耐药结核分枝杆菌PhoPR双组分系统基因表达的研究
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摘要
目的比较结核分枝杆菌(Mycobacterium tuberculosis,MTB)原始耐药菌株与经过含不同浓度培养基培养后的菌株中PhoPR双组分系统基因表达水平的差异,研究MTB PhoPR双组分系统与MTB耐药的相关性。方法分别在原始状态下和用低浓度含药培养基及高浓度含药培养基培养单纯耐异烟肼MTB临床分离株(INH-MTB)、单纯耐利福平的MTB临床分离株(RFP-MTB)、单纯耐链霉素MTB临床分离株(SM-MTB)、单纯耐乙胺丁醇的MTB临床分离株(EB-MTB)、耐多药MTB临床分离株(MDR)养至对数期,提取各组MTB总RNA,并进行纯度鉴定;运用SYBR GreenⅠ实时荧光定量PCR,检测各组MTB的PhoP基因和PhoR基因的表达,比较不同耐药的MTB临床分离株PhoP基因和PhoR基因表达水平的差异。结果在低浓度抗结核药物条件下培养的INH-MTB、RFP-MTB、SM-MTB和MDR组MTB与原始状态下MTB相比,PhoP基因表达分别上调2.19、2.04、1.72、2.73倍,PhoR基因分别上调1.57、2.57、1.56和2.83倍,差异有统计学意义(P<0.05);在高浓度抗结核药物条件下培养,INH-MTB、RFP-MTB、SMMTB、EB-MTB和MDR组MTB中的PhoP基因表达分别上调1.79、1.44、2.10、1.27和1.97倍,PhoR基因在INHMTB、RFP-MTB、SM-MTB和MDR组MTB分别上调1.05、1.91、1.76和2.13倍,差异有统计学意义(P<0.05)。结论 PhoP和PhoR基因的差异表达与MTB的耐药性相关,提示PhoPR双组分系统与MTB耐药具有相关性。
Objective To study the association between the Mycobacterium tuberculosis PhoP-PhoR two-component system and the drug resistance of M.tuberculosis by determining differences in levels of PhoP and PhoR expression in initially drug-resistant M.tuberculosis and M.tuberculosis cultured in media containing different drug concentrations. Methods Total RNA of M.tuberculosis was extracted from initially drug-resistant M.tuberculosis,M.tuberculosis cultured in media containing different concentrations of isoniazid(INH),rifampicin(RFP),streptomycin(SM),or ethambutol(EB),and multi drug-resistant(MDR)M.tuberculosis,and then the purity or those samples was determined.Reverse transcription was also performed.Expression of the PhoP and PhoR genes was quantified using SYBR Green I qRT-PCR,which sought to determine the difference in expression of the PhoP and PhoR genes between drug-resistant strains and susceptible strains. Results Expression of the PhoP gene was up-regulated 2.19-fold in M.tuberculosis cultured with a low concentration of INH,2.04-fold in M.tuberculosis cultured with a low concentration of RFP,1.72-fold in M.tuberculosis cultured with a low concentration of SM,and 2.73-fold in MDR M.tuberculosis compared to M.tuberculosis its initial state.Expression of the PhoR gene was up-regulated 1.57-fold in M.tuberculosis cultured with a low concentration of INH,2.57-fold in M.tuberculosis cultured with a low concentration of RFP,1.56-fold in M.tuberculosis cultured with a low concentration of SM,and 2.83-fold in MDR M.tuberculosis.Differences in the levels of expression were statistically significant(P<0.05).Expression of the PhoP gene was up-regulated 1.79-fold in M.tuberculosis cultured with a high concentration of INH,1.44-fold in M.tuberculosis cultured with a high concentration of RFP,2.10-fold in M.tu-berculosis cultured with a high concentration of SM,1.27-fold in M.tuberculosis cultured with a high concentration of EB,and 1.97-fold in MDR M.tuberculosis.Expression of the PhoR gene was up-regulated 1.05-fold in M.tuberculosis cultured with a high concentration of INH,1.91-fold in M.tuberculosis cultured with a high concentration of RFP,1.76-fold in M.tuberculosis cultured with a high concentration of SM,and 2.13-fold in MDR M.tuberculosis.Differences in the levels of expression were statistically significant(P<0.05). Conclusion The PhoR and PhoP genes may be associated with M.tuberculosis drug resistance and are expressed at different levels in drug-resistant strains.Therefore,the PhoPR two-component system is associated with M.tuberculosis drug resistance.
Objective To study the association between the Mycobacterium tuberculosis PhoP-PhoR two-component system and the drug resistance of M.tuberculosis by determining differences in levels of PhoP and PhoR expression in initially drug-resistant M.tuberculosis and M.tuberculosis cultured in media containing different drug concentrations. Methods Total RNA of M.tuberculosis was extracted from initially drug-resistant M.tuberculosis,M.tuberculosis cultured in media containing different concentrations of isoniazid(INH),rifampicin(RFP),streptomycin(SM),or ethambutol(EB),and multi drug-resistant(MDR)M.tuberculosis,and then the purity or those samples was determined.Reverse transcription was also performed.Expression of the PhoP and PhoR genes was quantified using SYBR Green I qRT-PCR,which sought to determine the difference in expression of the PhoP and PhoR genes between drug-resistant strains and susceptible strains. Results Expression of the PhoP gene was up-regulated 2.19-fold in M.tuberculosis cultured with a low concentration of INH,2.04-fold in M.tuberculosis cultured with a low concentration of RFP,1.72-fold in M.tuberculosis cultured with a low concentration of SM,and 2.73-fold in MDR M.tuberculosis compared to M.tuberculosis its initial state.Expression of the PhoR gene was up-regulated 1.57-fold in M.tuberculosis cultured with a low concentration of INH,2.57-fold in M.tuberculosis cultured with a low concentration of RFP,1.56-fold in M.tuberculosis cultured with a low concentration of SM,and 2.83-fold in MDR M.tuberculosis.Differences in the levels of expression were statistically significant(P<0.05).Expression of the PhoP gene was up-regulated 1.79-fold in M.tuberculosis cultured with a high concentration of INH,1.44-fold in M.tuberculosis cultured with a high concentration of RFP,2.10-fold in M.tu-berculosis cultured with a high concentration of SM,1.27-fold in M.tuberculosis cultured with a high concentration of EB,and 1.97-fold in MDR M.tuberculosis.Expression of the PhoR gene was up-regulated 1.05-fold in M.tuberculosis cultured with a high concentration of INH,1.91-fold in M.tuberculosis cultured with a high concentration of RFP,1.76-fold in M.tuberculosis cultured with a high concentration of SM,and 2.13-fold in MDR M.tuberculosis.Differences in the levels of expression were statistically significant(P<0.05). Conclusion The PhoR and PhoP genes may be associated with M.tuberculosis drug resistance and are expressed at different levels in drug-resistant strains.Therefore,the PhoPR two-component system is associated with M.tuberculosis drug resistance.
引文
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[23]孙燕妮,王一松,王海霞,等.潜伏期结核分枝杆菌HLA-A*0201限制性CD8+CTL表位的高通量筛选[J].中国病原生物学杂志,2013,8(1):26-9.
[24]李文娟,张万江.双组分系统与结核分枝杆菌致病性的相关性研究进展[J].中国病原生物学杂志,2013,8(6):656-8.
[2]Parish T,Smith DA,Roberts G,et al.The senX3-regX3twocomponent regulatory system of Mycobacterium tuberculosis is required for virulence[J].Microbiology,2003,149(6):1423-15.
[3]Gupta AK,Katoch VM,Chauhan DS,et al.Microarray analysis of efflux pump genes in multidrug-resistant Mycobacterium tuberculosis during stress induced by common anti-tuberculous drugs[J].Microb Drug Resist,2010,16:21-8
[4]Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2-[Delta][Delta]CT method[J].MetIlods,200l,25:402-8.
[5]Khue PM,Truffot-Pernot C,Texier-Maugein J,et al.A 10-year prospective surveillance of Mycobacterium tuberculosis drug resistance in France 1995-2004[J].Eur Respir J,2007,30(5):937-44.
[6]WHO.Global tuberculosis control:WHO report 2011[R].Geneva:WHO,2011:11.
[7]Zhao Y,Xu S,Wang L,et al.National survey of drug-resistant tuberculosis in China[J].N Engl J Med,2012,366(23):2161-70.
[8]王星,赵玉荣,宋守忠,等.第二次初治培阳结核患者结核菌耐药性调查[J].临床肺科杂志,2005,10(6):698-9.
[9]World Health Organization.Anti-tuberculosis drug resistance in the world.Report no.4.WHO/HTM/TB/2008.394.World Health Organization,Geneva,Switzerland.
[10]Piddock LJ.Multidrug-resistance efflux pumps-not just for resistance[J].Nat Rev Microbiol,2006,4(8):629-36.
[11]Pang Y,Lu J,Wang Y,et al.Study of the rifampin monoresistance mechanism in Mycobacterium tuberculosis[J].Antimicrob Agents Chemotherapy,2013,57(2):893-900.
[12]Rodrigues L,Villellas C,Bailo R,et al.Role of the Mmr efflux pump in drug resistance in Mycobacterium tuberculosis.[J].Antimicrob Agents Chemother,2013,57(2):751-7.
[13]Mena C,Christophe T,Amine N,et al.Identification of DNA binding motifs of the Mycobacterium tuberculosis PhoP/PhoR two-component signal transduction System[J].Plos One,2012,7(8):e42876.
[14]Jesus GA,Serge M,Jose HW,et al.PhoP:A missing piece in the intricate Puzzle of Mycobacterium tuberculosis virulence[J].PLoS One,2008,3(10):e3496.
[15]Tahlan K,Wilson R,Kastrinsky DB,et al.SQ109targets MmpL3,a membrane transporter of trehalose monomycolate involved in mycolic acid donation to the cell wall core of Mycobacterium tuberculosis[J].Antimicrobial Agents Chemotherapy,2012,56(4):1797-809.
[16]Park HD,Guinn KM,Harell MI,et al.Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis[J].Mol Microbiol,2003,48:833-43.
[17]Martin IV,Dirk S,Kevin CV,et al.Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program[J].J Exp Med,2003,198:705-13.
[18]Kumar A,Deshane JS,Crossman DK,et al.Heme oxygenase-1-derived carbon monoxide induces the Mycobacterium tuberculosis dormancy regulon[J].J Biol Chem,2008,283:18032-9.
[19]Taneja NK,Dhingra S,Mittal A,et al.Mycobacterium tuberculosis transcriptional adaptation,growth arrest and dormancy phenotype development is triggered by vitamin C[J].Plos One,2010,5:e10860.
[20]Chauhan S,Sharma D,Singh A,et al.Comprehensive insights into Mycobacterium tuberculosis DevR(DosR)regulon activation switch[J].Nucleic Acids Res,2011,39:7400-14.
[21]Cole ST,Brosch R,Parkhill J,et al.Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence[J].Nature,1998,393:537-44.
[22]Saini DK,Malhotra V,Dey D,et al.DevR-DevS is a bona fide two-component system of Mycobacterium tuberculosis that is hypoxia-responsive in the absence of the DNA-binding domain of DevR[J].Microbiology,2004,150(4):865-75.
[23]孙燕妮,王一松,王海霞,等.潜伏期结核分枝杆菌HLA-A*0201限制性CD8+CTL表位的高通量筛选[J].中国病原生物学杂志,2013,8(1):26-9.
[24]李文娟,张万江.双组分系统与结核分枝杆菌致病性的相关性研究进展[J].中国病原生物学杂志,2013,8(6):656-8.