Humans with a dominant negative mutation in STAT3 are susceptible to severe skin infections, suggesting an essential role for STAT3 signaling in defense against cutaneous pathogens.
To focus on innate antiviral defenses in keratinocytes, we used a standard model of cutaneous infection of severe combined immunodeficient mice with the current smallpox vaccine, ACAM-2000. In parallel, early events post-infection with the smallpox vaccine ACAM-2000 were investigated in cultured keratinocytes of human and mouse origin.
Mice treated topically with a STAT3 inhibitor (Stattic) developed larger vaccinia lesions with higher virus titers and died more rapidly than untreated controls. Cultured human and murine keratinocytes infected with ACAM-2000 underwent rapid necrosis, but when treated with Stattic or with inhibitors of RIP1 kinase or caspase-1, they survived longer, produced higher titers of virus, and showed reduced activation of type I interferon responses and inflammatory cytokines release. Treatment with inhibitors of RIP1 kinase and STAT3, but not caspase-1, also reduced the inflammatory response of keratinocytes to TLR ligands. Vaccinia growth properties in Vero cells, which are known to be defective in some antiviral responses, were unaffected by inhibition of RIP1K, caspase-1, or STAT3.
Our findings indicate that keratinocytes suppress the replication and spread of vaccinia virus by undergoing rapid programmed cell death, in a process requiring STAT3. These data offer a new framework for understanding susceptibility to skin infection in patients with STAT3 mutations. Interventions which promote prompt necroptosis/pyroptosis of infected keratinocytes may reduce risks associated with vaccination with live vaccinia virus.
The majority of North American adult females do not meet current physical activity recommendations (150 min of moderate-to-vigorous intensity physical activity (MVPA) per week accrued in ≥10 min bouts) ultimately placing themselves at increased risk of morbidity and mortality. Working-age females face particular challenges in meeting physical activity recommendations as they have multiple demands, including occupational, family and social demands. To develop effective interventions to increase MVPA among working-age females, it is necessary to identify and understand the strongest modifiable determinants influencing these behaviours. Therefore, the objective of this systematic review is to examine the available evidence to identify intrapersonal, social and environmental determinants of MVPA among working-age females.
Six electronic databases will be searched to identify all prospective cohort studies that report on intrapersonal, social and/or environmental determinants of MVPA in working-age females. Grey literature sources including theses, published conference abstracts and websites from relevant organizations will also be included. Articles that report on intrapersonal (e.g. health status, self-efficacy, socio-economic status (SES), stress, depression), social environmental (e.g. crime, safety, area SES, social support, climate and capital, policies), and environmental (e.g. weather, workplace, home, neighbourhood, recreation environment, active transportation) determinants of MVPA in a working-age (mean age 18–65 years) female population will be included. Risk of bias will be assessed within and across all included studies using the Tool to Assess Risk of Bias in Cohort Studies and the Grades of Recommendation, Assessment, Development and Evaluation approach. Harvest plots will be used to synthesize results across all determinants, and meta-analyses will be conducted where possible among studies with sufficient homogeneity.
This review will provide a comprehensive examination of evidence in this field and will serve to highlight gaps for future research on the determinants of MVPA in working-age females and ultimately inform intervention design.
Systematic review registration
Moderate-to-vigorous physical activity; Socio-ecological determinants; Working females
Lactobacillus casei strains are widely used in industry and the utility of this organism in these industrial applications is strain dependent. Hence, tools capable of predicting strain specific phenotypes would have utility in the selection of strains for specific industrial processes. Genome-scale metabolic models can be utilized to better understand genotype-phenotype relationships and to compare different organisms. To assist in the selection and development of strains with enhanced industrial utility, genome-scale models for L. casei ATCC 334, a well characterized strain, and strain 12A, a corn silage isolate, were constructed. Draft models were generated from RAST genome annotations using the Model SEED database and refined by evaluating ATP generating cycles, mass-and-charge-balances of reactions, and growth phenotypes. After the validation process was finished, we compared the metabolic networks of these two strains to identify metabolic, genetic and ortholog differences that may lead to different phenotypic behaviors. We conclude that the metabolic capabilities of the two networks are highly similar. The L. casei ATCC 334 model accounts for 1,040 reactions, 959 metabolites and 548 genes, while the L. casei 12A model accounts for 1,076 reactions, 979 metabolites and 640 genes. The developed L. casei ATCC 334 and 12A metabolic models will enable better understanding of the physiology of these organisms and be valuable tools in the development and selection of strains with enhanced utility in a variety of industrial applications.
Adults spend the majority of their time being sedentary, and evidence suggests that those who spend more of their day engaged in sedentary activities (TV viewing, sitting, screen-based activities) are at increased risk for morbidity and mortality, regardless of whether they exercise regularly. In order to develop effective interventions to reduce sedentary time, it is necessary to identify and understand the strongest modifiable factors of these behaviours. Therefore, the objective of this systematic review is to examine the available evidence in order to identify individual, social, environmental and policy correlates and determinants of sedentary behaviours (TV time, sitting time, screen time) and total sedentary time among adults.
Six electronic databases will be searched to identify all studies that report on individual, social and/or environmental correlates and determinants of sedentary behaviours and total sedentary time in adults. Grey literature sources including theses, published conference abstracts and websites from relevant organizations will also be included. Articles that report on modifiable individual (e.g. health behaviours and status, self-efficacy, socio-economic status), social (e.g. crime, safety, social support, climate and capital), environmental (e.g. weather, workplace, home, neighbourhood, recreation environment, transportation environment) and policy correlates and determinants (based on study design) of sedentary behaviours in an adult population (mean age ≥18 years) will be included. Study quality and risk of bias will be assessed within and across all included studies. Harvest plots will be used to synthesize results across all correlates, and meta-analyses will be conducted where possible among studies with sufficient homogeneity.
This review will provide a comprehensive examination of evidence in the field and will serve to highlight gaps for future research on the determinants of sedentary behaviours and inform intervention design.
Systematic review registration
Sedentary behaviour; Socio-ecological correlates; Adults
Symbiotic interactions between organisms are important for human health and biotechnological applications. Microbial mutualism is a widespread phenomenon and is important in maintaining natural microbial communities. Although cooperative interactions are prevalent in nature, little is known about the processes that allow their initial establishment, govern population dynamics and affect evolutionary processes. To investigate cooperative interactions between bacteria, we constructed, characterized, and adaptively evolved a synthetic community comprised of leucine and lysine Escherichia coli auxotrophs. The co-culture can grow in glucose minimal medium only if the two auxotrophs exchange essential metabolites — lysine and leucine (or its precursors). Our experiments showed that a viable co-culture using these two auxotrophs could be established and adaptively evolved to increase growth rates (by ∼3 fold) and optical densities. While independently evolved co-cultures achieved similar improvements in growth, they took different evolutionary trajectories leading to different community compositions. Experiments with individual isolates from these evolved co-cultures showed that changes in both the leucine and lysine auxotrophs improved growth of the co-culture. Interestingly, while evolved isolates increased growth of co-cultures, they exhibited decreased growth in mono-culture (in the presence of leucine or lysine). A genome-scale metabolic model of the co-culture was also constructed and used to investigate the effects of amino acid (leucine or lysine) release and uptake rates on growth and composition of the co-culture. When the metabolic model was constrained by the estimated leucine and lysine release rates, the model predictions agreed well with experimental growth rates and composition measurements. While this study and others have focused on cooperative interactions amongst community members, the adaptive evolution of communities with other types of interactions (e.g., commensalism, ammensalism or parasitism) would also be of interest.
Shewanella is a genus of facultatively anaerobic, Gram-negative bacteria that have highly adaptable metabolism which allows them to thrive in diverse environments. This quality makes them an attractive bacterial target for research in bioremediation and microbial fuel cell applications. Constraint-based modeling is a useful tool for helping researchers gain insights into the metabolic capabilities of these bacteria. However, Shewanella oneidensis MR-1 is the only strain with a genome-scale metabolic model constructed out of 21 sequenced Shewanella strains.
In this work, we updated the model for Shewanella oneidensis MR-1 and constructed metabolic models for three other strains, namely Shewanella sp. MR-4, Shewanella sp. W3-18-1, and Shewanella denitrificans OS217 which span the genus based on the number of genes lost in comparison to MR-1. We also constructed a Shewanella core model that contains the genes shared by all 21 sequenced strains and a few non-conserved genes associated with essential reactions. Model comparisons between the five constructed models were done at two levels – for wildtype strains under different growth conditions and for knockout mutants under the same growth condition. In the first level, growth/no-growth phenotypes were predicted by the models on various carbon sources and electron acceptors. Cluster analysis of these results revealed that the MR-1 model is most similar to the W3-18-1 model, followed by the MR-4 and OS217 models when considering predicted growth phenotypes. However, a cluster analysis done based on metabolic gene content revealed that the MR-4 and W3-18-1 models are the most similar, with the MR-1 and OS217 models being more distinct from these latter two strains. As a second level of comparison, we identified differences in reaction and gene content which give rise to different functional predictions of single and double gene knockout mutants using Comparison of Networks by Gene Alignment (CONGA). Here, we showed how CONGA can be used to find biomass, metabolic, and genetic differences between models.
We developed four strain-specific models and a general core model that can be used to do various in silico studies of Shewanella metabolism. The developed models provide a platform for a systematic investigation of Shewanella metabolism to aid researchers using Shewanella in various biotechnology applications.
Constraint-based model; Electron acceptors; Phenotype; FBA
The success of genome-scale metabolic modeling is contingent on a model's ability to accurately predict growth and metabolic behaviors. To date, little focus has been directed towards developing systematic methods of proposing, modifying and interrogating an organism's biomass requirements that are used in constraint-based models. To address this gap, the biomass modification and generation (BioMog) framework was created and used to generate lists of biomass components de novo, as well as to modify predefined biomass component lists, for models of Escherichia coli (iJO1366) and of Shewanella oneidensis (iSO783) from high-throughput growth phenotype and fitness datasets. BioMog's de novo biomass component lists included, either implicitly or explicitly, up to seventy percent of the components included in the predefined biomass equations, and the resulting de novo biomass equations outperformed the predefined biomass equations at qualitatively predicting mutant growth phenotypes by up to five percent. Additionally, the BioMog procedure can quantify how many experiments support or refute a particular metabolite's essentiality to a cell, and it facilitates the determination of inconsistent experiments and inaccurate reaction and/or gene to reaction associations. To further interrogate metabolite essentiality, the BioMog framework includes an experiment generation algorithm that allows for the design of experiments to test whether a metabolite is essential. Using BioMog, we correct experimental results relating to the essentiality of thyA gene in E. coli, as well as perform knockout experiments supporting the essentiality of protoheme. With these capabilities, BioMog can be a valuable resource for analyzing growth phenotyping data and component of a model developer's toolbox.
The clinical significance of Mycoplasma genitalium (MG) infection in adolescent women is poorly understood. We compared the prevalence of MG with that of other sexually transmitted organisms such as Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV) and assessed the associations of MG with sexual behaviors, genitourinary symptoms, physical and laboratory findings.
Women aged 14 to 21 years (n = 331) were recruited from an urban medical center. The subjects’ sexual behaviors, genitourinary symptoms, and physical findings were recorded. Endocervical swabs were collected for CT and NG testing and vaginal swabs for wet mount, Gram stain, TV and MG testing. MG infection was identified by nucleic acid amplification using a transcription-mediated amplification assay.
MG was detected in 74 (22.4%), CT in 79 (24.4%), TV in 60 (18.2%), and NG in 35 (10.7%) subjects. MG infection was not associated with vaginal symptoms, physical evidence of cervicitis, or findings on wet mount or Gram stain. In logistic regression, variables positively associated with MG were current CT [odds ratio (OR), 2.3; 95% confidence interval (CI), 1.4–4.4] and recent sexual contact (≤7 days) (OR, 2.0; CI, 1.1–3.2). Dysuria (OR, 0.44; CI, 0.2– 0.96) and use of hormonal contraception (OR, 0.55; CI, 0.3–1.0) were negatively associated with MG infection.
In adolescent women, MG infection was as common as chlamydial infection and trichomoniasis and more common than gonorrhea. MG was associated with CT and recent sexual contact but not with vaginal symptoms or signs of cervicitis.
BACKGROUND AND OBJECTIVE:
Inadequate follow-up of positive sexually transmitted infection (STI) test results is a gap in health care quality that contributes to the epidemic of STIs in adolescent women. The goal of this study was to improve our ability to contact adolescent women with positive STI test results after an emergency department visit.
We conducted an interventional quality improvement project at a pediatric emergency department. Phase 1 included plan-do-study-act cycles to test interventions such as provider education and system changes. Phase 2 was a planned experiment studying 2 interventions (study cell phone and patient activation card), using a 2 × 2 factorial design with 1 background variable and 2 replications. Outcomes were: (1) the proportion of women aged 14 to 21 years with STI testing whose confidential telephone number was documented in the electronic medical record; (2) the proportion of STI positive women successfully contacted within 7 days.
Phase 1 interventions increased the proportion of records with a confidential number from 24% to 58% and the proportion contacted from 45% to 65%, and decreased loss to follow-up from 40% to 24%. In phase 2, the proportion contacted decreased after the electronic medical record system changed and recording of the confidential number decreased. Study interventions (patient activation card and study cell phone) had a synergistic effect on successful contact, especially when confidential numbers were less reliably documented.
Feasible and sustainable interventions such as improved documentation of a confidential number worked synergistically to increase our ability to successfully contact adolescent women with their STI test results.
adolescent sexual behavior; outcomes research; quality improvement; sexually transmitted infection
Enterobacteriaceae diversified from an ancestral lineage ~300-500 million years ago (mya) into a wide variety of free-living and host-associated lifestyles. Nutrient availability varies across niches, and evolution of metabolic networks likely played a key role in adaptation.
Here we use a paleo systems biology approach to reconstruct and model metabolic networks of ancestral nodes of the enterobacteria phylogeny to investigate metabolism of ancient microorganisms and evolution of the networks. Specifically, we identified orthologous genes across genomes of 72 free-living enterobacteria (16 genera), and constructed core metabolic networks capturing conserved components for ancestral lineages leading to E. coli/Shigella (~10 mya), E. coli/Shigella/Salmonella (~100 mya), and all enterobacteria (~300-500 mya). Using these models we analyzed the capacity for carbon, nitrogen, phosphorous, sulfur, and iron utilization in aerobic and anaerobic conditions, identified conserved and differentiating catabolic phenotypes, and validated predictions by comparison to experimental data from extant organisms.
This is a novel approach using quantitative ancestral models to study metabolic network evolution and may be useful for identification of new targets to control infectious diseases caused by enterobacteria.
Constraint-based modeling; Enterobacteria; Metabolic network reconstruction; Ancient metabolism; Paleo systems biology; Ancestral core
Constraint-based modeling uses mass balances, flux capacity, and reaction directionality constraints to predict fluxes through metabolism. Although transcriptional regulation and thermodynamic constraints have been integrated into constraint-based modeling, kinetic rate laws have not been extensively used.
In this study, an in vivo kinetic parameter estimation problem was formulated and solved using multi-omic data sets for Escherichia coli. To narrow the confidence intervals for kinetic parameters, a series of kinetic model simplifications were made, resulting in fewer kinetic parameters than the full kinetic model. These new parameter values are able to account for flux and concentration data from 20 different experimental conditions used in our training dataset. Concentration estimates from the simplified kinetic model were within one standard deviation for 92.7% of the 790 experimental measurements in the training set. Gibbs free energy changes of reaction were calculated to identify reactions that were often operating close to or far from equilibrium. In addition, enzymes whose activities were positively or negatively influenced by metabolite concentrations were also identified. The kinetic model was then used to calculate the maximum and minimum possible flux values for individual reactions from independent metabolite and enzyme concentration data that were not used to estimate parameter values. Incorporating these kinetically-derived flux limits into the constraint-based metabolic model improved predictions for uptake and secretion rates and intracellular fluxes in constraint-based models of central metabolism.
This study has produced a method for in vivo kinetic parameter estimation and identified strategies and outcomes of kinetic model simplification. We also have illustrated how kinetic constraints can be used to improve constraint-based model predictions for intracellular fluxes and biomass yield and identify potential metabolic limitations through the integrated analysis of multi-omics datasets.
Metabolic engineering; Kinetics; Central metabolism; Constraint-based; FBA
Current computational tools can generate and improve genome-scale models based on
existing data; however, for many organisms, the data needed to test and refine such
models are not available. To facilitate model development, we created the forced
coupling algorithm, FOCAL, to identify genetic and environmental conditions such that
a reaction becomes essential for an experimentally measurable phenotype. This
reaction's conditional essentiality can then be tested experimentally to evaluate
whether network connections occur or to create strains with desirable phenotypes.
FOCAL allows network connections to be queried, which improves our understanding of
metabolism and accuracy of developed models.
Activation state-dependent secretion of alpha-1 proteinase inhibitor (A1PI) by monocytes and macrophages was first reported in 1985. Since then, monocytes and tissue macrophages have emerged as key sentinels of infection and tissue damage via activation of self-assembling pattern recognition receptors (inflammasomes), which trigger inflammation and cell death in a caspase-1 dependent process. These studies examine the relationship between A1PI expression in primary monocytes and monocytic cell lines, and inflammatory cytokine expression in response to inflammasome directed stimuli.
IL-1 β expression was examined in lung macrophages expressing wild type A1PI (A1PI-M) or disease-associated Z isoform A1PI (A1PI-Z). Inflammatory cytokine release was evaluated in THP-1 monocytic cells or THP-1 cells lacking the inflammasome adaptor ASC, transfected with expression vectors encoding A1PI-M or A1PI-Z. A1PI-M was localized within monocytes by immunoprecipitation in hypotonic cell fractions. Cell-free titration of A1PI-M was performed against recombinant active caspase-1 in vitro.
IL-1 β expression was elevated in lung macrophages expressing A1PI-Z. Overexpression of A1PI-M in THP-1 monocytes reduced secretion of IL-1β and TNF-α. In contrast, overexpression of A1PI-Z enhanced IL-1β and TNF- α secretion in an ASC dependent manner. A1PI-Z-enhanced cytokine release was inhibited by a small molecule caspase-1 inhibitor but not by high levels of exogenous wtA1PI. Cytosolic localization of A1PI-M in monocytes was not diminished with microtubule-inhibiting agents. A1PI-M co-localized with caspase-1 in gel-filtered cytoplasmic THP-1 preparations, and was co-immunoprecipitated with caspase 1 from nigericin-stimulated THP-1 cell lysate. Plasma-derived A1PI inhibited recombinant caspase-1 mediated conversion of a peptide substrate in a dose dependent manner.
Our results suggest that monocyte/macrophage-expressed A1PI-M antagonizes IL-1β secretion possibly via caspase-1 inhibition, a function which disease-associated A1PI-Z may lack. Therapeutic approaches which limit inflammasome responses in patients with A1PI deficiency, in combination with A1PI augmentation, may provide additional respiratory tissue-sparing benefits.
Regulation of the actin cytoskeleton is essential for epithelial cell polarity and protein trafficking within human uterine epithelium. The actin-binding protein cofilin is involved in regulation of actin dynamics by promoting actin branching and cytoskeleton reorganization. Dual immunohistochemical staining of cofilin and G-actin (represented by DNAse I staining) revealed cofilin-G-actin colocalization in the apical side of luminal epithelial cells of human uterine endometrium during the proliferative phase of the menstrual cycle. Interestingly, during the secretory phase of the menstrual cycle, cofilin was only present on the basolateral side. To determine whether the disease endometriosis causes a different pattern of actin remodeling, we investigated an established baboon model of induced endometriosis. The cofilin pattern in the secretory phase of baboons with endometriosis was similar to the proliferative phase in normal animals; cofilin was observed in the apical parts of luminal and glandular epithelium. A phosphatase regulating the activity of cofilin, slingshot (SSH1), revealed a similar staining pattern within these tissues. These patterns were confirmed through quantitative image analysis. Quantification of messenger RNA (mRNA) detected upregulated SSH1 and suggested a progesterone resistance-related pattern of nuclear steroid hormone receptors, but no change in membrane progesterone receptors (mPR alpha or mPR beta) was observed in endometriosis. Our data indicate that the severe dyssynchrony during menstrual cycle phases in endometriosis is connected with improper cytoskeleton rearrangements. We suggest that cofilin-mediated actin reorganization in uterine epithelial cells might be important in preparation for blastocyst implantation; dysregulation of this reorganization may lead to decreased fertility in endometriosis.
cofilin; slingshot; cytoskeleton; actin dynamics; endometriosis
Regulation of the actin cytoskeleton is essential for epithelial cell polarity and protein trafficking within human uterine epithelium. The actin-binding protein cofilin is involved in regulation of actin dynamics by promoting actin branching and cytoskeleton reorganization. Dual immunohistochemical staining of cofilin and G-actin (represented by DNAse I staining) revealed cofilin-G-actin colocalization in the apical side of luminal epithelial cells of human uterine endometrium during the proliferative phase of the menstrual cycle. Interestingly, during the secretory phase of the menstrual cycle cofilin was only present on the basolateral side. To determine whether the disease endometriosis causes a different pattern of actin remodeling, we investigated an established baboon model of induced endometriosis. The cofilin pattern in the secretory phase of baboons with endometriosis was similar to the proliferative phase in normal animals; cofilin was observed in the apical parts of luminal and glandular epithelium. A phosphatase regulating the activity of cofilin, slingshot (SSH1), revealed a similar staining pattern within these tissues. These patterns were confirmed through quantitative image analysis. Quantification of mRNA detected upregulated SSH1 and suggested a progesterone resistance related pattern of nuclear steroid hormone receptors, but no change in membrane progesterone receptors (mPR alpha or mPR beta) was observed in endometriosis. Our data indicate that the severe dyssynchrony during menstrual cycle phases in endometriosis is connected with improper cytoskeleton rearrangements. We suggest that cofilin-mediated actin reorganization in uterine epithelial cells might be important in preparation for blastocyst implantation; dysregulation of this reorganization may lead to decreased fertility in endometriosis.
cofilin; slingshot; cytoskeleton; actin dynamics; endometriosis
Predicting cellular responses to perturbations is an important task in systems biology. We report a new approach, RELATCH, which uses flux and gene expression data from a reference state to predict metabolic responses in a genetically or environmentally perturbed state. Using the concept of relative optimality, which considers relative flux changes from a reference state, we hypothesize a relative metabolic flux pattern is maintained from one state to another, and that cells adapt to perturbations using metabolic and regulatory reprogramming to preserve this relative flux pattern. This constraint-based approach will have broad utility where predictions of metabolic responses are needed.
A33 is a type II integral membrane protein expressed on the extracellular enveloped form of vaccinia virus (VACV). Passive transfer of A33-directed monoclonal antibodies or vaccination with an A33 subunit vaccine confers protection against lethal poxvirus challenge in animal models. Homologs of A33 are highly conserved among members of the Orthopoxvirus genus and are potential candidates for inclusion in vaccines or assays targeting extracellular enveloped virus activity. One monoclonal antibody directed against VACV A33, MAb-1G10, has been shown to target a conformation-dependent epitope. Interestingly, while it recognizes VACV A33 as well as the corresponding variola homolog, it does not bind to the monkeypox homolog. In this study, we utilized a random phage display library to investigate the epitope recognized by MAb-1G10 that is critical for facilitating cell-to-cell spread of the vaccinia virus.
By screening with linear or conformational random phage libraries, we found that phages binding to MAb-1G10 display the consensus motif CEPLC, with a disulfide bond formed between two cysteine residues required for MAb-1G10 binding. Although the phage motif contained no linear sequences homologous to VACV A33, structure modeling and analysis suggested that residue D115 is important to form the minimal epitope core. A panel of point mutants expressing the ectodomain of A33 protein was generated and analyzed by either binding assays such as ELISA and immunoprecipitation or a functional assessment by blocking MAb-1G10 mediated comet inhibition in cell culture.
These results confirm L118 as a component of the MAb-1G10 binding epitope, and further identify D115 as an essential residue. By defining the minimum conformational structure, as well as the conformational arrangement of a short peptide sequence recognized by MAb-1G10, these results introduce the possibility of designing small molecule mimetics that may interfere with the function of A33 in vivo. This information will also be useful for designing improved assays to evaluate the potency of monoclonal and polyclonal products that target A33 or A33-modulated EV dissemination.
Orthopoxviruses; Monoclonal antibody; B-cell epitope; Immunogen; Vaccinia; Phage display library
Constraint-based models of metabolism have been used in a variety of studies on drug discovery, metabolic engineering, evolution, and multi-species interactions. These genome-scale models can be generated for any sequenced organism since their main parameters (i.e., reaction stoichiometry) are highly conserved. Their relatively low parameter requirement makes these models easy to develop; however, these models often result in a solution space with multiple possible flux distributions, making it difficult to determine the precise flux state in the cell. Recent research efforts in this modeling field have investigated how additional experimental data, including gene expression, protein expression, metabolite concentrations, and kinetic parameters, can be used to reduce the solution space. This mini-review provides a summary of the data-driven computational approaches that are available for reducing the solution space and thereby improve predictions of intracellular fluxes by constraint-based models.
Shewanella oneidensis MR-1 is a facultative anaerobe that derives energy by coupling organic matter oxidation to the reduction of a wide range of electron acceptors. Here, we quantitatively assessed the lactate and pyruvate metabolism of MR-1 under three distinct conditions: electron acceptor-limited growth on lactate with O2, lactate with fumarate, and pyruvate fermentation. The latter does not support growth but provides energy for cell survival. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of that needed for growth depending on the electron acceptor nature and availability. While being indispensable for growth, the respiration of fumarate does not contribute significantly to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions, S. oneidensis MR-1 carried out incomplete substrate oxidation, whereby the tricarboxylic acid (TCA) cycle did not contribute significantly. Pyruvate dehydrogenase was not involved in lactate metabolism under conditions of O2 limitation but was required for anaerobic growth, likely by supplying reducing equivalents for biosynthesis. The results suggest that pyruvate fermentation by S. oneidensis MR-1 cells represents a combination of substrate-level phosphorylation and respiration, where pyruvate serves as an electron donor and an electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by a recently described new type of oxidative NAD(P)H-independent d-lactate dehydrogenase (Dld-II). The results further indicate that pyruvate reduction coupled to formate oxidation may be accompanied by the generation of proton motive force.
The physiology of ethanologenic Escherichia coli grown anaerobically in alkali-pretreated plant hydrolysates is complex and not well studied. To gain insight into how E. coli responds to such hydrolysates, we studied an E. coli K-12 ethanologen fermenting a hydrolysate prepared from corn stover pretreated by ammonia fiber expansion. Despite the high sugar content (∼6% glucose, 3% xylose) and relatively low toxicity of this hydrolysate, E. coli ceased growth long before glucose was depleted. Nevertheless, the cells remained metabolically active and continued conversion of glucose to ethanol until all glucose was consumed. Gene expression profiling revealed complex and changing patterns of metabolic physiology and cellular stress responses during an exponential growth phase, a transition phase, and the glycolytically active stationary phase. During the exponential and transition phases, high cell maintenance and stress response costs were mitigated, in part, by free amino acids available in the hydrolysate. However, after the majority of amino acids were depleted, the cells entered stationary phase, and ATP derived from glucose fermentation was consumed entirely by the demands of cell maintenance in the hydrolysate. Comparative gene expression profiling and metabolic modeling of the ethanologen suggested that the high energetic cost of mitigating osmotic, lignotoxin, and ethanol stress collectively limits growth, sugar utilization rates, and ethanol yields in alkali-pretreated lignocellulosic hydrolysates.
Genome-scale network reconstructions are useful tools for understanding cellular metabolism, and comparisons of such reconstructions can provide insight into metabolic differences between organisms. Recent efforts toward comparing genome-scale models have focused primarily on aligning metabolic networks at the reaction level and then looking at differences and similarities in reaction and gene content. However, these reaction comparison approaches are time-consuming and do not identify the effect network differences have on the functional states of the network. We have developed a bilevel mixed-integer programming approach, CONGA, to identify functional differences between metabolic networks by comparing network reconstructions aligned at the gene level. We first identify orthologous genes across two reconstructions and then use CONGA to identify conditions under which differences in gene content give rise to differences in metabolic capabilities. By seeking genes whose deletion in one or both models disproportionately changes flux through a selected reaction (e.g., growth or by-product secretion) in one model over another, we are able to identify structural metabolic network differences enabling unique metabolic capabilities. Using CONGA, we explore functional differences between two metabolic reconstructions of Escherichia coli and identify a set of reactions responsible for chemical production differences between the two models. We also use this approach to aid in the development of a genome-scale model of Synechococcus sp. PCC 7002. Finally, we propose potential antimicrobial targets in Mycobacterium tuberculosis and Staphylococcus aureus based on differences in their metabolic capabilities. Through these examples, we demonstrate that a gene-centric approach to comparing metabolic networks allows for a rapid comparison of metabolic models at a functional level. Using CONGA, we can identify differences in reaction and gene content which give rise to different functional predictions. Because CONGA provides a general framework, it can be applied to find functional differences across models and biological systems beyond those presented here.
Genome-scale metabolic models have proven useful for answering fundamental questions about metabolic capabilities of a variety of microorganisms, as well as informing their metabolic engineering. However, only a few models are available for oxygenic photosynthetic microorganisms, particularly in cyanobacteria in which photosynthetic and respiratory electron transport chains (ETC) share components. We addressed the complexity of cyanobacterial ETC by developing a genome-scale model for the diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142. The resulting metabolic reconstruction, iCce806, consists of 806 genes associated with 667 metabolic reactions and includes a detailed representation of the ETC and a biomass equation based on experimental measurements. Both computational and experimental approaches were used to investigate light-driven metabolism in Cyanothece sp. ATCC 51142, with a particular focus on reductant production and partitioning within the ETC. The simulation results suggest that growth and metabolic flux distributions are substantially impacted by the relative amounts of light going into the individual photosystems. When growth is limited by the flux through photosystem I, terminal respiratory oxidases are predicted to be an important mechanism for removing excess reductant. Similarly, under photosystem II flux limitation, excess electron carriers must be removed via cyclic electron transport. Furthermore, in silico calculations were in good quantitative agreement with the measured growth rates whereas predictions of reaction usage were qualitatively consistent with protein and mRNA expression data, which we used to further improve the resolution of intracellular flux values.
Cyanobacteria have been promoted as platforms for biofuel production due to their useful physiological properties such as photosynthesis, relatively rapid growth rates, ability to accumulate high amounts of intracellular compounds and tolerance to extreme environments. However, development of a computational model is an important step to synthesize biochemical, physiological and regulatory understanding of photoautotrophic metabolism (either qualitatively or quantitatively) at a systems level, to make metabolic engineering of these organisms tractable. When integrated with other genome-scale data (e.g., expression data), numerical simulations can provide experimentally testable predictions of carbon fluxes and reductant partitioning to different biosynthetic pathways and macromolecular synthesis. This work is the first to computationally explore the interactions between components of photosynthetic and respiratory systems in detail. In silico predictions obtained from model analysis provided insights into the effects of light quantity and quality upon fluxes through electron transport pathways, alternative pathways for reductant consumption and carbon metabolism. The model will not only serve as a platform to develop genome-scale metabolic models for other cyanobacteria, but also as an engineering tool for manipulation of photosynthetic microorganisms to improve biofuel production.
Carbon-13 (13C) analysis is a commonly used method for estimating reaction rates in biochemical networks. The choice of carbon labeling pattern is an important consideration when designing these experiments. We present a novel Monte Carlo algorithm for finding the optimal substrate input label for a particular experimental objective (flux or flux ratio). Unlike previous work, this method does not require assumption of the flux distribution beforehand.
Using a large E. coli isotopomer model, different commercially available substrate labeling patterns were tested computationally for their ability to determine reaction fluxes. The choice of optimal labeled substrate was found to be dependent upon the desired experimental objective. Many commercially available labels are predicted to be outperformed by complex labeling patterns. Based on Monte Carlo Sampling, the dimensionality of experimental data was found to be considerably less than anticipated, suggesting that effectiveness of 13C experiments for determining reaction fluxes across a large-scale metabolic network is less than previously believed.
While 13C analysis is a useful tool in systems biology, high redundancy in measurements limits the information that can be obtained from each experiment. It is however possible to compute potential limitations before an experiment is run and predict whether, and to what degree, the rate of each reaction can be resolved.
Despite the availability of numerous complete genome sequences from E. coli strains, published genome-scale metabolic models exist only for two commensal E. coli strains. These models have proven useful for many applications, such as engineering strains for desired product formation, and we sought to explore how constructing and evaluating additional metabolic models for E. coli strains could enhance these efforts.
We used the genomic information from 16 E. coli strains to generate an E. coli pangenome metabolic network by evaluating their collective 76,990 ORFs. Each of these ORFs was assigned to one of 17,647 ortholog groups including ORFs associated with reactions in the most recent metabolic model for E. coli K-12. For orthologous groups that contain an ORF already represented in the MG1655 model, the gene to protein to reaction associations represented in this model could then be easily propagated to other E. coli strain models. All remaining orthologous groups were evaluated to see if new metabolic reactions could be added to generate a pangenome-scale metabolic model (iEco1712_pan). The pangenome model included reactions from a metabolic model update for E. coli K-12 MG1655 (iEco1339_MG1655) and enabled development of five additional strain-specific genome-scale metabolic models. These additional models include a second K-12 strain (iEco1335_W3110) and four pathogenic strains (two enterohemorrhagic E. coli O157:H7 and two uropathogens). When compared to the E. coli K-12 models, the metabolic models for the enterohemorrhagic (iEco1344_EDL933 and iEco1345_Sakai) and uropathogenic strains (iEco1288_CFT073 and iEco1301_UTI89) contained numerous lineage-specific gene and reaction differences. All six E. coli models were evaluated by comparing model predictions to carbon source utilization measurements under aerobic and anaerobic conditions, and to batch growth profiles in minimal media with 0.2% (w/v) glucose. An ancestral genome-scale metabolic model based on conserved ortholog groups in all 16 E. coli genomes was also constructed, reflecting the conserved ancestral core of E. coli metabolism (iEco1053_core). Comparative analysis of all six strain-specific E. coli models revealed that some of the pathogenic E. coli strains possess reactions in their metabolic networks enabling higher biomass yields on glucose. Finally the lineage-specific metabolic traits were compared to the ancestral core model predictions to derive new insight into the evolution of metabolism within this species.
Our findings demonstrate that a pangenome-scale metabolic model can be used to rapidly construct additional E. coli strain-specific models, and that quantitative models of different strains of E. coli can accurately predict strain-specific phenotypes. Such pangenome and strain-specific models can be further used to engineer metabolic phenotypes of interest, such as designing new industrial E. coli strains.