Dissertation
Dissertation > Medicine, health > Chinese Medicine > Of Pharmacy > Pharmacology

Screening of Anxi-mycobacterium Tuberculosis Compounds&the Effect of Transcriptome of H37Rv under the Stress of Plumbagin

Author YeHaiQing
Tutor LiuZengShan
School Jilin University
Course Preventive Veterinary Medicine
Keywords Mycobacterium tuberculosis plumbagin microarray transcriptome screening
CLC R285
Type PhD thesis
Year 2013
Downloads 142
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Tuberculosis (TB) is an ancient infectious diseases, which is widely distributedin the world and serious harm to human health. In human history, TB was once widelypopular on the global scale, and there have been hundreds of millions of people diedof tuberculosis. After the1950s, due to the constant discovery of effective anti-TBdrugs, the TB epidemic has been effectively controlled. However, in recent years, dueto the combined effect of the emergence of extensively drug-resistant strains andhuman immunodeficiency virus (HIV), resulted the incidence of tuberculosis hasincreased both in developing and developed countries. How to defense TB activelyhas become the grim reality placed before mankind.For decades, mankind have developed20kinds of anti-TB drugs. But thewidespread disadvantage is that side effects and prone to drug resistance. Especially,in recent years, the emergence of multi-drug resistant and multi-drug-resistant strains,which made the programs of treatment tuberculosis with original first-lineanti-tuberculosis drugs and second-line anti-tuberculosis drug lose their effectiveness.However, the finding of anti-TB drugs from chemical compounds became more andmore difficult. Since2000years ago, there are many records on the treatment oftuberculosis of Chinese traditional herbal medicine. Therefore, Chinese scholarsbegan to focus on looking for a highly effective anti-TB drugs from the rich resourcesof Chinese herbal medicine in recent years, as well as in abroad. Compared to thecrude extracts of traditional Chinese medicine and the effective components,monomer compound has many advantages, such as clear structure, stability of theantibacterial effect and fixed action target. Therefore,there has a very importantsignificance of the selection of anti-TB drugs from Chinese medicine monomercompound, and its drug targets screening and confirmatory.In this study,microdilution susceptibility assay were used to detect the singleantibacterial activity and the synergistic antibacterial activity about29drugs, which have potential anti-TB activity. According to the single drug susceptibility test,MIC≦64μg/mL as judgment standard,6kinds of natural plants monomercomponents were selected, they are chelerythrine (64μg/mL), plumbagin (64μg/mL),oleanolic acid (16μg/mL), lupulone (32μg/mL), tryptanthrin (1μg/mL) and poonprime (4μg/mL). According to synergistic antibacterial test, four natural compoundswere verified have anti-TB activity synergistic with the first-line drugs, they arechelerythrine, plumbagin, oleanolic acid and lupulone. The results of MTT assayindicated that natural monomer compound have toxicity to Vero cells at highconcentrations (≧256μg/mL).In this paper, the affect of transcriptome of M. tuberculosis H37Rv (ATCC27294)under the stress of plumbagin were detected with cDNA microarray. This is the firsttime to explore the molecular mechanism from the molecular level of plumbagintreated to Mycobacterium tuberculosis.2-fold difference of gene expression were seenas a significance, the results showed that a total of274genes differentially expressedunder the stress of plumbagin, of which103genes were up-regulated, and171geneswere down-regulated. Among these genes, most were classified as having anunclassified role category not yet assigned (25.9%); and the others were classified asinvolved in conserved hypothetical proteins (16.1%); hypothetical proteins (14.6%);the cell envelope (5.1%); energy metabolism (5.5%); regulatory functions (4.4%); orunknown function (5.5%). Inference the possible mechanisms as:①To prevent theaggregation of newly synthesized proteins of Mycobacterium tuberculosis by raisedthe high expression of GroEL/GroES or DnaK/DnaJ;②A large number of PE andPPE gene were inhibited, and through this approach, the reasonable proportion of PEand PPE were damaged thus reduced the infectivity of Mycobacterium tuberculosis;③Through inhibited the expression of phthiocerol dimycocerosates related gene, thusincrease the permeability of the outer membrane of Mycobacterium tuberculosis, andas a result, reduced the resistance and activity of Mycobacterium tuberculosis;④Plumbagin has the chemical structure of the natural naphthoquinone, whenplumbagin appear it can induced competitive inhibition with MenB (one necessary enzyme of menaquinone biosynthesis of Mycobacterium tuberculosis), therebyinhibiting the activity of Mycobacterium tuberculosis;⑤Many of membrane proteingene and virulence genes were inhibited, such as yrbE4B, yrbE1B and mce1;⑥Triglyceride (TG) synthase gene is suppressed, thereby reduced the stress force ofMycobacterium tuberculosis. These data laid the foundation for the further study ofthe molecular mechanism of plumbagin treated to Mycobacterium tuberculosis.17differentially expressed genes were randomly selected and were verified withRT-PCR. The results of RT-PCR consistent with the results of microarray, whichindicated that the data of microarray are accurate and reliable.In this study, menB gene and mce1A gene were successfully cloned, expressionplasmid (pET-28a-menB and pET-28a-mce1A) were constructed, and MenB proteinMce1A protein were expressed in E. coli efficiently. Then Mce1A protein was used toimmunize rabbits to get polyclonal antibody. Western-blotting was used to detect theexpression of Mce1A protein of Mycobacterium tuberculosis. As a result shown that,when increased the drug concentration, then the expression of Mce1A proteindecreased. It can be inferred, plumbagin can weakened the ability of the bacteria intoanimal cells by inhibit the protein expression of Mce1A, thereby reducing itsinfectivity and enhance the antibacterial ability of the drug indirectly.

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