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Inhibitory activity of quinoloneagainst DNA gyrase of

Mycobacterium tuberculosis

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Purpose

To measure the inhibitory activity og quinoloneagainst Mycobacterium tuberculosisDNA gyrase

To know the effect of variation of quinolenes

concentration on its effect on DNA gyrasesupercoiling activity

To know what causes quinolone resistance in M.Tuberculosis

To understand the resistance to quinolone bymodify the GyrA and GyrB protein ins DNAgyrase

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Theory

Multi-drug-resisten tuberculosis posesserious problem around the world, andnew pharmaceutical are needed for control

of this disease.

Quinolones ihibit bacterial type IItopoisomerase, DNA gyrase and

topoisomerase IV.

But in M. tuberculosisgenome thetopoisomerase IV wasnt identified in data

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Theory

DNA gyrase would certainly be expectedto play an important role in drug resistance

Single missense mutation in gyrAconferred low-level quinolone resistance,and bacteria with high level resistancehad two missence mutations gyrA

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Methods

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Methods

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Result

Puryfication of DNA gyrase subunit protein

The gyrA and GyrB of DNA gyrase of M.TuberculosisH37Rv were purifiedseparately:

A factor Xa recognition site had beenintroduced into the fisuion protein and,after factor Xa disgestion, the GyrA

and GyrB proteins migrated atappoximately 90 and 70 kDa

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Result

GyrA and GyrBshowedsupercoiling activity

The optimumconcentration rangfor the potassiumcation was 20-200mM and for the

magnesiumwas>1mM

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Result

Comparison of inhibitory activities ofantibacterial agent agains DNA gyrase

Quinolone inhibits in concentration

dependent mann.er

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Result

Inhibitory avtivities ofquinolone againstaltered DNA gyrase

Altered DNA gyrasewith Val(GTG) forALA90(GCG) ordouble substitution witAsp94(GAC)

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Discussion

The GyrA and GyrB proteinstogetherresonstituted supercoiling activityof DNA gyrase.

Among the quinolone tested the inhibitoryactivity of sitafloxacin was especially high.

Reported that the level of quinoloneresistance is depends on Mycobacteriumtype.

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The resistance is related to amino acid atposition 90 (ser83 of gyrA in E.coli)

The studies was confirmed thar DNAgyrase which have alanin at position 83,were higher resistance.

Quinolones inhibitory activities to DNAgyrase with single change in GyrA were 28times weaker than wild type

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These result indicates that while themutatuin confferes quinolone resistance,an aditional target could also contribute to

quinolone resistance

The purified gyrA with substitution of va foral90 and gly for asp94 indicated that the

addition of a second mutatuion causedeven greater resistance

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