| Penicillin-class antibiotics, known as β-lactams, are one of medicine's most valuable weapons against bacterial disease. These drugs infiltrate susceptible bacteria and interrupt normal growth via interactions with a class of cell wall-synthesizing enzymes known as penicillin-binding proteins (PBPs). Some pathogens, however, can escape such a fate.; Beta-lactam antibiotics are not currently prescribed for the treatment of tuberculosis, although it has now been shown that commercially available lactams can bind to PBPs in membrane fractions from Mycobacterium tuberculosis (MTB), the causative agent.12 13 This lab has previously documented that PBPs form native protein complexes within H. influenzae and E. coli.14 15 If similar PBP complexes could be identified within mycobacteria, it would improve our understanding of the enzymology of cell wall biosynthesis in these organisms. Mycobacterial PBPs and the proteins with which they naturally interact are valuable targets for antibiotic research, especially in light of the emergence of multidrug-resistant tuberculosis (MDRTB) in certain populations worldwide.; The approach outlined here covalently labels PBPs within intact cells before protein isolation; the advantage provided by this scheme is the opportunity to cross-link PBPs in their native protein-protein associations. Cross-linking can take place after interaction with β-lactam tags, but before disruption of cells. This lab has previous experience exploring the topography of bacterial PBPs using cyanogen (ethanedinitrile) as a cross-linking agent.3 4 Cyanogen has been shown to permeate intact cells and to covalently crosslink native PBP complexes.; Dansylated penicillin has been used to covalently label purified serine protease enzymes, though to the best of our knowledge it has not been used to label mycobacterial proteins.16 When accompanied by β-lactamase inhibitors, β-lactams interfere with the growth of mycobacteria in cultures17 18 19 20 and intracellularly within macrophages.21 This project sought to label PBPs and associated enzymes in whole MTB cells with a fluorescent tag, then to isolate these proteins. Both a de novo monocyclic lactam and a commercially available penicillin were dansylated in order to investigate their effectiveness in labeling PBPs within whole mycobacterial cells. We found that mycobacterial PBPs could be fluorescently tagged and cross-linked to associated proteins within intact cells.; 12Chambers, H. F., Moreau, D., Yajko, D., Miick, C., Wagner, C., Hackbarth, C., Kocagoz, S., Rosenberg, E., Hadley, W., Nikaido, H. (1995) Antimicrob. Agents and Chemo. 39, 2620–2624. 13Basu, J., Chattopadhyay, R., Manikuntala, K., Chakrabarti, P. (1992) J. Bacteriol. 174, 4829–4832. 14Alaedini, A., Day, R A. (1999) Biochem. Biophys. Res. Commun. 264, 191–195. 15Bhardwaj, S., Day, R A. (1998) Techniques in Protein Chemistry VIII, Academic Press, San Diego: 469. 16Fink, A. L., Cartwright, S. J. (1989) Biochem. J. 263, 905–912. 17Sorg, T., Cynamon, M. (1987) J. Antimicrob. Chemother. 19(1), 59–64. 18Prabhakaran, K., Harris, E, Randhawa, B, Adams, L, Williams, D., Hastings, R. (1993) Microbios 76(309), 251–61. 19Chen, C., Yand, M., Lin, J., Lee, Y., Perng, R. (1995) Proc. Nat. Sci. Coun. Repub. China B. 19(2),80–84. 20Herbert, D., Paramasivan, C., Venkatesan, P., Kubendiran, G., Prabhakara... |
