M. leprae HSP18 suppresses copper (II) mediated ROS generation: Effect of redox stress on its structure and function.

Mycobacterium leprae, causative organism of leprosy, is known to counter redox stress generated by reactive oxygen species (ROS) during its survival inside host macrophages. But, the involvement of any antigenic protein(s) for countering such redox stress is still unknown. Interestingly, M. leprae HSP18, an important antigenic protein that helps in the growth and survival of M. leprae pathogen inside host macrophages, is induced under redox stress. Moreover, HSP18 also interacts with Cu2+. Copper (II) can induce redox stress via Fenton reaction. But, whether HSP18 suppresses Cu2+ mediated ROS generation, is still far from clear. Also, the effect of redox stress on its structure and function is not known. In this study, we show that HSP18 efficiently suppresses Cu2+ mediated generation of ROS and also prevents the redox mediated aggregation of a client protein (γD-crystallin). Upon exposure to substantial redox stress, irreversible perturbation in the secondary and tertiary structure of HSP18 and the tryptophan and tyrosine oxidation are evidenced. Interestingly, HSP18 retains a considerable amount of functionality even after being exposed to substantial redox stress. Perhaps, the redox scavenging ability as well as the chaperone function of HSP18 may possibly help M. leprae pathogen to counter redox stress inside host macrophages.

A comparative study of the redox-cycling of a quinone (rifamycin S) and a quinonimine (rifabutin) antibiotic by rat liver microsomes.

Rifamycin S and rifabutin are clinical drugs used to treat tuberculosis and leprosy. The formation of reactive oxygen species during the redox-cycling of rifamycin S (quinone) and rifabutin (quinonimine) was evaluated. The semiquinone (or semiquinonimine) and hydroquinone (or hydroquinonimine) formed during the reduction of the parent molecules by microsomal electron transfer in the presence of nicotinamide-adenine dinucleotide phosphate, reduced (NADPH) or nicotinamide-adenine dinucleotide, reduced (NADH) reoxidizes in air to generate superoxide radical and hydrogen peroxide. In the presence of added iron, hydroxyl radicals, formed by the Fenton reaction, were detected using 5,5'-dimethyl-1-pyroline-N-oxide as the spin-trap. Rifamycin S, a quinone, redox cycles more efficiently than rifabutin, a quinonimine, as approximately five times the concentration of hydroxyl radical adduct of 5,5'-dimethyl-1-pyroline-N-oxide (DMPO) was detected, when compared with rifabutin. The NADPH-dependent microsomal production of hydroxyl radical in the presence of rifamycin S was somewhat higher than the NADH-rifamycin S system with most iron chelators. However, with rifabutin, NADH-dependent microsomal production of hydroxyl radical was higher than that found with the NADPH-rifabutin system. An exception was the iron chelator, diethylene-triamine-pentacetic acid (DTPA), in which NADPH-dependent rates exceeded the rates with NADH with both antibiotics. Rat liver sub-mitochondrial particles also generated hydroxyl radical in the presence of NADH and either rifamycin S or rifabutin. The electron transport chain inhibitors such as rotenone and antimycin A enhanced the signal intensity of DMPO-OH, suggesting NADH dehydrogenase (complex I) as the major component involved in the reduction of rifamycin S. Rifamycin S was shown to be readily reduced to rifamycin SV, the corresponding hydroquinone by Fe(II); under similar conditions Fe(II) did not reduce rifabutin. Using optical spectroscopy, we determined that rifamycin S forms a complex with Fe(II). The stoichiometry of the complex was Fe(rifamycin S)3 in phosphate buffer at pH 7.4. Rifabutin did not form a detectable complex with Fe(II). The redox cycling of rifamycin S and rifabutin did not cause microsomal lipid peroxidation. In fact, the Fe:ATP induced lipid peroxidation was completely inhibited by these two molecules. These results indicate that rifamycin S and rifabutin can interact with rat liver microsomes to undergo redox-cycling, with the subsequent production of hydroxyl radicals when iron complexes are present. Compared to NADPH, NADH is almost as effective (rifamycin S) or even more effective (rifabutin) in promoting these interactions. These interactions may play a role in the hepatotoxicity associated with the use of these antibiotics.