| Mycobacterium tuberculosis (TB) is a global emergency, as declared by the World Health Organization (WHO). The emergence of multidrug-resistant TB (MDR-TB) poses an important threat to TB control as MDR-TB reduces response to standard short-course chemotherapy with anti-TB drugs. Prompting detection of new type of anti-TB drugs and searching molecular targets for controlling the development and spread of MDR-TB are in current focus. One of the approaches to develop a new drug is to elucidate the mechanism of a clinical drug and establish a screening assay for selection of lead compounds and design of a new drug.Ethambutol (EMB) is the most common first-line anti-TB drug, which is always known to rapidly inhibit biosynthesis of the arabinan component of the mycobacterial cell wall core polymer, arabinogalactan. Subsequent studies showed that EMB inhibits the polymerization of arabinan in arabinogalactan and lipoarabinomannan. Some genetic studies and mutation analysis indicated that the target of EMB is EmbB, arabinosyl transferase, which involved in synthesis of arabinogalactan. A series experiment by Alcaide et al indicated that mutations in the embB gene could increase the resistance of mycobacterium to EMB as well, Escuyer and co-workers demonstrated that mutations of embA, and embB could shorten the arabinogalactan molecules of Mycobacterium smegmatis. Although mutations in the embB gene result in high-level resistance in Mycobacterium tuberculosis, a recent study from Wu et al showed that the structure of embB gene has no changed at 44 (63.8%) of 69 EMB-resistant strains, raising some doubts about the significance of embB mutations in EMB resistance. Meanwhile, other evidences from Yi Xin suggested that EMB has no inhibitory activity on arabinosyl transferase. Other studies indicated that the effect of EMB seems tocorrelate with the increase of a unknown arabinan hydrolase . Thus, the direct action sites and the initial effect of EMB remain to be investigated.The objective of this study was to investigate the microbiostatic mechanism of ethambutol (EMB) with proteomic approach. The strategy was to utilize carrier ampholytes pH gradient two-dimensional electrophoresis (ISO-2DE) and fluorescence difference two-dimensional gel electrophoresis(2D-DIGE) to detect the protein expression of Mycobacterium smegmatis mc2 155 after the treatments with ethambutol and find the specific expressed proteins by analysis with PDQuest and DeCyder in-gel analysis software. The peptide sequences of these specific expressed proteins were identified by electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS), subsequently. The DNA sequences encoding these expressed proteins were determined by searching and BLAST the DNA sequence in databases of M. smegmatis mc2155 and M. tuberculosis H37Rv. The mRNA expression l of several selected genes for those detected proteins was measured by RT-PCR. The methods of this study were as follows: 1) To defined the optimal treatment time and treatment dose of EMB. For the growth curve, M. smegmatis mc2155 was grown in LB medium at 37℃. EMB at various concentrations (the concentrations tested were: 1.5μg /ml, 3.0μg /ml, 6.0μg /ml, 12.0μg /ml, 24.0μg /ml) were added to drug-treated cultures at the mid-log phase (OD600 0.7), respectively. At various time points, the growth of control cells or drug-treated cells was determined by optical density at 600nm to determine the optimal concentration and time of each drug treatment. To find the specific 2-DE pattern caused by EMB, the total proteins from mc2155 cells treated and untreated with ethambutol, isoniazid, and rifampin, were separate by ampholyte pH gradients–based two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were analyzed by using image analysis software PDQuest. 2) To detecte the total protein expressions from M. smegmatis mc2155 after the treatments with EMB based on the defined optimum conditions by using carrier ampholytes pH gradient two-dimensional electrophoresis and fluorescence difference two-dimensional gel electrophoresis, respectively. The specific expressed proteins were recognized by use of PDQuest and DeCyder differential in-gel analysis software. Once those proteins in gel were hydrolyzed with trypsin, the peptide fragments were identified by electrospray ionization massspectrometry. 3) To detect the membrane protein expression from M. smegmatis mc2155 after the treatments with EMB based on the defined optimum conditions by using carrier ampholytes pH gradient two-dimensional electrophoresis simultaneously. The specific expressed proteins were recognized as described previously except that peptide fragments were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry. 4) Based on the information on peptide sequence, the primers for RT-PCR were designed, and mRNA expression was detected by RT-PCR.Following results were obtained in this study:1) The growth of M. smegmatis mc2155 cells was inhibited after the treatments with EMB at variant concentration. The growth of M. smegmatis mc2155 cells was inhibited significantly after three-hour treatment with drug, and, at this turning point, the optimal concentrations of EMB were 24.0μg /ml. The inhibition rates were 26.43%. There was a significant difference at protein expression between untreated and ethambutol-, isoniazid- and rifampin- treated mc2155 cells.2) By using immobilized pH gradient two-dimensional electrophoresis and fluorescence differential two-dimensional gel electrophoresis, we found that the protein spots of control group and EMB-treated group mainly distributed in the pH range on 4.0 ~ 6.0, and in the range of relative molecular weight their distributions were correspondent basically as for EMB-treated group. The number of protein spots was less than those of control group in any range of relative molecular weight. Peptide mass mapping of the selected spots led to the identification of forty proteins specific expressed in the EMB-treated total proteins, among of which sixteen spots were up-regulated and twenty-four spots were down-regulated. All of the identified proteins were classified mainly into five categories: (a) heat-shock protein; (b) membrane-associated proteins; (c) factors involved in translation; (d) enzymes; (e) other proteins.3) By using carrier ampholytes pH gradient two-dimensional electrophoresis and PDQuest software analysis, five spots of membrane protein were found as differentially expressed in EMB-treated mc2155 cells. After MALDI-TOF/MS identification, the five differentially expressed membrane proteins were translation elongation factor Tu, molybdenum cofactor biosynthesis protein A, CG30165-PA, catechol 2, 3-dioxygenase and myosin heavy chain-like protein respectively.4) On mRNA expression, we found that brf and tpx genes were highlyexpressed in EMB-treated mc2155 cells and the expression of groL was low compared with control group.In summary, we have detected and identified certain differentially expressed proteins with ISO-2DE and 2D-DIGE and mass spectrometric determination after the EMB treatment, suggesting that the microbiostatic process of EMB is a complicated and diverse sites attraction which indicated that we should focus on a wide scale to investigate the initial effect of EMB, but also provide some potential drug targets for the rational design of more effective anti-mycobacterial agents. Several interesting proteins are likely related to the EMB treatment, however, most possible candidates are still remained to investigate after database research. Functional study should be followed to identify the direct targets of EMB.Future work:1) The possible role on arabinan biosynthesis from identified genes should be determined by gene knock-out and structural analysis on mutated arabinan.2) Exact function in the pathway will be further analyzed various approaches, such as enzyme assays, protein-protein interaction assays, and etc.
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