ABSTRACT
Objective:To explore the regulation of prokaryotic ubiquitin-like protein ( Pup )-proteasome system on the persistence of Mycobacterium tuberculosis by inducing Mycobacterium tuberculosis to being persistence state under hypoxia conditions and then analyzing the difference on the expression levels of Pup,Dop,PafA and Mpa gene at various time and different conditions. Methods:The total mRNA of the international standard virulent strains of Mycobacterium tuberculosis(H37Rv),which were cultured under hypoxia and aerobic conditions, were extracted from each group at various time and purity of the mRNA were identified.The expression of Pup,Dop,PafA and Mpa genes of M.tuberculosis strains in each group were quantified by SYBR Green I qRT-PCR,which aimed at finding the difference among the expression of Pup,Dop,PafA and Mpa genes at various time and different conditions.Results:The expression levels of Pup, Dop, PafA and Mpa genes in Mycobacterium tuberculosis under hypoxia conditions were measured at various times.The expression levels of Pup,Dop,PafA and Mpa genes:compared with the 0 d,the expression of the Pup gene was up-regulated 1.66,2.43 and 2.76-fold at 4 d,7 d,10 d respectively(P<0.05);the expression of the Dop gene was up-regulated 1.38, 1.91,2.54 and 3.28-fold at 2 d,4 d,7 d,10 d respectively(P<0.05);the expression of the PafA gene was up-regulated 1.22,1.75, 2.37 and 2.67-fold at 2 d,4 d,7 d,10 d respectively( P<0.05);the expression of the Mpa gene was up-regulated 1.66,2.21 and 2.63-fold at 4 d,7 d,10 d respectively(P<0.05).Take the aerobic conditions as control,under hypoxic conditions with the same culture time,the expression of the Pup gene was up-regulated 1.85,2.81 and 2.93-fold in 4 d,7 d,10 d respectively(P<0.05);the ex-pression of the Dop gene was up-regulated 1.20,1.76,2.01 and 3.01-fold in 2 d,4 d,7 d,10 d,respectively( P<0.05);the expression of the PafA gene was up-regulated 1.22,1.57,2.29 and 2.29-fold in 2 d,4 d,7 d,10 d,respectively(P<0.05);the expression of the Mpa gene was up-regulated 1.16,1.58,2.16 and 2.69-fold in 2 d,4 d,7 d,10 d,respectively(P<0.05).Conclusion:Under hypoxic conditions,there were significant differences on the expressions of Pup, Dop, PafA and Mpa genes at various times;what′s more, significant differences on the expressions of Pup,Dop,PafA and Mpa genes exist between hypoxic and aerobic conditions at the same time,Prokaryotic ubiquitin-like protein( Pup)-proteasome system plays a regulatory role on M.tuberculosis′s persistence.
ABSTRACT
Objective:To study the different gene expressions of Pup-proteasome system between the original drug resistance Mtb and the cultured Mtb( INH-Mtb,RFP-Mtb,SM-Mtb,EB-Mtb,MDR) which were at the different concentrations of the Mtb drug,and to explore whether the different Pup-proteasome system gene expression was relevent to the clinical isolates of Mtb-resistant which was widely spreaded in Xinjiang region. Methods:Culturing INH-Mtb,RFP-Mtb,SM-Mtb,EB-Mtb,MDR strains at Original drug resistance Medium,low concentrations of drug Medium and high concentrations of drug Medium to the logarithmic phase,and extract total RNA from each group of Mtb. Using SYBR GreenⅠreal-time PCR to detect the Pup-proteasome system expression level in different concen-trations of drug Mtb in each group of Mtb. Results: Compared with the original state of drug-resistant strains,Pup gene in INH-Mtb, RFP-Mtb,SM-Mtb and MDR strains group were down-regulated 0. 74,0. 23,0. 28,0. 57 times;Dop gene were up-regulated 1. 33,1. 63, 1. 14,2. 88 times;PafA gene were up-regulated 1. 69,1. 30,1. 58,1. 32 times;Mpa gene were up-regulated 3. 05,1. 79,1. 31,2. 27 times in low concentrations of Anti-Mtb drugs condition, the difference was statistically significant ( P<0. 05 ) . Compared with the original state of drug-resistant strains,Pup gene in INH-Mtb,RFP-Mtb,SM-Mtb,MDR strains group were down-regulate 0. 58,0. 37, 0. 43,0. 78 times;Dop gene were up-regulated 2. 62,2. 49,1. 69,2. 95 times;PafA gene were up-regulated 2. 16,1. 48,2. 02,2. 21 times;Mpa gene were up-regulated 1. 63,3. 22,1. 13,3. 94 times in the high concentrations of Anti-Mtb drugs condition,the difference was statistically significant(P<0. 05). Conclusion:Through the different concentrations of antibiotic selection pressure,these groups of Mtb strains expressions in the Pup-proteasome system of Pup gene,Dop gene,Mpa gene and PafA gene were different,The results reveal that Pup-proteasome system is associated with the drug resistance in Mtb which was spreaded in Xinjiang region.