Mycobacterium tuberculosis (Mtb) is an obligate aerobe that is capable of long-term persistence under conditions of low oxygen tension. Analysis of the Mtb genome predicts the existence of a branched aerobic respiratory chain terminating in a cytochrome bd system and a cytochrome aa3 system. Both chains can be initiated with type II NADH:menaquinone oxidoreductase. We present a detailed biochemical characterization of the aerobic respiratory chains from Mtb and show that phenothiazine analogs specifically inhibit NADH:menaquinone oxidoreductase activity. The emergence of drug-resistant strains of Mtb has prompted a search for antimycobacterial agents. Several phenothiazines analogs are highly tuberculocidal in vitro, suppress Mtb growth in a mouse model of acute infection, and represent lead compounds that may give rise to a class of selective antibiotics. Mycobacterium tuberculosis ͉ respiratory chainT he World Health Organization estimates that two billion people are infected with Mycobacterium tuberculosis (Mtb), and two million people die of the disease each year (1). Most individuals infected with the organism are latent carriers who have a 2-23% lifetime risk of developing reactivation tuberculosis (TB). The risk dramatically increases if the carrier's immune system is suppressed. Also, drug resistance is a serious concern; the isoniazid (INH)-resistance rate is Ϸ10%, and the rifampicin (RIF) resistance rate is Ϸ1%, with lower numbers in countries with effective TB programs and higher numbers in countries with deficient TB programs. The World Health Organization declared TB infections to be a global public health emergency (1), and the need to identify targets for antimicrobial therapy remains urgent.Mtb is capable of establishing persistent infection in the host by using a complex interplay between the host immune system and bacterial survival mechanisms. In the persistent infection, Mtb adapt to depletion of available oxygen and nutrients and enter a stage of nonreplicating persistence (NRP) in granulomatous or necrotic lesions. NRP Mtb are resistant to INH, ethambutol, and RIF, but they become sensitive to metronidazole in vitro (2). Given the critical role of oxygen in the generation of cellular energy and bacterial long-term survival, there is surprisingly little information on oxidative phosphorylation in Mtb. Clearly, oxidative phosphorylation is a central component in the production of ATP and the subsequent growth and pathogenesis of Mtb. Here, we characterize the aerobic respiratory pathway and show that NADH:menaquinone oxidoreductase (Ndh) is a key target for TB agents. Materials and MethodsMedia and Strains. Mtb H 37 R v was a gift from C. Imperatrice (Clinical Infectious Diseases, Hospital of the University of Pennsylvania) and Mycobacterium smegmatis Mc 2 155 was obtained from V. Mizrahi (National Health Laboratory Service, Johannesburg). Bacteria were cultured in 7H9 broth supplemented with 10% oleic acid-albumin-dextrose catalase͞0.5% glycerol͞0.05% Tween 80. Solid agar (15 g͞liter) was ad...

The dual-function Rel(Mtb) protein from Mycobacterium tuberculosis catalyzes both the synthesis and hydrolysis of (p)ppGpp, the effector of the stringent response. In our previous work [Avarbock, D., Avarbock, A., and Rubin, H. (2000) Biochemistry 39, 11640], we presented evidence that the Rel(Mtb) protein might catalyze its two opposing reactions at distinct active sites. In the study presented here, we purified and characterized fragments of the 738-amino acid Rel(Mtb) protein and confirmed the hypothesis that amino acid fragment 1-394 contains both synthesis and hydrolysis activities, amino acid fragment 87-394 contains only (p)ppGpp synthesis activity, and amino acid fragment 1-181 contains only (p)ppGpp hydrolysis activity. Mutation of specific residues within fragment 1-394 results in the loss of synthetic activity and retention of hydrolysis (G241E and H344Y) or loss of hydrolytic activity with retention of synthesis (H80A and D81A). The C-terminally cleaved Rel(Mtb) fragment proteins have basal activities similar to that of full-length Rel(Mtb), but are no longer regulated by the previously described Rel(Mtb) activating complex (RAC). Residues within the C-terminus of Rel(Mtb) (D632A and C633A) are shown to have a role in interaction with the RAC. Additionally, size exclusion chromatography indicates Rel(Mtb) forms trimers and removal of the C-terminus results in monomers. The C-terminal deletion, 1-394, which exists as a mixture of monomers and trimers, will dissociate from the trimer state upon the addition of substrate. Furthermore, the trimer state of fragment 1-394 appears to be a catalytically less efficient state than the monomer state.

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Congenital malalignment is the lateral deviation of the nail plate along the longitudinal axis due to the lateral rotation of the nail matrix. The nail plate grows out in ridges caused by repeated microtrauma to the nail. Common complications include onychomycosis, Pseudomonas infection and acute or chronic paronychia. Treatment options range from conservative management to surgical options including realignment and nail matrixectomy. Congenital malalignment usually presents in infancy or childhood, but we present two cases of acquired malalignment occurring in the teenage years.