Large Scale Screening And Identification Of Interaction Genes Between Pathogenic Rickettsia And Ticks

Research BackgroundAs obligate blood-sucking arthropods,ticks can transmit a wide range of viral,bacterial,and protozoan pathogens to their hosts through blood-feeding,resulting in a huge public health burden.Studies have shown that Lyme disease has spread throughout the United States,and the livestock industry in China has also been greatly affected by tick-borne African swine fever virus(ASFV).In recent years,with the sharp increase in the demand for efficient control of tick-borne diseases,the development of tick genomics,the successful establishment of tick cell lines and the widespread application of gene editing technologies RNA interference(RNAi)and CRISPR to study tick gene function,tick-borne pathogens develop in ticks,the interaction of reproduction,immunity and transmission,has become a research hotspot.At present,foreign research on tickpathogen interaction focuses on the role of Borrelia burgdorferi and Anaplasma phagocytophilum on related genes in ticks,while there are few basic researches on ticks in China.Rickettsia is a kind of strict G-intracellular parasite,which naturally parasitizes in arthropods such as ticks,mites and fleas.After ticks bite the host,the Rickettsia directly enters the human body at the bite site,causing zoonotic diseases such as endemic typhus,epidemic typhus and so on.Based on our available complete genome data of the six major tick genera,we found that Rickettsia was present in all major tick genera through analysis,comparison and annotation,but different tick genera carried Rickettsia in different abundance,and the abundance of Rickettsia carried by the same tick genus was also different.In order to continue to study the internal reasons,and consider the CTVM23 cell line in our lab,we compared the positive rate of Rickettsia between the evolutionary branches of different clades of ticks,then selected a number of genes related to the interaction between ticks and Rickettsia as the research object.Through knocking down the gene expression by RNAi technology,and observed its effect on the growth and replication of Rickettsia in CTVM23 cell lines、IDE8 cell lines and THP-1 cells.Research Objectives(1)Building an interactive gene screening scheme and technical system that combines bioinformatics and laboratory technology to establish a potential genetic library for the interaction between pathogenic Rickettsia and ticks;(2)Establishing an vitro RNAi experiment technique on various tick cells and human cell lines;(3)Identifing a group of genes or gene families related to the growth and replication of pathogenic Rickettsia by using RNA interference technology.Research content(1)Different clades of Rhipicephalus microplus(R.microplus)samples was carried out,and the differences positive rate of Rickettsia among different clades were also analyzed.The differential genes positively correlated with the positive rate of Rickettsia among different clades were screened.The interaction genes of Rickettsia raoultii(R.raoultii)were screened by CTVM23 cells in vitro,and the interaction gene library of R.raoultii and ticks was established;(2)dsRNA was designed for the above genes,and its expression at mRNA level was knocked down by RNAi.To observe the growth and replication of Rickettsia in tick cells after gene knockdown and explore its potential correlation.(3)Based on the experimental results of RNAi in CTVM23 cells,the effect of RNAi of other tick cells and human cells on the growth of Rickettsia was investigated.Research Methods(1)Cluster analysis was carried out on the genome of the existing R.microplus in our research team.After comparing the positive rates of Rickettsia among different clades,the differential genes were screened to establish an alternative gene library for RickettsiaR.microplus interaction genes.Then,the CTVM23 cells were infected with R.raoultii,and the expression levels of candidate genes at different time points were analyzed by q RT-PCR,and the genes with statistical differences were screened to establish R.raoultii-R.microplus potential interacting gene pool.(2)The dsRNA was designed for genes in the reciprocal gene pool,and the dsRNA was transfected into CTVM23 cells by transfection reagent to observe its knockdown effect on the target genes in cells.For genes that can be successfully knocked down,CTVM23 cells were infected by R.raoultii and then transfected with dsRNA into cells to observe the effect on the growth and replication of R.raoultii.(3)Referring to the experimental results in CTVM23 cells,genomic data in NCBI were used to search for homologous gene sequences between homo sapiens;Ixodes scapularis and R.microplus,mainly Calreticulin and Histone genes.After designing dsRNA/si RNA and successfully knocking down the target genes,we investigated the effect of RNAi on the growth and replication of Rickettsia in other tick cells and humanderived cell.Research Results(1)Screening and establishment of a R.raoultii-R.microplus cell potential interaction gene pool.The genotyping of R.microplus populations allowed us to classify R.microplus samples into three clades,and after differential analysis of Rickettsia positivity,99 genes were screened as positively associated with Rickettsia infection;after comparing the transcriptome second-generation sequencing data of CTVM23 cells with the genomic RNA Seq data of R.microplus samples,we obtained 46 genes that were simultaneously expressed in the CTVM23 cell genome and the adult tick genome of R.microplus.After in vitro screening of CTVM23 cells,a total of 28 gene sequences were found to have increased expression at different time points after R.raoultii infection in CTVM23 cells,which was statistically significant(P < 0.05),and the R.raoultii-R.microplus gene library was established.(2)After RNAi,the replication and growth of R.raoultii in CTVM 23 cells were reduced.A total of 17 pairs of dsRNA were synthesized through Snap Dragon design.Then dsRNA was transfected into tick cells by transfection reagent,the expression of gene mRNA was obtained by q RT-PCR.After statistical analysis,a total of 10 pairs of dsRNA could effectively knock down the expression of target gene at the mRNA level,which was statistically significant(P < 0.05).Next,CTVM 23 cells were infected with R.raoultii.After 12 hours,dsRNA was transfected into tick cells.The expression level of each target gene and the changes of growth and replication of R.raoultii in cells were measured by q RT-PCR.The experimental results showed that the six candidate tick genes effectively reduced the growth and replication of R.raoultii in tick cells(P < 0.05).(3)The Homologous Calreticulin and Histone genes in I.scapularis and Homo Sapiens reduce the growth and replication of R.raoultii in cells.NCBI Blast was used to find the homologous sequences in and Homo Sapiens which have been already confirmed of affecting the growth and replication of R.raoultii in CTVM23 cells,and it was found that Calreticulin and Histone were closely related to R.microplus.Based on these two sequences,dsRNA / si RNA was designed and proved by experiments.dsRNA / si RNA of the above genes effectively knocked down the mRNA expression level of endogenous target genes in IDE8 cells and THP-1 cells,which was statistically significant.In the same way,IDE8 cells and THP-1 cells were infected with R.raoultii to observe the effect of the above genes.The results showed that knocking down the expression of Calreticulin and Histone in IDE8 cells and THP-1 cells reduced the replication level of R.raoultii in cells,with statistical difference(P < 0.05).Research Conclusions(1)A total of 99 genes were obtained after large-scale genomic screening of R.microplus and comparing the differences in Rickettsia positivity rates among different clades.A total of 46 genes were simultaneously expressed in the genome of CTVM23 cells and the genome of adult R.microplus after the next generation sequencing.27 genes showed a significant increase in mRNA expression after CTVM23 cells’ infection with R.raoultii and were used as alternative genes for R.raoultii-R.microplus potential gene library.(2)Among the 17 pairs of dsRNAs designed for the gene sequence of R.microplus,10 pairs can significantly reduce the expression of endogenous genes of R.microplus at the mRNA level,but not necessarily at the protein level.In addition,6 pairs of dsRNAs affect the growth and replication of Rickettsia in tick cells after knocking down the gene expression level of R.microplus.(3)The knockdown of Calreticulin and Histones not only affected the replication of Rickettsia in CTVM23 cells,but reduced the proliferation of Rickettsia in IDE8 cells and THP-1 cells.It shows that Calreticulin and Histones play a greater role in the tickRickettsia-host interaction process,which is worth further exploration.Innovation Points1.Based on the metagenomic data and hypothesis obtained from the long-term work of our research group,we found that different clades of R.microplus carry different Rickettsia abundance,and we conducted a large-scale screening of pathogenic RickettsiaR.microplus reciprocal genes through a combination of bioinformatic analysis and in vitro experiments.Then we established an alternative library of R.raoultii-R.microplus interactive genes.2.In this study,characteristic double-stranded RNAs(dsRNAs)targeting endogenous genes of R.microplus were successfully designed,among which 10 pairs of dsRNAs effectively knocked down the mRNA expression of their endogenous target genes;3.This study found that the same tick species carried different Rickettsia abundances.Based on this,we screened out 6 genes that affect the growth and replication of Rickettsia in R.microplus,The interactions between these six genes and Rickettsia are not yet reported in the paper,and further clarification of the specific mechanisms of their interactions with Rickettsia is urgently needed;4.Based on the homology sequence between Homo Sapiens;I.scapularis and R.microplus,further explorations have been done that Histones not only affect Rickettsia replication in CTVM23 cells,but also in IDE8 cells and THP1 cells.Furthermore,although calreticulin did not affect Rickettsia replication in CTVM23,it reduced Rickettsia replication levels in IDE8 cells and THP1 cells.