation resulting from competitive binding of ALP to EGF.The unborn mouse,newborn mouse,and the adult mouse with cataract have a small amount of expressed FBLN7;however,none was detected in the brain and very little expressions were seen in the eyes of normal adult mice.The conclusion was that ALP played a role in S.eriocheiris infection process by competitive effecting the EGF/EGFR axis of the target cells.