Aconitases (Acns), the iron-sulfur proteins, are considered ancient enzymes in the evolution of metabolic pathways (5). The iron-sulfur clusters of these proteins not only participate in electron transport during reversible isomerization of citrate and isocitrate in citric acid cycle (7) but also serve as iron and oxygen sensors of the cell (6, 23). The binary activities are exerted through the assembly and disassembly of iron and sulfur clusters (6, 23). The protein with an intact 4Fe-4S cluster functions as Acn, whereas the protein with 3Fe-4S is an RNAbinding translational regulator (21,29). The stability and functionality of Acns as translation regulators are affected by not only iron levels but also oxidative stress, which induces these iron-regulatory proteins (IRPs) to bind to iron-responsive elements (IREs) and maintain iron homeostasis (15). IRPs maintain iron homeostasis by posttranscriptionally binding to conserved RNA stem-loop structures or IREs present at either the 5Ј or 3Ј ends of untranslated regions (UTRs) of mRNA. Depending on whether the IRE is present on the 3Ј or 5Ј end, binding of IRPs to IREs either protects the mRNA from degradation or inhibits its translation (1, 24). IRE-like sequences are present in the UTRs of at least two enzymes of the citric acid cycle, Acn and succinate dehydrogenase in mammals, implying that IRPs play an important role in mediating iron regulation of mitochondrial energy production (20). IRPs are also activated by both hydrogen peroxide and iron-mediated oxidative stress (27). The reactivity of H 2 O 2 with iron (Fenton reaction) intimately connects oxidative stress and cellular iron metabolism (28). Thus, recruitment of IRPs constitutes a highly effective strategy employed by pathogens for survival.Acns and IRPs are related with respect to the conserved amino acid residues across the family. This became evident when active-site residues identified in the pig heart mitochondria Acn crystal structure were found to be conserved across mammalian IRPs (16). It is worth mentioning that most of the knowledge on IRP binding to the IRE and the regulatory consequences has been collected from eukaryotic systems where partitioning between cytosolic Acn (IRPs) and mitochondrial Acn exists (references 14, 19, 26, 30, 31 and the references therein). However, only a few Acns have been reported so far from prokaryotes. Based on primary structure similarity, all bacterial Acns, including the ␣-proteobacterial Acns, are categorized mainly either into the Acn group similar to eukaryotic IRP or cytosolic Acn (AcnA/IRP group) or into the Acn group found only in bacteria (AcnB) (4, 37). Several bacteria, such as Escherichia coli, have two isoforms of Acn, AcnA and AcnB, with different physiological properties and expression profiles (22,34,36), while prokaryotes like Bacillus or Xanthomonas (1, 33, 38) have only one Acn. Bacillus Acn has been reported to bind to IRE-like sequences and therefore displays IRP properties (1). The M. tuberculosis energy cycle has separate oxidative and ...
Proteins released from Mycobacterium tuberculosis (Mtb) during late logarithmic growth phase are often considered candidate components of immunogenic or autolysis markers. One such protein is isocitrate dehydrogenase (ICD), a key regulatory enzyme in the citric acid cycle. We have evaluated the immunogenic properties of two isoforms of Mtb ICD and compared them with the control antigens heat-shock protein 60 and purified protein derivative (PPD). PPD lacks the sensitivity to distinguish between bacillus Calmette-Gué rin (BCG)-vaccinated and tuberculosis (TB)-infected populations, and, therefore, epidemiological relevance of PPD in BCG-vaccinated regions is debatable. We show that Mtb ICDs elicit a strong B cell response in TB-infected populations and can differentiate between healthy BCG-vaccinated populations and those with TB. The study population (n ؍ 215) was categorized into different groups, namely, patients with fresh infection (n ؍ 42), relapsed TB cases (n ؍ 32), patients with extrapulmonary TB (n ؍ 35), clinically healthy donors (n ؍ 44), nontuberculous mycobacteria patients (n ؍ 30), and non-TB patients (culture negative for acid-fast bacteria but carrying other infections, n ؍ 32). The Mtb ICDs showed statistically significant antigenic distinction between healthy BCG-vaccinated controls and TB patients (P < 0.0001) and those with other infections. Although extrapulmonary infections could not be discriminated from healthy controls by heat-shock protein 60 (P ؍ 0.2177), interestingly, the Mtb ICDs could significantly (P < 0.0001) do so. Our results highlight the immunodominant, immunosensitive, and immunospecific nature of Mtb ICDs and point to an unusual property of this tricarboxylic acid energy cycle enzyme. T uberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major threat to the human population, being responsible for Ϸ2-3 millions deaths every year worldwide (1-3). The secret of the pathogen's success is its ability to escape the host immune system and remain undetected in lungs for decades. In only 10% of infected people, the number being higher in immunocompromised patients, does TB erupt as a full-blown disease (4). Delay in diagnosis and treatment impedes the downstream management and control of the disease. With the increasing emergence of multidrug resistant strains and coinfection with HIV, the problem is further compounded (5-7). Early diagnosis, therefore, is a matter of utmost concern not just for TB disease management but also for epidemiological investigations (8). Current diagnostic tools for TB often lack sensitivity and can be time consuming. TB diagnosis in developing countries largely banks on tuberculin skin tests and staining and culture methods. The epidemiological relevance of the tuberculin test with purified protein derivative (PPD) is questionable in areas where bacillus Calmette-Guérin vaccination is compulsory because PPD is not sensitive enough to distinguish between vaccinated and infected individuals (9). Microscopic determination ...
BackgroundM.tb icd-1 and M.tb icd-2, have been identified in the Mycobacterium tuberculosis genome as probable isocitrate dehydrogenase (ICD) genes. Earlier we demonstrated that the two isoforms can elicit B cell response in TB patients and significantly differentiate TB infected population from healthy, BCG-vaccinated controls. Even though immunoassays suggest that these proteins are closely related in terms of antigenic determinants, we now show that M.tb icd-1 and M.tb icd-2 code for functional energy cycle enzymes and document the differences in their biochemical properties, oligomeric assembly and phylogenetic affiliation.ResultsFunctionally, both M.tb ICD-1 and ICD-2 proteins are dimers. Zn+2 can act as a cofactor for ICD-1 apart from Mg+2, but not for ICD-2. ICD-1 has higher affinity for metal substrate complex (Km (isocitrate) with Mg++:10 μM ± 5) than ICD-2 (Km (isocitrate) with Mg++:20 μM ± 1). ICD-1 is active across a wider pH range than ICD-2, retaining 33–35% activity in an acidic pH upto 5.5. Difference in thermal behaviour is also observed with ICD-2 being active across wider temperature range (20°C to 40°C) than ICD-1 (optimum temperature 40°C). The isozymes are NADP+ dependent with distinct phylogenetic affiliations; unlike M.tb ICD-2 that groups with bacterial ICDs, M.tb ICD-1 exhibits a closer lineage to eukaryotic NADP+ dependent ICDs.ConclusionThe data provide experimental evidence to show that the two open reading frames, Rv3339c (ICD-1) and Rv0066c (ICD-2), annotated as probable ICDs are functional TCA cycle enzymes with identical enzymatic function but different physio-chemical and kinetic properties. The differences in biochemical and kinetic properties suggest the possibility of differential expression of the two ICDs during different stages of growth, despite having identical metabolic function.
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