Targeting Mycobacterium Tuberculosis RmlA And Rv0228for TB Drug Development

Tuberculosis （TB） remains one of the deadliest threats to public health. The casesof multi-drug resistant TB, extensive-drug resistant TB as well as co-infection of M.tuberculosis with HIV are increasing fast in recent years. There is an urgent need fornew TB drugs. Target-based approach is one of the main strategies in TB drugdevelopment. For the targets that have been identified by experiments, high-throughputscreening of inhibitors from compound libraries can be performed with a suitable assay.For target candidates, their essentiality for mycobacterial growth should be confirmedfirst of all.D-glucose-1-phosphate thymidylyltransferase （RmlA） together with other threeenzymes, RmlB, RmlC and RmlD, catalyze the formation of dTDP-L-rhamnose, a sugardonor providing L-rhamnosyl residue in the synthesis of M. tuberculosis cell wall. Theprevious study on RmlA essentiality has proved RmlA as a potential target for TB drugdevelopment.M. tuberculosis Rv0228is a target candidate for TB drug development predictedthrough computational methods. Its essentiality and function for mycobacterial growthare still unknown.The objectives of this study are:（1） to develop a rapid enzyme assay forpotential drug target RmlA which is suitable for high-throughput screening of inhibitors,and using this assay, to determine the kinetic properties of M. tuberculosis RmlAincluding initial velocity, optimal temperature, optimal pH, the effect of Mg²⁺andkinetic parameters;（2） to test the essentiality of target candidate Rv0228formycobacterial growth. Using M. smegmatis as a model organism, MSMEG₀319（theortholog of Rv0228） gene knockout strain will be constructed via homologousrecombination method. Rv0228’s essentiality for mycobacterial growth will beconfirmed through growing MSMEG₀319knockout strain at different temperatures;（3） to analyze the function of target candidate Rv0228primarily. The function of Rv0228will be first predicted through bioinformatic analysis and further studied usingMSMEG₀319knockout strain as a model strain. An amount of MSMEG₀319knockout cells will be acquired through temperature shift experiment, and whether thefunction of Rv0228is related with the cell wall metabolism will be confirmed bycellular morphology observation and cell wall sugar analysis of MSMEG₀319knockout cells. For further function study of Rv0228, the expression of Rv0228proteinin Escherichia coli and M. smegmatis with different expression systems will also betried in this study.Followings are results we obtained in this study:1. Development of a colorimetric assay and kinetic analysis for M. tuberculosisRmlA.（1） Expression and purification of M. tuberculosis RmlA: RmlA protein with afused his-tag at N-terminus was expressed in E. coli BL21（DE3） cells and purified byNi²⁺affinity chromatography. SDS-PAGE and Western blot analysis confirmed that thepurified RmlA with an expected molecular weight of33.67kD and a perfecthomogeneity.（2） HPLC analysis of M. tuberculosis RmlA activity: The enzyme activity of thepurified RmlA was detected by HPLC analysis. The result confirmed that the purifiedRmlA had the D-glucose-1-phosphate thymidylyltransferase activity, which catalyzedsubstrate dTTP and Glc-1-P converting to dTDP-D-Glc and PPi.（3） Development of a colorimetric RmlA assay for high-throughput screening ofM. tuberculosis RmlA inhibitors: In this assay, pyrophosphatase was coupled tocatalyze the product PPi of RmlA reaction to Pi, and Pi molecules were finally detectedby the malachite green reagent. The results showed RmlA catalytic reaction had anapparent color change from yellow-green to blue-green. The assay had adequatesensitivity and reproducibility with the ratio of signal to background （S/B） above2.0and the Z’ value about0.70statistically, which was suitable for high-throughputscreening of M. tuberculosis RmlA inhibitors.（4） Kinetic study of M. tuberculosis RmlA: Using the colorimetric assay, thekinetic properties of M. tuberculosis RmlA was determined. The results showed M.tuberculosis RmlA had the relative maximal activity in the buffer of pH7.5containing5mM Mg²⁺at the temperature of37C. The Km and Vmax against dTTP were0.020±0.004mM and0.003±0.0003mM/min respectively, and against D-Glc-1-P were0.069 ±0.0050mM and0.002±0.0001mM/min respectively.（5） RmlA inhibitors screening: Twenty-three natural products were screenedby the colorimetric assay and none of them was found to inhibit the activity ofM. tuberculosis RmlA.2. The essentiality study of M. tuberculosis Rv0228for mycobacterial growth（1） Construction of MSMEG₀319gene knockout strainThe kanamycin resistant gene （kan^R） was inserted into M. smegmgtis MSMEG₀319gene, resulting in a mutant MSMEG₀319gene （MSMEG₀319::kan^R）.MSMEG₀319::kan^Rfragment was then cloned into pPR27-xylE plasmid, yielding a conditional replication plasmid pPR27-xylE-MSMEG₀319::kan^R（pMS-3）, which is sensitive to temperature.M. tuberculosis Rv0228gene was cloned into pET23b-Phsp60plasmid resultingin a plasmid pET23b-Phsp60-Rv0228. Phsp60-Rv0228was then cloned into pCG76plasmid, yielding a rescue plasmid pCG76-Phsp60-Rv0228（pMS-6）, which is alsosensitive to temperature.pMS-3plasmid was elctroporated to M. smegmatis mc²155competent cells. In theselective pressure of temperature and antibiotics, the first homologous recombinationoccurred between MSMEG₀319::kan^Rfragment in pMS-3plasmid and MSMEG₀319gene in mc²155genome, thus MSMEG₀319::kan^Rfragment was integrated intomc²155genome. Seven mc²155MS-1mutant strains with correct homologousrecombination were screened by Southern blot analysis, and one of them waselectroporated with pMS-6plasmid. In the selective pressure of sucrose, the secondhomologous recombination occurred within the genome of mc²155MS-1. The normalMSMEG₀319gene was deleted from the mc²155mutant genome and only the mutantMSMEG₀319（MSMEG₀319::kan^R） was reserved. Seven mc²155MS-2mutantstrains, i.e. MSMEG₀319knockout strains, were confirmed by Southern blot analysis.（2） Growth of MSMEG₀319knockout strains at different temperaturesAs the model strain for studying the essentiality of Rv0228for mycobacterialgrowth, MSMEG₀319knockout strain was grown at both30℃and42℃. The growthcurve showed that they could grow at30℃but not at42℃, while the mc²155carryingpCG76plasmid as a control could grow at both30℃and42℃. The result demonstratedthat Rv0228was essential for mycobacterial growth.3. The functional analysis of M. tuberculosis Rv0228（1） Bioinformatic analysis of Rv0228: The information of Rv0228and its neighboring genes in M. tuberculosis genome was understood in GenBank and TBdatabases, and the homologous proteins of Rv0228were confirmed through BLASTanalysis. The result showed Rv0228located in M. tuberculosis genome without formingan operon with other open reading frames, and its neighboring genes were mostinvolved in cell wall biosynthesis. The orthologs exist in mycobacteria and someactinobacterial bacteria, but not found in human and other prokaryotic organisms. It wasconcluded from these information that Rv0228was probably involved in cell wall（except for peptidoglycan） biosynthesis.（2） Acquirement of MSMEG₀319gene knockout cells as a model for functionalstudy of Rv0228. In the temperature shift experiment, MSMEG₀319knockout cellswere grown at30℃for20h, then the cells were transferred to a42℃incubator andgrown at42℃. The Rv0228protein expressed at30℃allowed the cells to grow at42℃for a certain period, however, with the Rv0228protein reduced gradually, the effects oflack of MSMEG₀319protein on the cells emerged. With this method, a certain amountof MSMEG₀319knockout cells were acquired for further function study of Rv0228.（3） Morphology of MSMEG₀319knockout cells: MSMEG₀319knockout cellsgrown at42℃for72h and144h in the temperature shift experiment were observed viaSEM. The cells significantly bulged at72h and some cells exhibited an irregularsurface and were even lysed at144h. TEM analysis of the MSMEG₀319knockoutcells grown at42℃for144h found that the cell wall ruptured and intracellular contentoverflowed. In contrast, the MSMEG₀319knockout cells grown at30℃exhibitednormal shape and integrated cell wall structure as wild type mc²155both in SEM andTEM. These results again confirmed that Rv0228was essential for mycobacterialgrowth, and indicated that the function of Rv0228related to the cell wall biosynthesis.（4） Sugar analysis of cell wall in MSMEG₀319knockout cells: Themycolyl-arabinogalactan-peptidoglycan （mAGP） complex, the core structure of cellwall, of the MSMEG₀319knockout cells in the temperature shift experiment wasprepared and the contents of galactose （Gal） and muramic acid （MuAc） were measuredby HPLC. The result showed that the ratio of Gal to MuAc decreased in MSMEG₀319knockout cells compared with the wild type mc²155. The result indicated that thefunction of Rv0228was involved in the formation of galactan in mycobacterial cell walldirectly or indirectly.（5） Expression of Rv0228protein: The expression plasmids pET29b-Rv0228,pET16b-Rv0228, pBAD-Rv0228-C and pBAD-Rv0228-N were constructed to express Rv0228protein in E. coli, and pVV16-Rv0228was constructed to express Rv0228protein in M. smegmatis. Dot blot analysis showed that pET16b-Rv0228/E. coliC41（DE3）, pET16b-Rv0228/E. coli ER2566and pBAD-RV0228-N/E. coli TOP10canexpress Rv0228protein effectively.Followings are conclusions we obtained from this study:1. M. tuberculosis RmlA protein was expressed in E. coli BL21（DE3） cells andpurified with Ni²⁺affinity chromatography.2. A colorimetric assay, which was coupled with pyrophosphatase and detectedwith malachite green reagent, was established to detect RmlA activity rapidly andsteadily. It was suitable for high-throughput screening of M. tuberculosis RmlAinhibitors.3. The kinetic properties of M. tuberculosis RmlA including optimal temperature,optimal pH, the effect of Mg²⁺and kinetic parameters Km and Vmax were determinedwith the established colorimetric assay.4. M. smegmatis MSMEG₀319gene knockout strain was constructed via homologous recombination strategy. It was a model strain for studying the essentiality of M. tuberculosis Rv0228gene （a target candidate） for mycobacterial growth. Through observing the growth of MSMEG₀319knockout strains at differenttemperatures, Rv0228gene was confirmed essential for mycobacterial growth.5. The function of Rv0228was predicted probably involved in the biosynthesis ofmycobacterial cell wall structure except for peptidoglycan through bioinformaticanalysis.6. MSMEG₀319knockout cells were acquired via temperature shift experiment,which could be used for function study of Rv0228. The morphology changes ofMSMEG₀319knockout cells were observed via SEM and TEM, and the content ratiochange of Gal to MuAc in the cell wall of MSMEG₀319knockout cells was analyzedby HPLC. The result demonstrated that Rv0228was involved in the formation ofgalactan in mycobacterial cell wall directly or indirectly.7. Though expressing Rv0228protein with different vectors in different host cells,three E. coli strains pET16b-Rv0228/E. coli C41（DE3）, pET16b-Rv0228/E. coliER2566and pBAD-Rv0228-N/E. coli TOP10, which could express Rv0228proteineffectively were obtained.Followings are further studies for the potential target RmlA:（1） Screening ofRmlA inhibitors from chemical compound libraries with the colorimetric assay established in this study.（2） Structural analysis of purified M. tuberculosis RmlAprotein for further structure-based screening of inhibitors or TB drug design.Followings are further studies for target candidate Rv0228:（1） Identify thefunction of Rv0228using MSMEG₀319knockout strain as model strain. The contentof other monosaccharide such as D-arabinose, L-rhamnose or N-acetylglucosamine inthe cell wall of MSMEG₀319knockout cells can be detected to confirm the functionof Rv0228.（2） Express Rv0228protein in large volume and purify Rv0228proteinthrough Ni²⁺affinity chromatography, and establish enzyme assay of acyltransferasemeanwhile. These works will also be helpful for identification of Rv0228’s functionfinally.