Expression Profile Analysis Of Host HeLa Cells Invasived By Shigella Flexneri 2a

The infectious disease is result of an intimate interaction between pathogen and host. Shigella is a major endemic health threat,and HeLa cells provide an ideal infection model for infection because the cell lines have been widely used for studying Shigella invasion.To uncover the mechanism by which Shigella induces diarrhea,and responsive mechanism of epithelial cells invaded by S.flexneri 2a,the changes of genes expression in HeLa cell during the invasion with Shigella species were analyzed by cDNA microarrays.At first,we constructed the invasion model of HeLa cell infection assay by using the invasive assay coupled with Fuchsin basic-Methylene blue stain.Approximately 106 HeLa cells were co-cultured with about 108 CFU S.flexneri 2a 2457T at 37℃for 3 h,the ratio of bacteria infection counted out was 50%.Under these conditions,the changes of genes expression in HeLa cell during the invasion with Shigella were analyzed by cDNA microarrays.The data of cDNA microarrays showed that the expression levels of 836 genes were altered twice or greater as compared with the control 517 of them were up-regulated,and 319 were down-regulated.Through bioinformative analysis,the different genes involved in several important intestinal functions,including DNA Replication;recombination and repair; DNA-binding transcription;Signal transduction mechanisms,related metabolism;Proteins translation;Posttranslational modification,protein turnover,chaperones;xenobiotic metabolism,and development.It was supposed that some signal pathways in HeLa cell were activated,such as cAMP,Ca²⁺-CaM,PKC,Ras and P38/MAPK,then many genes were induced.However,some signal pathway(PI3K and MKK4/MAPK ) were shut off.As a result,many specific proteins were induced,including enzymes,the cytoskeleton,cell factors,and at last comprehensive cell responses were produced,so that HeLa cell could prevent against Shigella species infection.To further investigate and test the differential mRNA expression of new ESTs within the HeLa epithelial cells(IECs) following infection with S.flexneri 2457T,hybrid selection on high-density cDNA microarrays was used to characterize the mRNA expression profile of～4000 ESTs,which seventy-six were determined as differential expression with＞3 fold or＜0.33 fold changes.Then these EST sequences were extended electronically with the software of Siclone.Tewnty-five extended sequences were identified as known genes involved in several important intestinal functions.Their major functional categories contributing most to the total of differentially regulated genes are those involved in the cytoskeleton,cell adhesion,transport,and cell apoptosis.Genes involved in ion transport, ion channel function,and cell apoptosis were over-represented in the set of up-regulated genes,potentially contributing to diarrhea.The largest group consisted of cytoskeleton associated proteins such as actin,talin,cofilin,and gelsolin,followed by ion transport proteins such as calpain-5,Nedd4 binding proteinâ… (NAP+ channel ubiquitination),and Calcium-transporting ATPaseâ… in the host epithelial cell during Shigella infection. Interestingly,three striking difference cDNA fragments from unknown ESTs were cloned, sequenced by RT-PCR,and analyzed expression by real time RT-PCR.We validated they were new human genes or pseudo genes,and had submitted their information to GeneBank accession nos.UAY776160U,UAY776161U,and UAY776162U.Based on the results of gene expression alterations,the molecular pathogenic mechanism of Shigella species could be greatly and deeply understood,and the strategy for prevention against and treatment for shigellosis would be formed.