Impact of vitamin B12 on rhamnose metabolism, stress defense and in-vitro virulence of Listeria monocytogenes.

Listeria monocytogenes is a facultative anaerobe which can cause a severe food-borne infection known as listeriosis. L. monocytogenes is capable of utilizing various nutrient sources including rhamnose, a naturally occurring deoxy sugar abundant in foods. L. monocytogenes can degrade rhamnose into lactate, acetate and 1,2-propanediol. Our previous study showed that addition of vitamin B12 stimulated anaerobic growth of L. monocytogenes on rhamnose due to the activation of bacterial microcompartments for 1,2-propanediol utilization (pdu BMC) with concomitant production of propionate and propanol. Notably, anaerobic 1,2-propanediol metabolism has been linked to virulence of enteric pathogens including Salmonella spp. and L. monocytogenes. In this study we investigated the impact of B12 and BMC activation on i) aerobic and anerobic growth of L. monocytogenes on rhamnose and ii) the level of virulence. We observed B12-induced pdu BMC activation and growth stimulation only in anaerobically grown cells. Comparative Caco-2 virulence assays showed that these pdu BMC-induced cells have significantly higher translocation efficiency compared to non-induced cells (anaerobic growth without B12; aerobic growth with or without B12), while adhesion and invasion capacity is similar for all cells. Comparative proteome analysis showed specific and overlapping responses linked to metabolic shifts, activation of stress defense proteins and virulence factors, with RNA polymerase sigma factor SigL, teichoic acid export ATP-binding protein TagH, DNA repair and protection proteins, RadA and DPS, and glutathione synthase GshAB, previously linked to activation of virulence response in L. monocytogenes, uniquely upregulated in anaerobically rhamnose grown pdu-induced cells. Our results shed light on possible effects of B12 on L. monocytogenes competitive fitness and virulence activation when utilizing rhamnose in anaerobic conditions encountered during transmission and the human intestine.

Dynamic genome-scale modeling of Saccharomyces cerevisiae unravels mechanisms for ester formation during alcoholic fermentation.

Fermentation employing Saccharomyces cerevisiae has produced alcoholic beverages and bread for millennia. More recently, S. cerevisiae has been used to manufacture specific metabolites for the food, pharmaceutical, and cosmetic industries. Among the most important of these metabolites are compounds associated with desirable aromas and flavors, including higher alcohols and esters. Although the physiology of yeast has been well-studied, its metabolic modulation leading to aroma production in relevant industrial scenarios such as winemaking is still unclear. Here we ask what are the underlying metabolic mechanisms that explain the conserved and varying behavior of different yeasts regarding aroma formation under enological conditions? We employed dynamic flux balance analysis (dFBA) to answer this key question using the latest genome-scale metabolic model (GEM) of S. cerevisiae. The model revealed several conserved mechanisms among wine yeasts, for example, acetate ester formation is dependent on intracellular metabolic acetyl-CoA/CoA levels, and the formation of ethyl esters facilitates the removal of toxic fatty acids from cells using CoA. Species-specific mechanisms were also found, such as a preference for the shikimate pathway leading to more 2-phenylethanol production in the Opale strain as well as strain behavior varying notably during the carbohydrate accumulation phase and carbohydrate accumulation inducing redox restrictions during a later cell growth phase for strain Uvaferm. In conclusion, our new metabolic model of yeast under enological conditions revealed key metabolic mechanisms in wine yeasts, which will aid future research strategies to optimize their behavior in industrial settings.

Modulating CRISPR-Cas Genome Editing Using Guide-Complementary DNA Oligonucleotides.

Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) has revolutionized genome editing and has great potential for many applications, such as correcting human genetic disorders. To increase the safety of genome editing applications, CRISPR-Cas may benefit from strict control over Cas enzyme activity. Previously, anti-CRISPR proteins and designed oligonucleotides have been proposed to modulate CRISPR-Cas activity. In this study, we report on the potential of guide-complementary DNA oligonucleotides as controlled inhibitors of Cas9 ribonucleoprotein complexes. First, we show that DNA oligonucleotides inhibit Cas9 activity in human cells, reducing both on- and off-target cleavage. We then used in vitro assays to better understand how inhibition is achieved and under which conditions. Two factors were found to be important for robust inhibition: the length of the complementary region and the presence of a protospacer adjacent motif-loop on the inhibitor. We conclude that DNA oligonucleotides can be used to effectively inhibit Cas9 activity both ex vivo and in vitro.

Underground metabolism as a rich reservoir for pathway engineering.

MOTIVATION: Bioproduction of value-added compounds is frequently achieved by utilizing enzymes from other species. However, expression of such heterologous enzymes can be detrimental due to unexpected interactions within the host cell. Recently, an alternative strategy emerged, which relies on recruiting side activities of host enzymes to establish new biosynthetic pathways. Although such low-level 'underground' enzyme activities are prevalent, it remains poorly explored whether they may serve as an important reservoir for pathway engineering. RESULTS: Here, we use genome-scale modeling to estimate the theoretical potential of underground reactions for engineering novel biosynthetic pathways in Escherichia coli. We found that biochemical reactions contributed by underground enzyme activities often enhance the in silico production of compounds with industrial importance, including several cases where underground activities are indispensable for production. Most of these new capabilities can be achieved by the addition of one or two underground reactions to the native network, suggesting that only a few side activities need to be enhanced during implementation. Remarkably, we find that the contribution of underground reactions to the production of value-added compounds is comparable to that of heterologous reactions, underscoring their biotechnological potential. Taken together, our genome-wide study demonstrates that exploiting underground enzyme activities could be a promising addition to the toolbox of industrial strain development. AVAILABILITY AND IMPLEMENTATION: The data and scripts underlying this article are available on GitHub at https://github.com/pappb/Kovacs-et-al-Underground-metabolism. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Metabolic flux sampling predicts strain-dependent differences related to aroma production among commercial wine yeasts.

BACKGROUND: Metabolomics coupled with genome-scale metabolic modeling approaches have been employed recently to quantitatively analyze the physiological states of various organisms, including Saccharomyces cerevisiae. Although yeast physiology in laboratory strains is well-studied, the metabolic states under industrially relevant scenarios such as winemaking are still not sufficiently understood, especially as there is considerable variation in metabolism between commercial strains. To study the potential causes of strain-dependent variation in the production of volatile compounds during enological conditions, random flux sampling and statistical methods were used, along with experimental extracellular metabolite flux data to characterize the differences in predicted intracellular metabolic states between strains. RESULTS: It was observed that four selected commercial wine yeast strains (Elixir, Opale, R2, and Uvaferm) produced variable amounts of key volatile organic compounds (VOCs). Principal component analysis was performed on extracellular metabolite data from the strains at three time points of cell cultivation (24, 58, and 144 h). Separation of the strains was observed at all three time points. Furthermore, Uvaferm at 24 h, for instance, was most associated with propanol and ethyl hexanoate. R2 was found to be associated with ethyl acetate and Opale could be associated with isobutanol while Elixir was most associated with phenylethanol and phenylethyl acetate. Constraint-based modeling (CBM) was employed using the latest genome-scale metabolic model of yeast (Yeast8) and random flux sampling was performed with experimentally derived fluxes at various stages of growth as constraints for the model. The flux sampling simulations allowed us to characterize intracellular metabolic flux states and illustrate the key parts of metabolism that likely determine the observed strain differences. Flux sampling determined that Uvaferm and Elixir are similar while R2 and Opale exhibited the highest degree of differences in the Ehrlich pathway and carbon metabolism, thereby causing strain-specific variation in VOC production. The model predictions also established the top 20 fluxes that relate to phenotypic strain variation (e.g. at 24 h). These fluxes indicated that Opale had a higher median flux for pyruvate decarboxylase reactions compared with the other strains. Conversely, R2 which was lower in all VOCs, had higher median fluxes going toward central metabolism. For Elixir and Uvaferm, the differences in metabolism were most evident in fluxes pertaining to transaminase and hexokinase associated reactions. The applied analysis of metabolic divergence unveiled strain-specific differences in yeast metabolism linked to fusel alcohol and ester production. CONCLUSIONS: Overall, this approach proved useful in elucidating key reactions in amino acid, carbon, and glycerophospholipid metabolism which suggest genetic divergence in activity in metabolic subsystems among these wine strains related to the observed differences in VOC formation. The findings in this study could steer more focused research endeavors in developing or selecting optimal aroma-producing yeast stains for winemaking and other types of alcoholic fermentations.

Anaerobic Growth of Listeria monocytogenes on Rhamnose Is Stimulated by Vitamin B12 and Bacterial Microcompartment-Dependent 1,2-Propanediol Utilization.

The foodborne pathogen Listeria monocytogenes can form proteinaceous organelles called bacterial microcompartments (BMCs) that optimize the utilization of substrates, such as 1,2-propanediol, and confer an anaerobic growth advantage. Rhamnose is a deoxyhexose sugar abundant in a range of environments, including the human intestine, and can be degraded in anaerobic conditions into 1,2-propanediol, next to acetate and lactate. Rhamnose-derived 1,2-propanediol was found to link with BMCs in some human pathogens such as Salmonella enterica, but the involvement of BMCs in rhamnose metabolism and potential physiological effects on L. monocytogenes are still unknown. In this study, we first test the effect of rhamnose uptake and utilization on anaerobic growth of L. monocytogenes EGDe without or with added vitamin B12, followed by metabolic analysis. We show that vitamin B12-dependent activation of pdu stimulates metabolism and anaerobic growth of L. monocytogenes EGDe on rhamnose via 1,2-propanediol degradation into 1-propanol and propionate. Transmission electron microscopy of pdu-induced cells shows that BMCs are formed, and additional proteomics experiments confirm expression of pdu BMC shell proteins and enzymes. Finally, we discuss the physiological effects and energy efficiency of L. monocytogenes pdu BMC-driven anaerobic rhamnose metabolism and the impact on competitive fitness in environments such as the human intestine. IMPORTANCE Listeria monocytogenes is a foodborne pathogen causing severe illness and, as such, it is crucial to understand the molecular mechanisms contributing to its survival strategy and pathogenicity. Rhamnose is a deoxyhexose sugar abundant in a range of environments, including the human intestine, and can be degraded in anaerobic conditions into 1,2-propanediol. In our previous study, the utilization of 1,2-propanediol (pdu) in L. monocytogenes was proved to be metabolized in bacterial microcompartments (BMCs), which are self-assembling subcellular proteinaceous structures and analogs of eukaryotic organelles. Here, we show that the vitamin B12-dependent activation of pdu stimulates metabolism and anaerobic growth of L. monocytogenes EGDe on rhamnose via BMC-dependent 1,2-propanediol utilization. Combined with metabolic and proteomics analysis, our discussion on the physiological effects and energy efficiency of BMC-driven rhamnose metabolism shed new light to understand the impact on L. monocytogenes competitive fitness in ecosystems such as the human intestine.

Nitrogenous Compound Utilization and Production of Volatile Organic Compounds among Commercial Wine Yeasts Highlight Strain-Specific Metabolic Diversity.

Genetic background and environmental conditions affect the production of sensory impact compounds by Saccharomyces cerevisiae. The relative importance of the strain-specific metabolic capabilities for the production of volatile organic compounds (VOCs) remains unclear. We investigated which amino acids contribute to VOC production and whether amino acid-VOC relations are conserved among yeast strains. Amino acid consumption and production of VOCs during grape juice fermentation was investigated using four commercial wine yeast strains: Elixir, Opale, R2, and Uvaferm. Principal component analysis of the VOC data demonstrated that Uvaferm correlated with ethyl acetate and ethyl hexanoate production, R2 negatively correlated with the acetate esters, and Opale positively correlated with fusel alcohols. Biomass formation was similar for all strains, pointing to metabolic differences in the utilization of nutrients to form VOCs. Partial least-squares linear regression showed that total aroma production is a function of nitrogen utilization (R2 = 0.87). We found that glycine, tyrosine, leucine, and lysine utilization were positively correlated with fusel alcohols and acetate esters. Mechanistic modeling of the yeast metabolic network via parsimonious flux balance analysis and flux enrichment analysis revealed enzymes with crucial roles, such as transaminases and decarboxylases. Our work provides insights in VOC production in wine yeasts. IMPORTANCE Saccharomyces cerevisiae is widely used in grape juice fermentation to produce wines. Along with the genetic background, the nitrogen in the environment in which S. cerevisiae grows impacts its regulation of metabolism. Also, commercial S. cerevisiae strains exhibit immense diversity in their formation of aromas, and a desirable aroma bouquet is an essential characteristic for wines. Since nitrogen affects aroma formation in wines, it is essential to know the extent of this connection and how it leads to strain-dependent aroma profiles in wines. We evaluated the differences in the production of key aroma compounds among four commercial wine strains. Moreover, we analyzed the role of nitrogen utilization on the formation of various aroma compounds. This work illustrates the unique aroma-producing differences among industrial yeast strains and suggests more intricate, nitrogen-associated routes influencing those aroma-producing differences.

Bacterial Microcompartment-Dependent 1,2-Propanediol Utilization of Propionibacterium freudenreichii.

Bacterial microcompartments (BMCs) are proteinaceous prokaryotic organelles that enable the utilization of substrates such as 1,2-propanediol and ethanolamine. BMCs are mostly linked to the survival of particular pathogenic bacteria by providing a growth advantage through utilization of 1,2-propanediol and ethanolamine which are abundantly present in the human gut. Although a 1,2-propanediol utilization cluster was found in the probiotic bacterium Propionibacterium freudenreichii, BMC-mediated metabolism of 1,2-propanediol has not been demonstrated experimentally in P. freudenreichii. In this study we show that P. freudenreichii DSM 20271 metabolizes 1,2-propanediol in anaerobic conditions to propionate and 1-propanol. Furthermore, 1,2-propanediol induced the formation of BMCs, which were visualized by transmission electron microscopy and resembled BMCs found in other bacteria. Proteomic analysis of 1,2-propanediol grown cells compared to L-lactate grown cells showed significant upregulation of proteins involved in propanediol-utilization (pdu-cluster), DNA repair mechanisms and BMC shell proteins while proteins involved in oxidative phosphorylation were down-regulated. 1,2-Propanediol utilizing cells actively produced vitamin B12 (cobalamin) in similar amounts as cells growing on L-lactate. The ability to metabolize 1,2-propanediol may have implications for human gut colonization and modulation, and can potentially aid in delivering propionate and vitamin B12 in situ.

Bacterial Microcompartments Coupled with Extracellular Electron Transfer Drive the Anaerobic Utilization of Ethanolamine in Listeria monocytogenes.

Ethanolamine (EA) is a valuable microbial carbon and nitrogen source derived from cell membranes. EA catabolism is suggested to occur in a cellular metabolic subsystem called a bacterial microcompartment (BMC), and the activation of EA utilization (eut) genes is linked to bacterial pathogenesis. Despite reports showing that the activation of eut is regulated by a vitamin B12-binding riboswitch and that upregulation of eut genes occurs in mice, it remains unknown whether EA catabolism is BMC dependent in Listeria monocytogenes Here, we provide evidence for BMC-dependent anaerobic EA utilization via metabolic analysis, proteomics, and electron microscopy. First, we show vitamin B12-induced activation of the eut operon in L. monocytogenes coupled to the utilization of EA, thereby enabling growth. Next, we demonstrate BMC formation connected with EA catabolism with the production of acetate and ethanol in a molar ratio of 2:1. Flux via the ATP-generating acetate branch causes an apparent redox imbalance due to the reduced regeneration of NAD+ in the ethanol branch resulting in a surplus of NADH. We hypothesize that the redox imbalance is compensated by linking eut BMCs to anaerobic flavin-based extracellular electron transfer (EET). Using L. monocytogenes wild-type, BMC mutant, and EET mutant strains, we demonstrate an interaction between BMCs and EET and provide evidence for a role of Fe3+ as an electron acceptor. Taken together, our results suggest an important role of BMC-dependent EA catabolism in L. monocytogenes growth in anaerobic environments like the human gastrointestinal tract, with a crucial role for the flavin-based EET system in redox balancing.IMPORTANCE Listeria monocytogenes is a foodborne pathogen causing severe illness, and as such, it is crucial to understand the molecular mechanisms contributing to pathogenicity. One carbon source that allows L. monocytogenes to grow in humans is ethanolamine (EA), which is derived from phospholipids present in eukaryotic cell membranes. It is hypothesized that EA utilization occurs in bacterial microcompartments (BMCs), self-assembling subcellular proteinaceous structures and analogs of eukaryotic organelles. Here, we demonstrate that BMC-driven utilization of EA in L. monocytogenes results in increased energy production essential for anaerobic growth. However, exploiting BMCs and the encapsulated metabolic pathways also requires the balancing of oxidative and reductive pathways. We now provide evidence that L. monocytogenes copes with this by linking BMC activity to flavin-based extracellular electron transfer (EET) using iron as an electron acceptor. Our results shed new light on an important molecular mechanism that enables L. monocytogenes to grow using host-derived phospholipid degradation products.

Stimulation of cholesterol biosynthesis in mitochondrial complex I-deficiency lowers reductive stress and improves motor function and survival in mice.

The majority of cellular energy is produced by the mitochondrial oxidative phosphorylation (OXPHOS) system. Failure of the first OXPHOS enzyme complex, NADH:ubiquinone oxidoreductase or complex I (CI), is associated with multiple signs and symptoms presenting at variable ages of onset. There is no approved drug treatment yet to slow or reverse the progression of CI-deficient disorders. Here, we present a comprehensive human metabolic network model of genetically characterized CI-deficient patient-derived fibroblasts. Model calculations predicted that increased cholesterol production, export, and utilization can counterbalance the surplus of reducing equivalents in patient-derived fibroblasts, as these pathways consume considerable amounts of NAD(P)H. We show that fibrates attenuated increased NAD(P)H levels and improved CI-deficient fibroblast growth by stimulating the production of cholesterol via enhancement of its cellular efflux. In CI-deficient (Ndufs4-/-) mice, fibrate treatment resulted in prolonged survival and improved motor function, which was accompanied by an increased cholesterol efflux from peritoneal macrophages. Our results shine a new light on the use of compensatory biological pathways in mitochondrial dysfunction, which may lead to novel therapeutic interventions for mitochondrial diseases for which currently no cure exists.

Suboptimal Global Transcriptional Response Increases the Harmful Effects of Loss-of-Function Mutations.

The fitness impact of loss-of-function mutations is generally assumed to reflect the loss of specific molecular functions associated with the perturbed gene. Here, we propose that rewiring of the transcriptome upon deleterious gene inactivation is frequently nonspecific and mimics stereotypic responses to external environmental change. Consequently, transcriptional response to gene deletion could be suboptimal and incur an extra fitness cost. Analysis of the transcriptomes of ∼1,500 single-gene deletion Saccharomyces cerevisiae strains supported this scenario. First, most transcriptomic changes are not specific to the deleted gene but are rather triggered by perturbations in functionally diverse genes. Second, gene deletions that alter the expression of dosage-sensitive genes are especially harmful. Third, by elevating the expression level of downregulated genes, we could experimentally mitigate the fitness defect of gene deletions. Our work shows that rewiring of genomic expression upon gene inactivation shapes the harmful effects of mutations.

Bacterial Microcompartment-Dependent 1,2-Propanediol Utilization Stimulates Anaerobic Growth of Listeria monocytogenes EGDe.

Bacterial microcompartments (BMCs) are proteinaceous organelles that optimize specific metabolic pathways referred to as metabolosomes involving transient production of toxic volatile metabolites such as aldehydes. Previous bioinformatics analysis predicted the presence of BMCs in 23 bacterial phyla including foodborne pathogens and a link with gene clusters for the utilization of host-derived substrates such as 1,2-propanediol utilization, i.e., the Pdu cluster. Although, transcriptional regulation of the Pdu cluster and its role in Listeria monocytogenes virulence in animal models have recently been reported, the experimental identification and the physiological role of BMCs in L. monocytogenes is still unexplored. Here, we ask whether BMCs could enable utilization of 1,2-propanediol (Pd) in L. monocytogenes under anaerobic conditions. Using L. monocytogenes EGDe as a model strain, we could demonstrate efficient utilization of Pd with concomitant production of 1-propanol and propionate after 24 h of anaerobic growth, while the utilization was significantly reduced in aerobic conditions. In line with this, expression of genes encoding predicted shell proteins and the signature enzyme propanediol dehydratase is upregulated more than 20-fold in cells anaerobically grown in Pdu-induced versus non-induced control conditions. Additional proteomics analysis confirmed the presence of BMC shell proteins and Pdu enzymes in cells that show active degradation of Pd. Furthermore, using transmission electron microscopy, BMC structures have been detected in these cells linking gene expression, protein composition, and BMCs to activation of the Pdu cluster in anaerobic growth of L. monocytogenes. Studies in defined minimal medium with Pd as an energy source showed a significant increase in cell numbers, indicating that Pdu and the predicted generation of ATP in the conversion of propionyl-phosphate to the end product propionate can support anaerobic growth of L. monocytogenes. Our findings may suggest a role for BMC-dependent utilization of Pd in L. monocytogenes growth, transmission, and interaction with the human host.

Enzyme promiscuity shapes adaptation to novel growth substrates.

Evidence suggests that novel enzyme functions evolved from low-level promiscuous activities in ancestral enzymes. Yet, the evolutionary dynamics and physiological mechanisms of how such side activities contribute to systems-level adaptations are not well characterized. Furthermore, it remains untested whether knowledge of an organism's promiscuous reaction set, or underground metabolism, can aid in forecasting the genetic basis of metabolic adaptations. Here, we employ a computational model of underground metabolism and laboratory evolution experiments to examine the role of enzyme promiscuity in the acquisition and optimization of growth on predicted non-native substrates in Escherichia coli K-12 MG1655. After as few as approximately 20 generations, evolved populations repeatedly acquired the capacity to grow on five predicted non-native substrates-D-lyxose, D-2-deoxyribose, D-arabinose, m-tartrate, and monomethyl succinate. Altered promiscuous activities were shown to be directly involved in establishing high-efficiency pathways. Structural mutations shifted enzyme substrate turnover rates toward the new substrate while retaining a preference for the primary substrate. Finally, genes underlying the phenotypic innovations were accurately predicted by genome-scale model simulations of metabolism with enzyme promiscuity.

CRISPR-Directed Microbiome Manipulation across the Food Supply Chain.

The advent of CRISPR-based technologies has revolutionized genetics over the past decade, and genome editing is now widely implemented for diverse medical and agricultural applications, such as correcting genetic disorders and improving crop and livestock breeding. CRISPR-based technologies are also of great potential to alter the genetic content of food bacteria in order to control the composition and activity of microbial populations across the food supply chain, from the farm to consumer products. Advancing the food supply chain is of great societal importance as it involves optimizing fermentation processes to enhance taste and sensory properties of food products, as well as improving food quality and safety by controlling spoilage bacteria and pathogens. Here, we discuss the various CRISPR technologies that can alter bacterial functionalities and modulate the composition of microbial communities in foods. We illustrate how these applications can be harnessed along the food supply chain to manipulate microbiomes that encompass spoilage and pathogenic bacteria as well as desirable starter cultures and health-promoting probiotics.

Delivery of genome editing tools by bacterial extracellular vesicles.

Here we propose to use bacterial cells as factories that generate EVs harboring both the RNP complex (or any other CRISPR-mediated tool) and a specific ligand molecule. The ligand would allow specific binding of EVs to cells with the matching receptors, adding a level of specificity to the delivery, hence stimulating precise genome editing.

CRISPR-Cas genome engineering of esterase activity in Saccharomyces cerevisiae steers aroma formation.

OBJECTIVE: Saccharomyces cerevisiae is used worldwide for the production of ale-type beers. This yeast is responsible for the production of the characteristic fruity aroma compounds. Esters constitute an important group of aroma active secondary metabolites produced by S. cerevisiae. Previous work suggests that esterase activity, which results in ester degradation, may be the key factor determining the abundance of fruity aroma compounds. Here, we test this hypothesis by deletion of two S. cerevisiae esterases, IAH1 and TIP1, using CRISPR-Cas9 genome editing and by studying the effect of these deletions on esterase activity and extracellular ester pools. RESULTS: Saccharomyces cerevisiae mutants were constructed lacking esterase IAH1 and/or TIP1 using CRISPR-Cas9 genome editing. Esterase activity using 5-(6)-carboxyfluorescein diacetate (cFDA) as substrate was found to be significantly lower for ΔIAH1 and ΔIAH1ΔTIP1 mutants compared to wild type (WT) activity (P < 0.05 and P < 0.001, respectively). As expected, we observed an increase in relative abundance of acetate and ethyl esters and an increase in ethyl esters in ΔIAH1 and ΔTIP1, respectively. Interestingly, the double gene disruption mutant ΔIAH1ΔTIP1 showed an aroma profile comparable to WT levels, suggesting the existence and activation of a complex regulatory mechanism to compensate multiple genomic alterations in aroma metabolism.

Tissue Metabolic Changes Drive Cytokine Responses to Mycobacterium tuberculosis.

Cellular metabolism can influence host immune responses to Mycobacterium tuberculosis. Using a systems biology approach, differential expression of 292 metabolic genes involved in glycolysis, glutathione, pyrimidine, and inositol phosphate pathways was evident at the site of a human tuberculin skin test challenge in patients with active tuberculosis infection. For 28 metabolic genes, we identified single nucleotide polymorphisms that were trans-acting for in vitro cytokine responses to M. tuberculosis stimulation, including glutathione and pyrimidine metabolism genes that alter production of Th1 and Th17 cytokines. Our findings identify novel therapeutic targets in host metabolism that may shape protective immunity to tuberculosis.

Cerebral tryptophan metabolism and outcome of tuberculous meningitis: an observational cohort study.

BACKGROUND: Immunopathology contributes to the high mortality of tuberculous meningitis, but the biological pathways involved are mostly unknown. We aimed to compare cerebrospinal fluid (CSF) and serum metabolomes of patients with tuberculous meningitis with that of controls without tuberculous meningitis, and assess the link between metabolite concentrations and mortality. METHODS: In this observational cohort study at the Hasan Sadikin Hospital (Bandung, Indonesia) we measured 425 metabolites using liquid chromatography-mass spectrometry in CSF and serum from 33 HIV-negative Indonesian patients with confirmed or probable tuberculous meningitis and 22 control participants with complete clinical data between March 12, 2009, and Oct 27, 2013. Associations of metabolite concentrations with survival were validated in a second cohort of 101 patients from the same centre. Genome-wide single nucleotide polymorphism typing was used to identify tryptophan quantitative trait loci, which were used for survival analysis in a third cohort of 285 patients. FINDINGS: Concentrations of 250 (70%) of 351 metabolites detected in CSF were higher in patients with tuberculous meningitis than in controls, especially in those who died during follow-up. Only five (1%) of the 390 metobolites detected in serum differed between patients with tuberculous meningitis and controls. CSF tryptophan concentrations showed a pattern different from most other CSF metabolites; concentrations were lower in patients who survived compared with patients who died (9-times) and to controls (31-times). The association of low CSF tryptophan with patient survival was confirmed in the validation cohort (hazard ratio 0·73; 95% CI 0·64-0·83; p<0·0001; per each halving). 11 genetic loci predictive for CSF tryptophan concentrations in tuberculous meningitis were identified (p<0·00001). These quantitative trait loci predicted survival in a third cohort of 285 HIV-negative patients in a prognostic index including age and sex, also after correction for possible confounders (p=0·0083). INTERPRETATION: Cerebral tryptophan metabolism, which is known to affect Mycobacterium tuberculosis growth and CNS inflammation, is important for the outcome of tuberculous meningitis. CSF tryptophan concentrations in tuberculous meningitis are under strong genetic influence, probably contributing to the variable outcomes of tuberculous meningitis. Interventions targeting tryptophan metabolism could improve outcomes of tuberculous meningitis. FUNDING: Royal Dutch Academy of Arts and Sciences; Netherlands Foundation for Scientific Research; Radboud University; National Academy of Sciences; Ministry of Research, Technology, and Higher Education, Indonesia; European Research Council; and PEER-Health.

Underground metabolism: network-level perspective and biotechnological potential.

A key challenge in molecular systems biology is understanding how new pathways arise during evolution and how to exploit them for biotechnological applications. New pathways in metabolic networks often evolve by recruiting weak promiscuous activities of pre-existing enzymes. Here we describe recent systems biology advances to map such 'underground' activities and to predict and analyze their contribution to new metabolic functions. Underground activities are prevalent in cellular metabolism and can form novel pathways that either enable evolutionary adaptation to new environments or provide bypass to genetic lesions. We also illustrate the potential of integrating computational models of underground metabolism and experimental approaches to study the evolution of novel metabolic phenotypes and advance the field of biotechnology.

Rewiring monocyte glucose metabolism via C-type lectin signaling protects against disseminated candidiasis.

Monocytes are innate immune cells that play a pivotal role in antifungal immunity, but little is known regarding the cellular metabolic events that regulate their function during infection. Using complementary transcriptomic and immunological studies in human primary monocytes, we show that activation of monocytes by Candida albicans yeast and hyphae was accompanied by metabolic rewiring induced through C-type lectin-signaling pathways. We describe that the innate immune responses against Candida yeast are energy-demanding processes that lead to the mobilization of intracellular metabolite pools and require induction of glucose metabolism, oxidative phosphorylation and glutaminolysis, while responses to hyphae primarily rely on glycolysis. Experimental models of systemic candidiasis models validated a central role for glucose metabolism in anti-Candida immunity, as the impairment of glycolysis led to increased susceptibility in mice. Collectively, these data highlight the importance of understanding the complex network of metabolic responses triggered during infections, and unveil new potential targets for therapeutic approaches against fungal diseases.