Identifying metabolic adaptations characteristic of cardiotoxicity using paired transcriptomics and metabolomics data integrated with a computational model of heart metabolism.

Improvements in the diagnosis and treatment of cancer have revealed long-term side effects of chemotherapeutics, particularly cardiotoxicity. Here, we present paired transcriptomics and metabolomics data characterizing in vitro cardiotoxicity to three compounds: 5-fluorouracil, acetaminophen, and doxorubicin. Standard gene enrichment and metabolomics approaches identify some commonly affected pathways and metabolites but are not able to readily identify metabolic adaptations in response to cardiotoxicity. The paired data was integrated with a genome-scale metabolic network reconstruction of the heart to identify shifted metabolic functions, unique metabolic reactions, and changes in flux in metabolic reactions in response to these compounds. Using this approach, we confirm previously seen changes in the p53 pathway by doxorubicin and RNA synthesis by 5-fluorouracil, we find evidence for an increase in phospholipid metabolism in response to acetaminophen, and we see a shift in central carbon metabolism suggesting an increase in metabolic demand after treatment with doxorubicin and 5-fluorouracil.

Multidimensional Clinical Surveillance of Pseudomonas aeruginosa Reveals Complex Relationships between Isolate Source, Morphology, and Antimicrobial Resistance.

Antimicrobial susceptibility in Pseudomonas aeruginosa is dependent on a complex combination of host and pathogen-specific factors. Through the profiling of 971 clinical P. aeruginosa isolates from 590 patients and collection of paired patient metadata, we show that antimicrobial resistance is associated with not only patient-centric factors (e.g., cystic fibrosis and antipseudomonal prescription history) but also microbe-specific phenotypes (e.g., mucoid colony morphology). Additionally, isolates from different sources (e.g., respiratory tract, urinary tract) displayed rates of antimicrobial resistance that were correlated with source-specific antimicrobial prescription strategies. Furthermore, isolates from the same patient often displayed a high degree of heterogeneity, highlighting a key challenge facing personalized treatment of infectious diseases. Our findings support novel relationships between isolate and patient-level data sets, providing a potential guide for future antimicrobial treatment strategies. IMPORTANCE P. aeruginosa is a leading cause of nosocomial infection and infection in patients with cystic fibrosis. While P. aeruginosa infection and treatment can be complicated by a variety of antimicrobial resistance and virulence mechanisms, pathogen virulence is rarely recorded in a clinical setting. In this study, we discovered novel relationships between antimicrobial resistance, virulence-linked morphologies, and isolate source in a large and variable collection of clinical P. aeruginosa isolates. Our work motivates the clinical surveillance of virulence-linked P. aeruginosa morphologies as well as the tracking of source-specific antimicrobial prescription and resistance patterns.

Untargeted Metabolomics Reveals Species-Specific Metabolite Production and Shared Nutrient Consumption by Pseudomonas aeruginosa and Staphylococcus aureus.

While bacterial metabolism is known to impact antibiotic efficacy and virulence, the metabolic capacities of individual microbes in cystic fibrosis lung infections are difficult to disentangle from sputum samples. Here, we show that untargeted metabolomic profiling of supernatants of multiple strains of Pseudomonas aeruginosa and Staphylococcus aureus grown in monoculture in synthetic cystic fibrosis media (SCFM) reveals distinct species-specific metabolic signatures despite intraspecies metabolic variability. We identify a set of 15 metabolites that were significantly consumed by both P. aeruginosa and S. aureus, suggesting that nutrient competition has the potential to impact community dynamics even in the absence of other pathogen-pathogen interactions. Finally, metabolites that were uniquely produced by one species or the other were identified. Specifically, the virulence factor precursor anthranilic acid, as well as the quinoline 2,4-quinolinediol (DHQ), were robustly produced across all tested strains of P. aeruginosa. Through the direct comparison of the extracellular metabolism of P. aeruginosa and S. aureus in a physiologically relevant environment, this work provides insight toward the potential for metabolic interactions in vivo and supports the development of species-specific diagnostic markers of infection. IMPORTANCE Interactions between P. aeruginosa and S. aureus can impact pathogenicity and antimicrobial efficacy. In this study, we aim to better understand the potential for metabolic interactions between P. aeruginosa and S. aureus in an environment resembling the cystic fibrosis lung. We find that S. aureus and P. aeruginosa consume many of the same nutrients, suggesting that metabolic competition may play an important role in community dynamics during coinfection. We further identify metabolites uniquely produced by either organism with the potential to be developed into species-specific biomarkers of infection in the cystic fibrosis lung.

Predicting changes in renal metabolism after compound exposure with a genome-scale metabolic model.

The kidneys are metabolically active organs with importance in several physiological tasks such as the secretion of soluble wastes into the urine and synthesizing glucose and oxidizing fatty acids for energy in fasting (non-fed) conditions. Once damaged, the metabolic capability of the kidneys becomes altered. Here, we define metabolic tasks in a computational modeling framework to capture kidney function in an update to the iRno network reconstruction of rat metabolism using literature-based evidence. To demonstrate the utility of iRno for predicting kidney function, we exposed primary rat renal proximal tubule epithelial cells to four compounds with varying levels of nephrotoxicity (acetaminophen, gentamicin, 2,3,7,8-tetrachlorodibenzodioxin, and trichloroethylene) for six and twenty-four hours, and collected transcriptomics and metabolomics data to measure the metabolic effects of compound exposure. For the transcriptomics data, we observed changes in fatty acid metabolism and amino acid metabolism, as well as changes in existing markers of kidney function such as Clu (clusterin). The iRno metabolic network reconstruction was used to predict alterations in these same pathways after integrating transcriptomics data and was able to distinguish between select compound-specific effects on the proximal tubule epithelial cells. Genome-scale metabolic network reconstructions with coupled omics data can be used to predict changes in metabolism as a step towards identifying novel metabolic biomarkers of kidney function and dysfunction.

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.

Increased Urinary Trimethylamine N-Oxide Following Cryptosporidium Infection and Protein Malnutrition Independent of Microbiome Effects.

Cryptosporidium infections have been associated with growth stunting, even in the absence of diarrhea. Having previously detailed the effects of protein deficiency on both microbiome and metabolome in this model, we now describe the specific gut microbial and biochemical effects of Cryptosporidium infection. Protein-deficient mice were infected with Cryptosporidium parvum oocysts for 6-13 days and compared with uninfected controls. Following infection, there was an increase in the urinary excretion of choline- and amino-acid-derived metabolites. Conversely, infection reduced the excretion of the microbial-host cometabolite (3-hydroxyphenyl)propionate-sulfate and disrupted metabolites involved in the tricarboxylic acid (TCA) cycle. Correlation analysis of microbial and biochemical profiles resulted in associations between various microbiota members and TCA cycle metabolites, as well as some microbial-specific degradation products. However, no correlation was observed between the majority of the infection-associated metabolites and the fecal bacteria, suggesting that these biochemical perturbations are independent of concurrent changes in the relative abundance of members of the microbiota. We conclude that cryptosporidial infection in protein-deficient mice can mimic some metabolic changes seen in malnourished children and may help elucidate our understanding of long-term metabolic consequences of early childhood enteric infections.

Protein- and zinc-deficient diets modulate the murine microbiome and metabolic phenotype.

BACKGROUND: Environmental enteropathy, which is linked to undernutrition and chronic infections, affects the physical and mental growth of children in developing areas worldwide. Key to understanding how these factors combine to shape developmental outcomes is to first understand the effects of nutritional deficiencies on the mammalian system including the effect on the gut microbiota. OBJECTIVE: We dissected the nutritional components of environmental enteropathy by analyzing the specific metabolic and gut-microbiota changes that occur in weaned-mouse models of zinc or protein deficiency compared with well-nourished controls. DESIGN: With the use of a 1H nuclear magnetic resonance spectroscopy-based metabolic profiling approach with matching 16S microbiota analyses, the metabolic consequences and specific effects on the fecal microbiota of protein and zinc deficiency were probed independently in a murine model. RESULTS: We showed considerable shifts within the intestinal microbiota 14-24 d postweaning in mice that were maintained on a normal diet (including increases in Proteobacteria and striking decreases in Bacterioidetes). Although the zinc-deficient microbiota were comparable to the age-matched, well-nourished profile, the protein-restricted microbiota remained closer in composition to the weaned enterotype with retention of Bacteroidetes. Striking increases in Verrucomicrobia (predominantly Akkermansia muciniphila) were observed in both well-nourished and protein-deficient mice 14 d postweaning. We showed that protein malnutrition impaired growth and had major metabolic consequences (much more than with zinc deficiency) that included altered energy, polyamine, and purine and pyrimidine metabolism. Consistent with major changes in the gut microbiota, reductions in microbial proteolysis and increases in microbial dietary choline processing were observed. CONCLUSIONS: These findings are consistent with metabolic alterations that we previously observed in malnourished children. The results show that we can model the metabolic consequences of malnutrition in the mouse to help dissect relevant pathways involved in the effects of undernutrition and their contribution to environmental enteric dysfunction.

Zinc deficiency alters host response and pathogen virulence in a mouse model of enteroaggregative Escherichia coli-induced diarrhea.

Enteroaggregative Escherichia coli (EAEC) is increasingly recognized as a major cause of diarrheal disease globally. In the current study, we investigated the impact of zinc deficiency on the host and pathogenesis of EAEC. Several outcomes of EAEC infection were investigated including weight loss, EAEC shedding and tissue burden, leukocyte recruitment, intestinal cytokine expression, and virulence expression of the pathogen in vivo. Mice fed a protein source defined zinc deficient diet (dZD) had an 80% reduction of serum zinc and a 50% reduction of zinc in luminal contents of the bowel compared to mice fed a protein source defined control diet (dC). When challenged with EAEC, dZD mice had significantly greater weight loss, stool shedding, mucus production, and, most notably, diarrhea compared to dC mice. Zinc deficient mice had reduced infiltration of leukocytes into the ileum in response to infection suggesting an impaired immune response. Interestingly, expression of several EAEC virulence factors were increased in luminal contents of dZD mice. These data show a dual effect of dietary zinc in benefitting the host while impairing virulence of the pathogen. The study demonstrates the critical importance of zinc and may help elucidate the benefits of zinc supplementation in cases of childhood diarrhea and malnutrition.

The micronutrient zinc inhibits EAEC strain 042 adherence, biofilm formation, virulence gene expression, and epithelial cytokine responses benefiting the infected host.

Enteroaggregative Escherichia coli (EAEC) is a major pathogen worldwide, associated with diarrheal disease in both children and adults, suggesting the need for new preventive and therapeutic treatments. We investigated the role of the micronutrient zinc in the pathogenesis of an E. coli strain associated with human disease. A variety of bacterial characteristics-growth in vitro, biofilm formation, adherence to IEC-6 epithelial cells, gene expression of putative EAEC virulence factors as well as EAEC-induced cytokine expression by HCT-8 cells-were quantified. At concentrations (≤ 0.05 mM) that did not alter EAEC growth (strain 042) but that are physiologic in serum, zinc markedly decreased the organism's ability to form biofilm (P<0.001), adhere to IEC-6 epithelial cells (P<0.01), and express putative EAEC virulence factors (aggR, aap, aatA, virK) (P<0.03). After exposure of the organism to zinc, the effect on virulence factor generation was prolonged (> 3 h). Further, EAEC-induced IL-8 mRNA and protein secretion by HCT-8 epithelial cells were significantly reduced by 0.05 mM zinc (P<0.03). Using an in vivo murine model of diet-induced zinc-deficiency, oral zinc supplementation (0.4 µg/mouse daily) administered after EAEC challenge (10 (10) CFU/mouse) significantly abrogated growth shortfalls (by>90%; P<0.01); furthermore, stool shedding was reduced (days 9-11) but tissue burden of organisms in the intestine was unchanged. These findings suggest several potential mechanisms whereby physiological levels of zinc alter pathogenetic events in the bacterium (reducing biofilm formation, adherence to epithelium, virulence factor expression) as well as the bacterium's effect on the epithelium (cytokine response to exposure to EAEC) to alter EAEC pathogenesis in vitro and in vivo. These effects may help explain and extend the benefits of zinc in childhood diarrhea and malnutrition.

Systems analysis of the transcriptional response of human ileocecal epithelial cells to Clostridium difficile toxins and effects on cell cycle control.

BACKGROUND: Toxins A and B (TcdA and TcdB) are Clostridium difficile's principal virulence factors, yet the pathways by which they lead to inflammation and severe diarrhea remain unclear. Also, the relative role of either toxin during infection and the differences in their effects across cell lines is still poorly understood. To better understand their effects in a susceptible cell line, we analyzed the transciptome-wide gene expression response of human ileocecal epithelial cells (HCT-8) after 2, 6, and 24 hr of toxin exposure. RESULTS: We show that toxins elicit very similar changes in the gene expression of HCT-8 cells, with the TcdB response occurring sooner. The high similarity suggests differences between toxins are due to events beyond transcription of a single cell-type and that their relative potencies during infection may depend on differential effects across cell types within the intestine. We next performed an enrichment analysis to determine biological functions associated with changes in transcription. Differentially expressed genes were associated with response to external stimuli and apoptotic mechanisms and, at 24 hr, were predominately associated with cell-cycle control and DNA replication. To validate our systems approach, we subsequently verified a novel G1/S and known G2/M cell-cycle block and increased apoptosis as predicted from our enrichment analysis. CONCLUSIONS: This study shows a successful example of a workflow deriving novel biological insight from transcriptome-wide gene expression. Importantly, we do not find any significant difference between TcdA and TcdB besides potency or kinetics. The role of each toxin in the inhibition of cell growth and proliferation, an important function of cells in the intestinal epithelium, is characterized.

Shiga toxin 2 targets the murine renal collecting duct epithelium.

Hemolytic-uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli infection is a leading cause of pediatric acute renal failure. Bacterial toxins produced in the gut enter the circulation and cause a systemic toxemia and targeted cell damage. It had been previously shown that injection of Shiga toxin 2 (Stx2) and lipopolysaccharide (LPS) caused signs and symptoms of HUS in mice, but the mechanism leading to renal failure remained uncharacterized. The current study elucidated that murine cells of the glomerular filtration barrier were unresponsive to Stx2 because they lacked the receptor glycosphingolipid globotriaosylceramide (Gb(3)) in vitro and in vivo. In contrast to the analogous human cells, Stx2 did not alter inflammatory kinase activity, cytokine release, or cell viability of the murine glomerular cells. However, murine renal cortical and medullary tubular cells expressed Gb(3) and responded to Stx2 by undergoing apoptosis. Stx2-induced loss of functioning collecting ducts in vivo caused production of increased dilute urine, resulted in dehydration, and contributed to renal failure. Stx2-mediated renal dysfunction was ameliorated by administration of the nonselective caspase inhibitor Q-VD-OPH in vivo. Stx2 therefore targets the murine collecting duct, and this Stx2-induced injury can be blocked by inhibitors of apoptosis in vivo.

p38 mitogen-activated protein kinase mediates lipopolysaccharide and tumor necrosis factor alpha induction of shiga toxin 2 sensitivity in human umbilical vein endothelial cells.

Escherichia coli O157:H7 Shiga toxin 2 (Stx2), one of the causative agents of hemolytic-uremic syndrome, is toxic to endothelial cells, including primary cultured human umbilical vein endothelial cells (HUVEC). This sensitivity of cells to Stx2 can be increased with either lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-alpha). The goal of the present study was to identify the intracellular signaling pathway(s) by which LPS and TNF-alpha sensitize HUVEC to the cytotoxic effects of Stx2. To identify these pathways, specific pharmacological inhibitors and small interfering RNAs were tested with cell viability endpoints. A time course and dose response experiment for HUVEC exposure to LPS and TNF-alpha showed that a relatively short exposure to either agonist was sufficient to sensitize the cells to Stx2 and that both agonists stimulated intracellular signaling pathways within a short time. Cell viability assays indicated that the p38 mitogen-activated protein kinase (MAPK) inhibitors SB202190 and SB203580 and the general protein synthesis inhibitor cycloheximide inhibited both the LPS and TNF-alpha sensitization of HUVEC to Stx2, while all other inhibitors tested did not inhibit this sensitization. Additionally, SB202190 reduced the cellular globotriaosylceramide content under LPS- and TNF-alpha-induced conditions. In conclusion, our results show that LPS and TNF-alpha induction of Stx2 sensitivity in HUVEC is mediated through a pathway that includes p38 MAPK. These results indicate that inhibition of p38 MAPK in endothelial cells may protect a host from the deleterious effects of Stx2.