Tag Archives: Mycobacterium tuberculosis
Mutation of katG in a clinical isolate of Mycobacterium tuberculosis: effects on catalase-peroxidase for isoniazid activation
Purkan1, Ihsanawati2, D. Natalia2, Y. M. Syah2,
D. S. Retnoningrum3, H. S. Kusuma4
1Biochemistry Research Division, Department of Chemistry,
Faculty of Sciences and Technology, Airlangga University; Surabaya, Indonesia;
e-mail: purkan@fst.unair.ac.id;
2Biochemistry Research Division, Faculty of Mathematics and Natural Sciences,
Bandung Institute of Technology, Bandung, Indonesia;
3School of Pharmacy, Bandung Institute of Technology, Bandung, Indonesia;
4Department of Chemical Engineering, Institut Teknologi
Sepuluh Nopember, Surabaya, Indonesia;
e-mail: heriseptyakusuma@gmail.com
Mutations in katG gene are often associated with isoniazid (INH) resistance in Mycobacterium tuberculosis strain. This research was perfomed to identify the katG mutation in clinical isolate (L8) that is resistant to INH at 1 μg/ml. In addition to characterize the catalase-peroxidase of KatG L8 and perform the ab initio structural study of the protein to get a more complete understanding in drug activation and the resistance mechanism. The katG gene was cloned and expressed in Escherichia coli, then followed by characterization of catalase-peroxidase of KatG. The structure modelling was performed to know a basis of alterations in enzyme activity. A substitution of A713G that correspond to Asn238Ser replacement was found in the L8 katG. The Asn238Ser modification leads to a decline in the activity of catalase-peroxidase and INH oxidation of the L8 KatG protein. The catalytic efficiency (Kcat/KM) of mutant KatGAsn238Ser respectively decreases to 41 and 52% for catalase and peroxidase. The mutant KatGAsn238Ser also shows a decrease of 62% in INH oxidation if compared to a wild type KatG (KatGwt). The mutant Asn238Ser might cause instability in the substrate binding site of KatG, because of removal of a salt bridge connecting the amine group of Asn238 to the carboxyl group of Glu233, which presents in KatGwt. The lost of the salt bridge in the substrate binding site in mutant KatGAsn238Ser created changes unfavorable for enzyme activities, which in turn emerge as INH resistance in the L8 isolate of M. tuberculosis.
Mycobacterium tuberculosis antigens MPT63 and MPT83 increase phagocytic activity of murine peritoneal macrophages
A. A. Siromolot1,2, O. S. Oliinyk2, D. V. Kolibo2,1, S. V. Komisarenko2
1Educational and Scientific Centre Institute of Biology,
Taras Shevchenko National University of Kyiv, Ukraine;
2Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: saa0205@ukr.net
Macrophages (MΦ) are the most described and characterized target and host of mycobacteria. Like other cells of innate immunity MΦ have a wide range of receptor molecules which interact with different pathogen associated molecular patterns (PAMPs). Immunodominant proteins MPT63 and MPT83 that are synthesized in abundance by Mycobacterium bovis or Mycobacterium tuberculosis strains could be involved in development of tuberculosis infection. The aim of this study was to search for effects of these mycobacterial antigens on target cells. For this aim full-sized sequences of MPT83 (rMPT83full) and MPT63 antigens were cloned into plasmid pET24a(+). The increase of phagocytic activity of murine peritoneal macrophages was demonstrated, but not of macrophage-like cells from J774 cell line, which were treated by rMPT63 and rMPT83full proteins for 24 h. This effect of such antigens can be considered as a way to facilitate the consumption of mycobacterial cells by macrophages to avoid other effector mechanisms of innate and adaptive immunity.