Transcriptome Analysis Of Biofilm Formation In Mycobacterium Tubercolusis And Development Of Quantitative Real Time PCR Detection Method

Tuberculosis (TB) remains one of the world’s deadliest communicablediseases(WHO). Mycobacterium tuberculosis(M. tuberculosis), the causative agent ofhuman TB which is an ancient companion of man since before the neolithic age, tilltoday, makes an estimated9million people developed TB and kills about1.5millionpeople annually. The treatment of M. tuberculosis’s infection requires6-9months ofmultidrug chemotherapy since M. tuberculosis is crafty and resistant. However, theworldwide appearance of multidrug-resistant (MDR) tuberculosis and extensivelydrug-resistant (XDR) tuberculosis makes the multidrug chemotherapy uneffective andcostly in past decade.The former studies have shown that, M. tuberculosis with other bacteria likePseudomonas aeruginosa can form the biofilm in vitro and the biofilm-formingabilities of different strains of M. tuberculosis are different. Drug resistance andpathogenicity of M. tuberculosis involve the biofilm-forming abilities, and theformation of biofilm can improve the drug resistance of bacteria by10-1000times.Bioflm formation presents a serious challenge that is treatment of TB and as anestimated65–80%of all infections is thought to be bioflm-related.In this study,.we used high throughput sequencing technology to compare thetranscriptome of M. tuberculosis bioflim with the transcriptome of planktonicpopulations. we found a lot of differentially expressed genes(DEGs), these genes werecompared, screened and verified. We developed a quantitative real time PCR detection method which based on Rv3519, the ability of biofilm formation can bejudged by the method.The work was carried out in this study.1.We improved the culturing method of biofilm that Ojha had built, and solvedthe long-time, poor repeatability problem of M. tuberculosis biofilm culture in vitro.In order to unearth the marker genes in the formation process of M. tuberculosisbiofilm, Illumina sequencing technology was firstly used to catalog global geneexpression profiling and functional network analysis of M. tuberculosis bioflim andplanktonic populations in the world. The results showed that, we used50ml serumbottle to establish bioflms in modified Sauton medium costs much shorter time thanin the former study and bioflims growing in good state. Obtained clean reads wereassembled into4550unigenes.4007unigenes were matched the known sequence andwe discovered543novel genes. There were a total of437known genes identified asdifferentially expressed in biofilm cells versus planktonic cells, of which153weredown-regulated (2fold) and284were up-regulated (2fold).343differentiallyexpressed genes(DEGs) were grouped in GO categories, and114differentiallyexpressed genes had a specific KEGG pathway annotation. DEGs involved inbiosynthesis and metabolism of sulfur metabolism, steroid degradation, atrazinedegradation, and ABC transporters. Our results suggested that the regulation of thegenes may result in enhance amounts of sulfate transport system and sulfate-sulfurassimilation and steroid degradation, alonging with response to nutrient starvation andhypoxia. Genes involved in sulfur metabolism and steroid degradation may regardedas novel potential antibiotic targets for curing TB.2.Rv3519was chosen according to the RNA-seq results and former study. Inorder to explore the relationship between Rv3519gene expression and the level of their biofilm formation ability. Biofilm formation ability of M. tuberculosis wasmeasured by crystal violet staining. Quantitative real time polymerase chain reaction(qRT-PCR) was used to detect the levels of expression of Rv3519gene in the biofilmsof different M. tuberculosis stains. Results showed that the stains with strongertendency to form biofilm can express more Rv3519mRNA, in contrary, which have alow expression. In short, there was a correlation between the biofilm-forming abilityand Rv3519gene expression level M. tuberculosis strains, and Rv3519gene can beregarded as a potential molecular diagnostic marker for biofilm-forming ability of M.tuberculosis.In total, we used high throughput sequencing technology to analysis thetranscriptome of M. tuberculosis bioflim and the transcriptome of planktonicpopulations. We screened the biofilm-forming ability related marker genes Rv3519ofM. tuberculosis and developed qRT-PCR detection method. The utilization of thismethod may provide reference for biofilm-related resistant clinical medication.