Comparative analyses of parasites with a comprehensive database of genome-scale metabolic models.

Protozoan parasites cause diverse diseases with large global impacts. Research on the pathogenesis and biology of these organisms is limited by economic and experimental constraints. Accordingly, studies of one parasite are frequently extrapolated to infer knowledge about another parasite, across and within genera. Model in vitro or in vivo systems are frequently used to enhance experimental manipulability, but these systems generally use species related to, yet distinct from, the clinically relevant causal pathogen. Characterization of functional differences among parasite species is confined to post hoc or single target studies, limiting the utility of this extrapolation approach. To address this challenge and to accelerate parasitology research broadly, we present a functional comparative analysis of 192 genomes, representing every high-quality, publicly-available protozoan parasite genome including Plasmodium, Toxoplasma, Cryptosporidium, Entamoeba, Trypanosoma, Leishmania, Giardia, and other species. We generated an automated metabolic network reconstruction pipeline optimized for eukaryotic organisms. These metabolic network reconstructions serve as biochemical knowledgebases for each parasite, enabling qualitative and quantitative comparisons of metabolic behavior across parasites. We identified putative differences in gene essentiality and pathway utilization to facilitate the comparison of experimental findings and discovered that phylogeny is not the sole predictor of metabolic similarity. This knowledgebase represents the largest collection of genome-scale metabolic models for both pathogens and eukaryotes; with this resource, we can predict species-specific functions, contextualize experimental results, and optimize selection of experimental systems for fastidious species.

Enhancing Microbiome Research through Genome-Scale Metabolic Modeling.

Construction and analysis of genome-scale metabolic models (GEMs) is a well-established systems biology approach that can be used to predict metabolic and growth phenotypes. The ability of GEMs to produce mechanistic insight into microbial ecological processes makes them appealing tools that can open a range of exciting opportunities in microbiome research. Here, we briefly outline these opportunities, present current rate-limiting challenges for the trustworthy application of GEMs to microbiome research, and suggest approaches for moving the field forward.

Megasphaera in the Stool Microbiota Is Negatively Associated With Diarrheal Cryptosporidiosis.

BACKGROUND: The protozoan parasites in the Cryptosporidium genus cause both acute diarrheal disease and subclinical (ie, nondiarrheal) disease. It is unclear if the microbiota can influence the manifestation of diarrhea during a Cryptosporidium infection. METHODS: To characterize the role of the gut microbiota in diarrheal cryptosporidiosis, the microbiome composition of both diarrheal and surveillance Cryptosporidium-positive fecal samples from 72 infants was evaluated using 16S ribosomal RNA gene sequencing. Additionally, the microbiome composition prior to infection was examined to test whether a preexisting microbiome profile could influence the Cryptosporidium infection phenotype. RESULTS: Fecal microbiome composition was associated with diarrheal symptoms at 2 timepoints. Megasphaera was significantly less abundant in diarrheal samples compared with subclinical samples at the time of Cryptosporidium detection (log2 [fold change] = -4.3; P = 10-10) and prior to infection (log2 [fold change] = -2.0; P = 10-4); this assigned sequence variant was detected in 8 children who had diarrhea and 30 children without diarrhea. Random forest classification also identified Megasphaera abundance in the pre- and postexposure microbiota as predictive of a subclinical infection. CONCLUSIONS: Microbiome composition broadly, and specifically low Megasphaera abundance, was associated with diarrheal symptoms prior to and at the time of Cryptosporidium detection. This observation suggests that the gut microenvironment may play a role in determining the severity of a Cryptosporidium infection. Clinical Trials Registration. NCT02764918.

Medusa: Software to build and analyze ensembles of genome-scale metabolic network reconstructions.

Uncertainty in the structure and parameters of networks is ubiquitous across computational biology. In constraint-based reconstruction and analysis of metabolic networks, this uncertainty is present both during the reconstruction of networks and in simulations performed with them. Here, we present Medusa, a Python package for the generation and analysis of ensembles of genome-scale metabolic network reconstructions. Medusa builds on the COBRApy package for constraint-based reconstruction and analysis by compressing a set of models into a compact ensemble object, providing functions for the generation of ensembles using experimental data, and extending constraint-based analyses to ensemble scale. We demonstrate how Medusa can be used to generate ensembles and perform ensemble simulations, and how machine learning can be used in conjunction with Medusa to guide the curation of genome-scale metabolic network reconstructions. Medusa is available under the permissive MIT license from the Python Packaging Index (https://pypi.org) and from github (https://github.com/opencobra/Medusa), and comprehensive documentation is available at https://medusa.readthedocs.io/en/latest.

Publisher Correction: MEMOTE for standardized genome-scale metabolic model testing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Guiding the Refinement of Biochemical Knowledgebases with Ensembles of Metabolic Networks and Machine Learning.

Mechanistic models explicitly represent hypothesized biological knowledge. As such, they offer more generalizability than data-driven models. However, identifying model curation efforts that improve performance for mechanistic models is nontrivial. Here, we develop a solution to this problem for genome-scale metabolic models. We generate an ensemble of models, each equally consistent with experimental data, then perform simulations with them. We apply machine learning to the simulation output to identify model structure variation that maximally influences simulations. These variants are high-priority candidates for curation through removal, addition, or reannotation in the model. We apply this approach, automated metabolic model ensemble-driven elimination of uncertainty with statistical learning (AMMEDEUS), to 29 bacterial species to improve gene essentiality predictions. We explore targets for individual species and compile pan-species targets to improve the database used during model construction. AMMEDEUS is an automated and performance-driven recommendation system that complements intuition during curation of biochemical knowledgebases.

Inferring Metabolic Mechanisms of Interaction within a Defined Gut Microbiota.

The diversity and number of species present within microbial communities create the potential for a multitude of interspecies metabolic interactions. Here, we develop, apply, and experimentally test a framework for inferring metabolic mechanisms associated with interspecies interactions. We perform pairwise growth and metabolome profiling of co-cultures of strains from a model mouse microbiota. We then apply our framework to dissect emergent metabolic behaviors that occur in co-culture. Based on one of the inferences from this framework, we identify and interrogate an amino acid cross-feeding interaction and validate that the proposed interaction leads to a growth benefit in vitro. Our results reveal the type and extent of emergent metabolic behavior in microbial communities composed of gut microbes. We focus on growth-modulating interactions, but the framework can be applied to interspecies interactions that modulate any phenotype of interest within microbial communities.

A novel mouse model of Campylobacter jejuni enteropathy and diarrhea.

Campylobacter infections are among the leading bacterial causes of diarrhea and of 'environmental enteropathy' (EE) and growth failure worldwide. However, the lack of an inexpensive small animal model of enteric disease with Campylobacter has been a major limitation for understanding its pathogenesis, interventions or vaccine development. We describe a robust standard mouse model that can exhibit reproducible bloody diarrhea or growth failure, depending on the zinc or protein deficient diet and on antibiotic alteration of normal microbiota prior to infection. Zinc deficiency and the use of antibiotics create a niche for Campylobacter infection to establish by narrowing the metabolic flexibility of these mice for pathogen clearance and by promoting intestinal and systemic inflammation. Several biomarkers and intestinal pathology in this model also mimic those seen in human disease. This model provides a novel tool to test specific hypotheses regarding disease pathogenesis as well as vaccine development that is currently in progress.

Influential Parameters for the Analysis of Intracellular Parasite Metabolomics.

Metabolomics is increasingly popular for the study of pathogens. For the malaria parasite Plasmodium falciparum, both targeted and untargeted metabolomics have improved our understanding of pathogenesis, host-parasite interactions, and antimalarial drug treatment and resistance. However, purification and analysis procedures for performing metabolomics on intracellular pathogens have not been explored. Here, we purified in vitro-grown ring-stage intraerythrocytic P. falciparum parasites for untargeted metabolomics studies; the small size of this developmental stage amplifies the challenges associated with metabolomics studies as the ratio between host and parasite biomass is maximized. Following metabolite identification and data preprocessing, we explored multiple confounding factors that influence data interpretation, including host contamination and normalization approaches (including double-stranded DNA, total protein, and parasite numbers). We conclude that normalization parameters have large effects on differential abundance analysis and recommend the thoughtful selection of these parameters. However, normalization does not remove the contribution from the parasite's extracellular environment (culture media and host erythrocyte). In fact, we found that extraparasite material is as influential on the metabolome as treatment with a potent antimalarial drug with known metabolic effects (artemisinin). Because of this influence, we could not detect significant changes associated with drug treatment. Instead, we identified metabolites predictive of host and medium contamination that could be used to assess sample purification. Our analysis provides the first quantitative exploration of the effects of these factors on metabolomics data analysis; these findings provide a basis for development of improved experimental and analytical methods for future metabolomics studies of intracellular organisms.IMPORTANCE Molecular characterization of pathogens such as the malaria parasite can lead to improved biological understanding and novel treatment strategies. However, the distinctive biology of the Plasmodium parasite, including its repetitive genome and the requirement for growth within a host cell, hinders progress toward these goals. Untargeted metabolomics is a promising approach to learn about pathogen biology. By measuring many small molecules in the parasite at once, we gain a better understanding of important pathways that contribute to the parasite's response to perturbations such as drug treatment. Although increasingly popular, approaches for intracellular parasite metabolomics and subsequent analysis are not well explored. The findings presented in this report emphasize the critical need for improvements in these areas to limit misinterpretation due to host metabolites and to standardize biological interpretation. Such improvements will aid both basic biological investigations and clinical efforts to understand important pathogens.

Novel co-culture plate enables growth dynamic-based assessment of contact-independent microbial interactions.

Interactions between microbes are central to the dynamics of microbial communities. Understanding these interactions is essential for the characterization of communities, yet challenging to accomplish in practice. There are limited available tools for characterizing diffusion-mediated, contact-independent microbial interactions. A practical and widely implemented technique in such characterization involves the simultaneous co-culture of distinct bacterial species and subsequent analysis of relative abundance in the total population. However, distinguishing between species can be logistically challenging. In this paper, we present a low-cost, vertical membrane, co-culture plate to quantify contact-independent interactions between distinct bacterial populations in co-culture via real-time optical density measurements. These measurements can be used to facilitate the analysis of the interaction between microbes that are physically separated by a semipermeable membrane yet able to exchange diffusible molecules. We show that diffusion across the membrane occurs at a sufficient rate to enable effective interaction between physically separate cultures. Two bacterial species commonly found in the cystic fibrotic lung, Pseudomonas aeruginosa and Burkholderia cenocepacia, were co-cultured to demonstrate how this plate may be implemented to study microbial interactions. We have demonstrated that this novel co-culture device is able to reliably generate real-time measurements of optical density data that can be used to characterize interactions between microbial species.

Cross-modulation of pathogen-specific pathways enhances malnutrition during enteric co-infection with Giardia lamblia and enteroaggregative Escherichia coli.

Diverse enteropathogen exposures associate with childhood malnutrition. To elucidate mechanistic pathways whereby enteric microbes interact during malnutrition, we used protein deficiency in mice to develop a new model of co-enteropathogen enteropathy. Focusing on common enteropathogens in malnourished children, Giardia lamblia and enteroaggregative Escherichia coli (EAEC), we provide new insights into intersecting pathogen-specific mechanisms that enhance malnutrition. We show for the first time that during protein malnutrition, the intestinal microbiota permits persistent Giardia colonization and simultaneously contributes to growth impairment. Despite signals of intestinal injury, such as IL1α, Giardia-infected mice lack pro-inflammatory intestinal responses, similar to endemic pediatric Giardia infections. Rather, Giardia perturbs microbial host co-metabolites of proteolysis during growth impairment, whereas host nicotinamide utilization adaptations that correspond with growth recovery increase. EAEC promotes intestinal inflammation and markers of myeloid cell activation. During co-infection, intestinal inflammatory signaling and cellular recruitment responses to EAEC are preserved together with a Giardia-mediated diminishment in myeloid cell activation. Conversely, EAEC extinguishes markers of host energy expenditure regulatory responses to Giardia, as host metabolic adaptations appear exhausted. Integrating immunologic and metabolic profiles during co-pathogen infection and malnutrition, we develop a working mechanistic model of how cumulative diet-induced and pathogen-triggered microbial perturbations result in an increasingly wasted host.

Systems-level metabolism of the altered Schaedler flora, a complete gut microbiota.

The altered Schaedler flora (ASF) is a model microbial community with both in vivo and in vitro relevance. Here we provide the first characterization of the ASF community in vitro, independent of a murine host. We compared the functional genetic content of the ASF to wild murine metagenomes and found that the ASF functionally represents wild microbiomes better than random consortia of similar taxonomic composition. We developed a chemically defined medium that supported growth of seven of the eight ASF members. To elucidate the metabolic capabilities of these ASF species-including potential for interactions such as cross-feeding-we performed a spent media screen and analyzed the results through dynamic growth measurements and non-targeted metabolic profiling. We found that cross-feeding is relatively rare (32 of 3570 possible cases), but is enriched between Clostridium ASF356 and Parabacteroides ASF519. We identified many cases of emergent metabolism (856 of 3570 possible cases). These data will inform efforts to understand ASF dynamics and spatial distribution in vivo, to design pre- and probiotics that modulate relative abundances of ASF members, and will be essential for validating computational models of ASF metabolism. Well-characterized, experimentally tractable microbial communities enable research that can translate into more effective microbiome-targeted therapies to improve human health.

Metabolic network modeling of microbial communities.

Genome-scale metabolic network reconstructions and constraint-based analyses are powerful methods that have the potential to make functional predictions about microbial communities. Genome-scale metabolic networks are used to characterize the metabolic functions of microbial communities via several techniques including species compartmentalization, separating species-level and community-level objectives, dynamic analysis, the 'enzyme-soup' approach, multiscale modeling, and others. There are many challenges in the field, including a need for tools that accurately assign high-level omics signals to individual community members, the need for improved automated network reconstruction methods, and novel algorithms for integrating omics data and engineering communities. As technologies and modeling frameworks improve, we expect that there will be corresponding advances in the fields of ecology, health science, and microbial community engineering.