Minding the gaps: metabolomics mends functional genomics

Two recent studies in PNAS and Nat Chem Biol highlight the power of modern mass-spectrometry techniques for enzyme discovery applied to microbiology. In so doing, they have uncovered new potential targets for the treatment of tuberculosis.Proc Natl Acad Sci USA (2013) 110 28, 11320–11325 doi: 10.1073/pnas.1221597110Nat Chem Biol (2013). doi:10.1038/nchembio.1355. Advance online publication 29 September 2013Many have come to regard metabolism as a well-understood housekeeping activity of all cells, functionally compartmentalized away from other biological processes. However, growing reports of unexpected links between a diverse range of disease states and specific metabolic enzymes or pathways have begun to challenge this view. In doing so, such discoveries have exposed more glaring, and neglected, deficiencies in our understanding of cellular metabolism, triggering a broad resurgence of interest in metabolism.“Metabolomics […] offers a global window into the biochemical state of a cell or organism…”Metabolomics is the newest of the systems-level disciplines and seeks to reveal the physiological state of a given cell or organism through the global and unbiased study of its small-molecule metabolites [1]. Metabolites are the final products of enzymes and enzyme networks, the substrates and products of which often cannot be deduced from genetic information and the levels of which reflect the integrated product of the genome, proteome and environment [2]. Metabolomics thus offers a global window into the biochemical state of a cell or organism, made experimentally possible by the unprecedented discriminatory power and sensitivity of modern mass-spectrometry-based technologies (Fig 1). Two recent reports from the Carvalho and Neyrolles groups, published recently in Proceedings of the National Academy of Science USA and Nature Chemical Biology [3,4], exemplify the rapidly growing impact of metabolomics-based approaches on tuberculosis research.Open in a separate windowFigure 1Modern mass spectrometry illuminates bacterial metabolism. A comparison of activity-based metabolomic profiling with classic metabolic tracing. See the text for details.Within the field of infectious diseases, the deficiencies in our understanding of microbial metabolism have emerged most prominently in the area of tuberculosis research. Despite the development of the first chemotherapies more than 50 years ago, tuberculosis remains the leading bacterial cause of death worldwide, due in part to a failure to keep pace with the emergence of drug resistance [5]. The causes of this shortfall are multifactorial. However, a key contributing factor is our incomplete understanding of the metabolic properties of Mycobacterium tuberculosis (Mtb), its aetiological agent. Unlike most bacterial pathogens, Mtb infects humans as its only known host and reservoir, within whom it resides largely isolated from other microbes. Mtb has thus evolved its metabolism to serve interdependent physiological and pathogenic roles. Yet, more than a century after Koch''s initial discovery of Mtb and 15 years after the first publication of its genome sequence, knowledge of Mtb''s metabolic network remains surprisingly incomplete [6,7,8].“…tuberculosis remains the leading bacterial cause of death worldwide…”As for almost all sequenced microbial genomes, homology-based in silico approaches have failed to suggest a function for nearly 40% of Mtb genes that, presumably, include a significant number of orphan enzyme activities for which no gene has been ascribed [8]. Such approaches have further neglected the impact of evolutionary selection and its ability to dissociate sequence conservation from biochemical activity and physiological function, in order to help optimize the fitness of a given organism within its specific niche. For Mtb, such genes and enzymes represent an especially promising and biologically selective, but untapped, source of potential drug targets.In the study from the Carvalho group, successful application of a recently developed metabolomics assay—known as activity-based metabolomic profiling (ABMP)—allowed the authors to reassign a putatively annotated nucleotide phosphatase (Rv1692) as a D,L-glycerol 3-phosphate phosphatase [3,9]. ABMP was specifically developed to identify enzymatic activities for genes of unknown function by leveraging the analytical discriminatory power of liquid-chromatography-coupled high-resolution mass spectrometry (LC-MS) to analyse the impact of a recombinant enzyme and potential co-factors on a highly concentrated, small-molecule extract derived from the homologous organism (Fig 1). By monitoring for the matched time and enzyme-dependent depletion and accumulation of putative substrates and products, this assay enables the discovery of catalytic activities—rather than simple binding—by using the cellular metabolome as arguably the most physiological chemical library of potential substrates that can be tested, in a label and synthesis-free manner. Moreover, candidate activities assigned by this method can be confirmed by using independent biochemical approaches—such as reconstitution with purified components—and genetic techniques—such as wild-type and genetic knockout, knockdown or overexpression strains. In reassigning Rv1692 as a glycerol phosphate phosphatase, rather than a nucleotide phosphatase, Carvalho and colleagues demonstrate the potential of ABMP to overcome the biochemical challenge of assigning substrate specificity to a member of a large enzyme superfamily—in this case, the haloacid dehydrogenase superfamily. But, perhaps more significantly, they also direct new biological attention to the largely neglected area of Mtb membrane homeostasis, in which Rv1692 might play an important role in glycerophospholipid recycling and catabolism.“…knowledge of Mtb''s metabolic network remains surprisingly incomplete”Neyrolles and colleagues make use of the same metabolomics platform to perform metabolite-tracing studies by using stable-isotope-labelled precursors, which led them to reassign a putatively annotated asparagine transporter (AnsP1) as an aspartate transporter. AnsP1 bears 55% sequence identity and 70% similarity to an orthologue in Salmonella that belongs to the amino acid transporter family 2.A.3.1, whereas aspartate transporters are typically members of the dicarboxylate amino acid:cation symporter family 2.A.23 [4]. This study demonstrates the ability of metabolomic platforms to not only characterize the activity of a given protein within its natural physiological milieu, but also revive classical experimental methods by using modern technologies. The availability of stable (non-radioactive) isotopically labelled precursors has now made it possible to resolve their specific metabolic fates. In this case, such an approach revealed that Mtb can use aspartate as both a carbon and nitrogen source, after its uptake through AnsP1. Looking beyond the specific biochemical assignment of AnsP1 as an aspartate—rather than asparagine—transporter, this study illustrates the potential impact of such discoveries on downstream paths of investigation. In this case, the remarkable application of high-resolution dynamic secondary ion mass spectroscopy to provide the first direct biochemical images of the nutritional environment of the Mtb-infected phagosome.New technologies are often developed in the context of specific needs. However, their impact is usually not realized until extended beyond such contexts, sometimes resulting in major paradigm shifts. The above examples highlight just two emerging possibilities of how metabolomics technologies can be extended beyond the context of global comparisons and provide unique biological insights. To the extent that the analytical power of these platforms can be adapted to other functional approaches, metabolomics promises to pay handsome biochemical and physiological dividends.     

Environmental footprinting of hospitals: Organizational life cycle assessment of a Canadian hospital

Healthcare is a critical and complex service sector with direct and indirect greenhouse gas (GHG) emissions amounting to 5%–10% of the national total in developed economies like Canada and the United States. Along with a growing, albeit sporadic, set of life cycle assessment (LCA) (and “carbon footprinting”) studies of specific medical products and procedures, there is growing interest in “environmental footprinting” of hospitals. In this article, we advance this rapidly evolving area through a comprehensive organizational LCA of a 40-bed hospital in British Columbia, Canada, in its 2019 fiscal year. Our results indicate that the total environmental footprint of the hospital includes, among other things, global warming potential of 3500–5000 t CO₂ eq. (with 95% confidence). “Hotspots” in this footprint are attributable to energy and water use (and wastewater released), releases of anesthetic gases (which are potent GHGs), and the upstream production of the thousands of materials, chemicals, pharmaceuticals, and other products used in the hospital. The generalizability and comparability of these results are limited by inconsistencies across the few environmental footprinting studies of hospitals conducted to date. Nonetheless, our novel methodological approach, in which we compiled new LCA data for 200 goods and services used in healthcare—strategically selected to statistically represent the 2927 unique products in the hospital's “supply-chains”—has broad applicability in healthcare and beyond.     

Genetic rescue in a plant polyploid complex: Case study on the importance of genetic and trait data for conservation management

Knowledge of the biology of rare plant species is indispensable to aid their survival and to inform efficient conservation actions, but in many cases relevant data are lacking. In addition, while studies of conservation genetics have provided a wealth of information on the considerations arising from inbreeding, mate limitation, or local adaptation, the impact of intraspecific polyploidy remains understudied. In this study, we examined the breeding system of the rare Australian daisy Rutidosis lanata (Asteraceae) and screened ten of its populations for their ploidy level to develop recommendations for management actions, in particular, with regard to seed sourcing and genetic rescue. We found R. lanata to represent a polyploid complex, with tetraploid, pentaploid and hexaploid individuals coexisting in the same species. Crossing experiments confirmed R. lanata to be self‐incompatible. Mate availability varied from c. 49% to c. 76% across populations. Most populations showed mate availability of c. 50%–70%, suggesting that mate limitation resulting from a lack of local genetic diversity may cause or at least contribute to reduced seed set. Crossing between populations resulted in significantly higher reproductive success for all populations except one, suggesting the possibility of genetic rescue through population mixing. However, the crossing experiments also showed that pentaploids suffer from a severely reduced paternal reproductive fitness. Any additional hybrids between tetraploids and pentaploids, as would be created by mixing populations with different genome copy numbers during conservation work, would consequently exacerbate mate limitation and thus reduce population viability. We conclude that seed set and thus population viability can be maximized by mixing populations with the same number of genome copies, but that populations with different numbers should be kept spatially separated. The case of Rutidosis lanata provides an example and a potential template for examining the conservation genetics of other species that may constitute polyploid complexes.     

Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis.

We have isolated a yeast conditional mutant which rapidly ceases synthesis of mRNA when subjected to the nonpermissive temperature. This mutant (rpb1-1) was constructed by replacing the wild-type chromosomal copy of the gene encoding the largest subunit of RNA polymerase II with one mutagenized in vitro. The rapid cessation of mRNA synthesis in vivo and the lack of RNA polymerase II activity in crude extracts indicate that the mutant possesses a functionally defective, rather than an assembly-defective, RNA polymerase II. The shutdown in mRNA synthesis in the rpb1-1 mutant has pleiotropic effects on the synthesis of other RNAs and on the heat shock response. This mutant provides direct evidence that the RPB1 protein has a functional role in mRNA synthesis.     

Retinoic acid induction of stress proteins in fetal mouse limb buds

Retinoic acid (RA) is teratogenic in rodent embryos. Several teratogens have been shown to induce the synthesis of a subset of heat shock proteins (stress proteins) in Drosophila. To determine if RA induces the synthesis of these proteins in rodent embryos, pregnant ICR mice were dosed with 100 mg/kg RA on Day 11 of gestation. Forelimb buds were removed from embryos 2.5 hr post-RA-treatment and nuclei were isolated, stained, and sorted from stages of the cell cycle. Nuclear proteins were extracted and analyzed by two-dimensional polyacrylamide gel electrophoresis. Nuclear proteins with molecular weights of approximately 84 and 25 kDa were synthesized in embryos in the G0 + G1 phase after pregnant dams were treated with RA. Isoelectric points, molecular weights, immunochemical blotting, and polypeptide mapping demonstrated that these proteins are indistinguishable from stress proteins isolated under a variety of conditions from rat submaxillary glands and mouse lymphoma cells. These results suggest that treatment with RA induces the synthesis of a subset of stress proteins; the role of these proteins in the teratogenic effects of RA is not known.     

Ozone response in wild type and radiation-sensitive mutants of Saccharomyces cerevisiae.

Summary The effect of ozone exposure on Saccharomyces cerevisiae was studied. Factors such as ozone concentration, treatment time, media, initial cell concentration and growth phase were shown to influence ozone response in this organism. Logarithmic phase cells were much more sensitive than stationary phase cells to the lethal effect of ozone.The radiation-sensitive mutants rad3, rad6, rad51 and rad52 of S. cerevisiae were exposed, in water, to 50 ppm of ozone for 30 min. On comparing their survival curves, the rad51 and the rad52 mutants showed a greater sensitivity to ozone exposure than the wild type.     

Airway epithelial cells initiate the allergen response through transglutaminase 2 by inducing IL-33 expression and a subsequent Th2 response

Background

Transglutaminase 2 (TG2) is a post-translational protein-modifying enzyme that catalyzes the transamidation reaction, producing crosslinked or polyaminated proteins. Increased TG2 expression and activity have been reported in various inflammatory conditions, such as rheumatoid arthritis, inflammation-associated pulmonary fibrosis, and autoimmune encephalitis. In particular, TG2 from epithelial cells is important during the initial inflammatory response in the lung. In this study, we evaluated the role of TG2 in the pathogenesis of allergic asthma, particularly whether TG2 affects initial activation signaling leading to Th2 differentiation against antigens.

Methods

We induced allergic asthma by ovalbumin sensitization and intranasal challenge in wild-type (WT) BALB/c and TG2-deficient mice. Broncheoalveolar lavage fluid cells and intracellular cytokine production were analyzed by flow cytometry. Interleukin (IL)-33 and TG2 expression in lung epithelial cells was detected by confocal microscopy.

Results

Airway responsiveness was attenuated in TG2-deficient mice compared to that in the WT control. In addition, recruitment of eosinophils and Th2 and Th17 differentiation decreased in TG2-deficient mice. Treatment with cysteamine, a transglutaminase inhibitor, also reduced airway hypersensitivity, inflammatory cell recruitment, and T helper cell differentiation. TG2-deficient mice showed reduced IL-33 expression following induction of allergic asthma compared to those in the WT control.

Conclusions

We found that pulmonary epithelial cells damaged by allergens triggered TG2-mediated IL-33 expression leading to type 2 responses by recruiting both innate and adaptive arms of the immune system.     

The ACh-evoked,Ca2+-activated Whole-cell K+ Current in Mouse Mandibular Secretory Cells. Whole-cell and Fluorescence Studies

In our previous studies on sheep parotid secretory cells, we showed that the K⁺ current evoked by acetylcholine (ACh) was not carried by the high-conductance voltage- and Ca²⁺-activated K⁺ (BK) channel which is so conspicuous in unstimulated cells, notwithstanding that the BK channel is activated by ACh. Since several studies from other laboratories had suggested that the BK channel did carry the ACh-evoked K⁺ current in the secretory cells of the mouse mandibular gland, and that the current could be blocked with tetraethylammonium (TEA), a known blocker of BK channels, we decided to investigate the ACh-evoked K⁺ current in mouse cells more closely. We studied whether the ACh-evoked K⁺ current in the mouse is inhibited by TEA and quinine. Using the whole-cell patch-clamp technique and microspectrofluorimetric measurement of intracellular Ca²⁺, we found that TEA and quinine do inhibit the ACh-evoked K⁺ current but that the effect is due to inhibition of the increase in intracellular Ca²⁺ evoked by ACh, not to blockade of a K⁺ conductance. Furthermore, we found that the K⁺ conductance activated when ionomycin is used to increase intracellular free Ca²⁺ was inhibited only by quinine and not by TEA. We conclude that the ACh-evoked K⁺ current in mouse mandibular cells does not have the blocker sensitivity pattern that would be expected if it were being carried by the high-conductance, voltage- and Ca²⁺-activated K⁺ (BK) channel. The properties of this current are, however, consistent with those of a 40 pS K⁺ channel that we have reported to be activated by ACh in these cells [16]. Received: 9 January 1996/Revised: 17 April 1996