Probing the mechanism of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III mtFabH: factors influencing catalysis and substrate specificity

Mycolic acids are the dominant feature of the Mycobacterium tuberculosis cell wall. These alpha-alkyl, beta-hydroxy fatty acids are formed by the condensation of two fatty acids, a long meromycolic acid and a shorter C(24)-C(26) fatty acid. The component fatty acids are produced via a combination of type I and II fatty acid synthases (FAS) with FAS-I products being elongated by FAS-II toward meromycolic acids. The beta-ketoacyl-acyl carrier protein (ACP) synthase III encoded by mtfabH (mtFabH) links FAS-I and FAS-II, catalyzing the condensation of FAS-I-derived acyl-CoAs with malonyl-acyl carrier protein (ACP). The acyl-CoA chain length specificity of mtFabH was assessed in vitro; the enzyme extended longer, physiologically relevant acyl-CoA primers when paired with AcpM, its natural partner, than with Escherichia coli ACP. The ability of the enzyme to use E. coli ACP suggests that a similar mode of binding is likely with both ACPs, yet it is clear that unique factors inherent to AcpM modulate the substrate specificity of mtFabH. Mutation of proposed key mtFabH residues was used to define their catalytic roles. Substitution of supposed acyl-CoA binding residues reduced transacylation, with double substitutions totally abrogating activity. Mutation of Arg(46) revealed its more critical role in malonyl-AcpM decarboxylation than in the acyl-CoA binding role. Interestingly, this effect was suppressed intragenically by Arg(161) --> Ala substitution. Our structural studies suggested that His(258), previously implicated in malonyl-ACP decarboxylation, also acts as an anchor point for a network of water molecules that we propose promotes deprotonation and transacylation of Cys(122).     

The Mycobacterium tuberculosis iniA gene is essential for activity of an efflux pump that confers drug tolerance to both isoniazid and ethambutol

Little is known about the intracellular events that occur following the initial inhibition of Mycobacterium tuberculosis by the first-line antituberculosis drugs isoniazid (INH) and ethambutol (EMB). Understanding these pathways should provide significant insights into the adaptive strategies M. tuberculosis undertakes to survive antibiotics. We have discovered that the M. tuberculosis iniA gene (Rv 0342) participates in the development of tolerance to both INH and EMB. This gene is strongly induced along with iniB and iniC (Rv 0341 and Rv 0343) by treatment of Mycobacterium bovis BCG or M. tuberculosis with INH or EMB. BCG strains overexpressing M. tuberculosis iniA grew and survived longer than control strains upon exposure to inhibitory concentrations of either INH or EMB. An M. tuberculosis strain containing an iniA deletion showed increased susceptibility to INH. Additional studies showed that overexpression of M. tuberculosis iniA in BCG conferred resistance to ethidium bromide, and the deletion of iniA in M. tuberculosis resulted in increased accumulation of intracellular ethidium bromide. The pump inhibitor reserpine reversed both tolerance to INH and resistance to ethidium bromide in BCG. These results suggest that iniA functions through an MDR-pump like mechanism, although IniA does not appear to directly transport INH from the cell. Analysis of two-dimensional crystals of the IniA protein revealed that this predicted transmembrane protein forms multimeric structures containing a central pore, providing further evidence that iniA is a pump component. Our studies elucidate a potentially unique adaptive pathway in mycobacteria. Drugs designed to inhibit the iniA gene product may shorten the time required to treat tuberculosis and may help prevent the clinical emergence of drug resistance.     

Insight into the role of DL-α-lipoic acid against cyclophosphamide induced alterations in calcium sensitivity of cardiac myofilaments

Cyclophosphamide (CP), a potent antitumor drug, is known to cause severe cardiotoxicity. The present study is aimed at evaluating the role of DL-α-Lipoic acid (LA) on the calcium responsiveness of cardiac myofilaments isolated from CP treated rats. Adult male Wistar rats were divided into four treatment groups. Two groups received single intraperitoneal injection of CP (200 mg/kg b.wt.) to induce cardiotoxicity, one of these groups received LA treatment (25 mg/kg b.wt. for 10 days). A vehicle treated control group and a LA drug control were also included. Cardiotoxicity was evident from increased levels of cardiac Troponin I in serum of CP treated rats. The pCa-actomyosin ATPase relationship of myofilaments demonstrated a rightward shift indicating diminished responsiveness in CP treated rats. The hill coefficient was reduced and the myofibrillar myosin Ca²⁺-ATPase and K⁺-(EDTA) activities were also significantly (P < 0.05) reduced. Ultrastuctural observations were also in agreement with the above abnormal changes, wherein loss of myofilaments occurred. LA effectively normalized these abnormalities and restored the cardiac function in CP administered rats.     

Lupeol and its ester ameliorate the cyclophosphamide provoked cardiac lysosomal damage studied in rat

Cyclophosphamide (CP), an alkylating agent widely used in cancer chemotherapy causes fatal cardiotoxicity. Lupeol, a pentacyclic triterpene, isolated from Crataeva nurvala stem bark and its ester, lupeol linoleate possess a wide range of medicinal properties. The effect of lupeol and its ester was evaluated in CP-induced myocardial toxicity in rats. Male albino rats of Wistar strain were categorized into six groups. Group I served as control. Rats in groups II, V and VI animals were injected intraperitoneally with a single dose of CP (200 mg/kg body weight) dissolved in saline. CP-treated groups V and VI received lupeol and lupeol linoleate (50 mg/kg body weight), respectively, dissolved in olive oil for 10 days by oral gavage. CP-administered rats showed a significant increase (p < 0.001) in the activities of lysosomal hydrolases in serum and heart, a decrease (p < 0.001) in the levels of cellular thiols and myofibres were swollen with loss of myofilaments in electron microscopical analysis in heart. Lupeol and its ester showed reversal of the above alterations induced by CP. These findings demonstrate that the supplementation with lupeol and its ester could preserve lysosomal integrity, improve thiol levels, highlighting their protective effect against CP-induced cardiotoxicity.