ABSTRACT
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.
Subject(s)
Ligases/chemistry , Multienzyme Complexes/chemistry , Mycobacterium tuberculosis/enzymology , Pyrophosphatases/chemistry , Binding Sites , Catalytic Domain , Enzyme Stability , Hydrolysis , Ligases/biosynthesis , Ligases/genetics , Ligases/metabolism , Magnesium/chemistry , Multienzyme Complexes/biosynthesis , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Mycobacterium tuberculosis/genetics , Peptide Fragments/chemistry , Peptide Fragments/genetics , Pyrophosphatases/biosynthesis , Pyrophosphatases/genetics , Pyrophosphatases/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
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 aa(3) 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.
Subject(s)
Antitubercular Agents/pharmacology , Enzyme Inhibitors/pharmacology , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Quinone Reductases/antagonists & inhibitors , Animals , Antitubercular Agents/chemistry , Electron Transport/genetics , Electron Transport Complex III/metabolism , Enzyme Inhibitors/chemistry , Escherichia coli/genetics , Female , Genes, Bacterial , Mice , Mice, Inbred BALB C , Mycobacterium tuberculosis/genetics , Oxygen Consumption/drug effects , Phenothiazines/chemistry , Phenothiazines/pharmacology , Quinone Reductases/genetics , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Tuberculosis, Pulmonary/drug therapy , Tuberculosis, Pulmonary/microbiologyABSTRACT
Long-term survival of nonreplicating Mycobacterium tuberculosis (Mtb) is ensured by the coordinated shutdown of active metabolism through a broad transcriptional program called the stringent response. In Mtb, this response is initiated by the enzymatic action of RelMtb and deletion of relMtb produces a strain (H37RvDeltarelMtb) severely compromised in the maintenance of long-term viability. Although aerosol inoculation of mice with H37RvDeltarelMtb results in normal initial bacterial growth and containment, the ability of this strain to sustain chronic infection is severely impaired. Significant histopathologic differences were noted in lungs and spleens of mice infected with H37RvDeltarelMtb compared with controls throughout the course of the infection. Microarray analysis revealed that H37RvDeltarelMtb suffers from a generalized alteration of the transcriptional apparatus, as well as specific changes in the expression of virulence factors, cell-wall biosynthetic enzymes, heat shock proteins, and secreted antigens that may alter immune recognition of the recombinant organism. Thus, RelMtb is critical for the successful establishment of persistent infection in mice by altering the expression of antigenic and enzymatic factors that may contribute to successful latent infection.
