Global effects of inactivation of the pyruvate kinase gene in the Mycobacterium tuberculosis complex.

To better understand the global effects of "natural" lesions in genes involved in the pyruvate metabolism in Mycobacterium bovis, null mutations were made in the Mycobacterium tuberculosis H37Rv ald and pykA genes to mimic the M. bovis situation. Like M. bovis, the M. tuberculosis DeltapykA mutant yielded dysgonic colonies on solid medium lacking pyruvate, whereas colony morphology was eugonic on pyruvate-containing medium. Global effects of the loss of the pykA gene, possibly underlying colony morphology, were investigated by using proteomics on cultures grown in the same conditions. The levels of Icd2 increased and those of Icl and PckA decreased in the DeltapykA knockout. Proteomics suggested that the synthesis of enzymes involved in fatty acid and lipid biosynthesis were decreased, whereas those involved in beta-oxidation were increased in the M. tuberculosis DeltapykA mutant, as confirmed by direct assays for these activities. Thus, the loss of pykA from M. tuberculosis results in fatty acids being used principally for energy production, in contrast to the situation in the host when carbon from fatty acids is conserved through the glyoxylate cycle and gluconeogenesis; when an active pykA gene was introduced into M. bovis, the opposite effects occurred. Proteins involved in oxidative stress-AhpC, KatG, and SodA-showed increased synthesis in the DeltapykA mutant, and iron-regulated proteins were also affected. Ald levels were decreased in the DeltapykA knockout, explaining why an M. tuberculosis DeltapykA Deltaald double mutant showed little additional phenotypic effect. Overall, these data show that the loss of the pykA gene has powerful, global effects on proteins associated with central metabolism.

Functional analysis of a clonal deletion in an epidemic strain of Mycobacterium bovis reveals a role in lipid metabolism.

Previous work on the population structure of Mycobacterium bovis strains in Great Britain has identified highly successful clones which are expanding across the country. One such clone, designated M. bovis type 17, differs from all other members of the Mycobacterium tuberculosis complex in having a region of deletion, termed RDbovis(d)_0173, of seven genes between Mb1963c and Mb1971. Three of these genes have functions annotated in lipid metabolism. To explore the molecular basis for the success of this clone, we examined the impact of this deletion on lipid metabolism. While type 17 isolates had similar lipid composition to other M. bovis strains, their ability to incorporate propanoate into mycolic acids was remarkably low. When expressed as a reciprocal (the ratio of incorporation of label from acetate : propanoate into mycolic acids) the ratio was higher for all three type 17 field strains tested (mean: 18.90) than the values of 7.30 to 7.61 for other field strains (P < 0.002) and values < 6.50 for all other strains in the M. tuberculosis complex tested. The label from propanoate was diverted to pyruvate, at significantly higher levels in M. bovis type 17 than all other strains (P < 0.021). Complementation of M. bovis type 17 with an integrating cosmid, IE471, carrying the M. tuberculosis orthologues of Mb1963c-Mb1971 resulted in the ability of the recombinant strain to incorporate label from propanoate into mycolic acids in a manner similar to other strains. M. bovis type 17 : : IE471 labelled pyruvate from propanoate about four times more slowly than the parent strain. Thus, RDbovis(d)_0173 results in a profound effect on carbon metabolism, providing the ability to compensate for the inactivation of the ald and pykA genes, involved in pyruvate metabolism, that is seen in M. bovis (but not in M. tuberculosis). This shift in carbon metabolism may be a factor in the extraordinary clonal expansion reported for M. bovis type 17.

Physiologically-based pharmacokinetic modeling of genistein in rats, Part I: Model development.

Genistein is a phytoestrogen-a plant-derived compound that binds to and activates the estrogen receptor-occurring at high levels in soy beans and food products, leading to widespread human exposure. The numerous scientific publications available describing genistein's dosimetry, mechanisms of action, and identified or putative health effects in both experimental animals and humans make it ideal for examination as an example of endocrine-active compound (EAC). We developed a physiologically-based pharmacokinetic (PBPK) model to quantify the internal, target-tissue dosimetry of genistein in adult rats. Complexities of the model include enterohepatic circulation, binding of both genistein and its conjugates to plasma proteins, and the multiple compartments used to describe transport through the bile duct and gastrointestinal tract. Other aspects of the model are simple perfusion-limited transport to the tissue groups and first-order rates of metabolism, uptake, and excretion. We describe here the model structure and initial calibration of the model by fitting to a large data set for Wistar rats. The model structure can be readily extrapolated to describe genistein dosimetry in humans or modified to describe the dosimetry of other phytoestrogens and phenolic EACs. The model does a fair job of capturing the pharmacokinetics. Although it does not describe the interindividual variability and we have not identified a single set of parameters that provide a good fit to the data for both oral and intravenous exposures, we believe it provides a good initial attempt at PBPK modeling for genistein, which can serve as a template for other phytoestrogens and in the design of future experiments and research that can be used to fill data gaps and better estimate model parameters.

Functional demonstration of reverse transsulfuration in the Mycobacterium tuberculosis complex reveals that methionine is the preferred sulfur source for pathogenic Mycobacteria.

Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex although it is not obvious from examination of the genome annotation how these bacteria utilize methionine. Given that genome annotation is a largely predictive process, key challenges are to validate these predictions and to fill in gaps for known functions for which genes have not been annotated. We have addressed these issues by functional analysis of methionine metabolism. Transport, followed by metabolism of (35)S methionine into the cysteine adduct mycothiol, demonstrated the conversion of exogenous methionine to cysteine. Mutational analysis and cloning of the Rv1079 gene showed it to encode the key enzyme required for this conversion, cystathionine gamma-lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine gamma-synthase (CGS), but demonstration of a gamma-elimination reaction with cystathionine as well as the gamma-replacement reaction yielding cystathionine showed it encodes a bifunctional CGL/CGS enzyme. Consistent with this, a Rv1079 mutant could not incorporate sulfur from methionine into cysteine, while a cysA mutant lacking sulfate transport and a methionine auxotroph was hypersensitive to the CGL inhibitor propargylglycine. Thus, reverse transsulfuration alone, without any sulfur recycling reactions, allows M. tuberculosis to use methionine as the sole sulfur source. Intracellular cysteine was undetectable so only the CGL reaction occurs in intact mycobacteria. Cysteine desulfhydrase, an activity we showed to be separable from CGL/CGS, may have a role in removing excess cysteine and could explain the ability of M. tuberculosis to recycle sulfur from cysteine, but not methionine.