Dataset Information

Characterization of alpha-isopropylmalate synthases containing different copy numbers of tandem repeats in Mycobacterium tuberculosis.

ABSTRACT: BACKGROUND: Alpha-isopropylmalate synthase (alpha-IPMS) is the key enzyme that catalyzes the first committed step in the leucine biosynthetic pathway. The gene encoding alpha-IPMS in Mycobacterium tuberculosis, leuA, is polymorphic due to the insertion of 57-bp repeat units referred to as Variable Number of Tandem Repeats (VNTR). The role of the VNTR found within the M. tuberculosis genome is unclear. To investigate the role of the VNTR in leuA, we compared two alpha-IPMS proteins with different numbers of amino acid repeats, one with two copies and the other with 14 copies. We have cloned leuA with 14 copies of the repeat units into the pET15b expression vector with a His6-tag at the N-terminus, as was previously done for the leuA gene with two copies of the repeat units. RESULTS: The recombinant His6-alpha-IPMS proteins with two and 14 copies (alpha-IPMS-2CR and alpha-IPMS-14CR, respectively) of the repeat units were purified by immobilized metal ion affinity chromatography and gel filtration. Both enzymes were found to be dimers by gel filtration. Both enzymes work well at pH values of 7-8.5 and temperatures of 37-42 degrees C. However, alpha-IPMS-14CR tolerates pH values and temperatures outside of this range better than alpha-IPMS-2CR does. alpha-IPMS-14CR has higher affinity than alpha-IPMS-2CR for the two substrates, alpha-ketoisovalerate and acetyl CoA. Furthermore, alpha-IPMS-2CR was feedback inhibited by the end product l-leucine, whereas alpha-IPMS-14CR was not. CONCLUSION: The differences in the kinetic properties and the l-leucine feedback inhibition between the two M. tuberculosis alpha-IPMS proteins containing low and high numbers of VNTR indicate that a large VNTR insertion affects protein structure and function. Demonstration of l-leucine binding to alpha-IPMS-14CR would confirm whether or not alpha-IPMS-14CR responds to end-product feedback inhibition.

SUBMITTER: Yindeeyoungyeon W

PROVIDER: S-EPMC2704214 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Characterization of alpha-isopropylmalate synthases containing different copy numbers of tandem repeats in Mycobacterium tuberculosis.

Yindeeyoungyeon Wandee W Likitvivatanavong Supaporn S Palittapongarnpim Prasit P

BMC microbiology 20090609

<h4>Background</h4>Alpha-isopropylmalate synthase (alpha-IPMS) is the key enzyme that catalyzes the first committed step in the leucine biosynthetic pathway. The gene encoding alpha-IPMS in Mycobacterium tuberculosis, leuA, is polymorphic due to the insertion of 57-bp repeat units referred to as Variable Number of Tandem Repeats (VNTR). The role of the VNTR found within the M. tuberculosis genome is unclear. To investigate the role of the VNTR in leuA, we compared two alpha-IPMS proteins with di ...[more]

PMID: 19505340

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Project description:Genomic tools have revealed genetically diverse pathogens within some hosts. Within-host pathogen diversity, which we refer to as "complex infection", is increasingly recognized as a determinant of treatment outcome for infections like tuberculosis. Complex infection arises through two mechanisms: within-host mutation (which results in clonal heterogeneity) and reinfection (which results in mixed infections). Estimates of the frequency of within-host mutation and reinfection in populations are critical for understanding the natural history of disease. These estimates influence projections of disease trends and effects of interventions. The genotyping technique MLVA (multiple loci variable-number tandem repeats analysis) can identify complex infections, but the current method to distinguish clonal heterogeneity from mixed infections is based on a rather simple rule. Here we describe ClassTR, a method which leverages MLVA information from isolates collected in a population to distinguish mixed infections from clonal heterogeneity. We formulate the resolution of complex infections into their constituent strains as an optimization problem, and show its NP-completeness. We solve it efficiently by using mixed integer linear programming and graph decomposition. Once the complex infections are resolved into their constituent strains, ClassTR probabilistically classifies isolates as clonally heterogeneous or mixed by using a model of tandem repeat evolution. We first compare ClassTR with the standard rule-based classification on 100 simulated datasets. ClassTR outperforms the standard method, improving classification accuracy from 48% to 80%. We then apply ClassTR to a sample of 436 strains collected from tuberculosis patients in a South African community, of which 92 had complex infections. We find that ClassTR assigns an alternate classification to 18 of the 92 complex infections, suggesting important differences in practice. By explicitly modeling tandem repeat evolution, ClassTR helps to improve our understanding of the mechanisms driving within-host diversity of pathogens like Mycobacterium tuberculosis.

Project description:Mixed infections of Mycobacterium tuberculosis, defined as the coexistence of multiple genetically distinct strains within a single host, have been associated with unfavorable treatment outcomes. Different methods have been used to detect mixed infections, but their performances have not been carefully evaluated. To compare the sensitivity of whole-genome sequencing (WGS) and variable-number tandem repeats (VNTR) typing to detect mixed infections, we prepared 10 artificial samples composed of DNA mixtures from two strains in different proportions and retrospectively collected 1,084 clinical isolates. The limit of detection (LOD) for the presence of a minor strain was 5% for both WGS and VNTR typing. The overall clinical detection rate of mixed infections was 3.7% (40/1,084) for the two methods combined, WGS identified 37/1,084 (3.4%), and VNTR typing identified 14/1,084 (1.3%), including 11 also identified by WGS. Multivariate analysis demonstrated that retreatment patients had a 2.7 times (95% confidence interval [CI], 1.2 to 6.0) higher risk of mixed infections than new cases. Collectively, WGS is a more reliable tool to identify mixed infections than VNTR typing, and mixed infections are more common in retreated patients. IMPORTANCE Mixed infections of M. tuberculosis have the potential to render treatment regimens ineffective and affect the transmission dynamics of the disease. VNTR typing, currently the most widely used method for the detection of mixed infections, detects mixed infections only by interrogating a small fraction of the M. tuberculosis genome, which necessarily limits sensitivity. With the introduction of WGS, it became possible to study the entire genome, but no quantitative comparison has yet been undertaken. Our systematic comparison of the ability of WGS and VNTR typing to detect mixed infections, using both artificial samples and clinical isolates, revealed the superior performance of WGS at a high sequencing depth (~100×) and found that mixed infections are more common in patients being retreated for tuberculosis (TB) in the populations studied. This provides valuable information for the application of WGS in the detection of mixed infections and the implications of mixed infections for tuberculosis control.

Dataset Information

Characterization of alpha-isopropylmalate synthases containing different copy numbers of tandem repeats in Mycobacterium tuberculosis.

Publications

Characterization of alpha-isopropylmalate synthases containing different copy numbers of tandem repeats in Mycobacterium tuberculosis.

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