Deep longitudinal multi-omics analysis of Bordetella pertussis cultivated in bioreactors highlights medium starvations and transitory metabolisms, associated to vaccine antigen biosynthesis variations and global virulence regulation.

Bordetella pertussis is the bacterial causative agent of whooping cough, a serious respiratory illness. An extensive knowledge on its virulence regulation and metabolism is a key factor to ensure pertussis vaccine manufacturing process robustness. The aim of this study was to refine our comprehension of B. pertussis physiology during in vitro cultures in bioreactors. A longitudinal multi-omics analysis was carried out over 26 h small-scale cultures of B. pertussis. Cultures were performed in batch mode and under culture conditions intending to mimic industrial processes. Putative cysteine and proline starvations were, respectively, observed at the beginning of the exponential phase (from 4 to 8 h) and during the exponential phase (18 h 45 min). As revealed by multi-omics analyses, the proline starvation induced major molecular changes, including a transient metabolism with internal stock consumption. In the meantime, growth and specific total PT, PRN, and Fim2 antigen productions were negatively affected. Interestingly, the master virulence-regulating two-component system of B. pertussis (BvgASR) was not evidenced as the sole virulence regulator in this in vitro growth condition. Indeed, novel intermediate regulators were identified as putatively involved in the expression of some virulence-activated genes (vags). Such longitudinal multi-omics analysis applied to B. pertussis culture process emerges as a powerful tool for characterization and incremental optimization of vaccine antigen production.

A standardized gnotobiotic mouse model harboring a minimal 15-member mouse gut microbiota recapitulates SOPF/SPF phenotypes.

Mus musculus is the classic mammalian model for biomedical research. Despite global efforts to standardize breeding and experimental procedures, the undefined composition and interindividual diversity of the microbiota of laboratory mice remains a limitation. In an attempt to standardize the gut microbiome in preclinical mouse studies, here we report the development of a simplified mouse microbiota composed of 15 strains from 7 of the 20 most prevalent bacterial families representative of the fecal microbiota of C57BL/6J Specific (and Opportunistic) Pathogen-Free (SPF/SOPF) animals and the derivation of a standardized gnotobiotic mouse model called GM15. GM15 recapitulates extensively the functionalities found in the C57BL/6J SOPF microbiota metagenome, and GM15 animals are phenotypically similar to SOPF or SPF animals in two different facilities. They are also less sensitive to the deleterious effects of post-weaning malnutrition. In this work, we show that the GM15 model provides increased reproducibility and robustness of preclinical studies by limiting the confounding effect of fluctuation in microbiota composition, and offers opportunities for research focused on how the microbiota shapes host physiology in health and disease.

An integrated transcriptomics and metabolomics study of the immune response of newly hatched chicks to the cytosine-phosphate-guanine oligonucleotide stimulation.

The immunological immaturity of the innate immune system during the first-week post-hatch enables pathogens to infect chickens, leading to the death of the animals. Current preventive solutions to improve the resistance of chicks to infections include vaccination, breeding, and sanitation. Other prophylactic solutions have been investigated, such as the stimulation of animal health with immunostimulants. Recent studies showed that administration of immune-modulators to one-day-old chicks, or in ovo, significantly reduces mortality in experimental bacterial or viral infection challenge models. Owing to a lack of molecular biomarkers required to evaluate chicken immune responses and assess the efficacy of vaccines or immune-modulators, challenge models are still used. One way to reduce challenge experiments is to define molecular signatures through omics approaches, resulting in new methodologies to rapidly screen candidate molecules or vaccines. This study aims at identifying a dual transcriptomics and metabolomics blood signature after administration of CpG-ODN (cytosine-phosphate-guanine oligodeoxynucleotides), a reference immune-stimulatory molecule. A clinical study was conducted with chicks and transcriptomics and metabolomics analyses were performed on whole-blood and plasma samples, respectively. Differentially expressed genes and metabolites with different abundance were identified in chicks treated with CpG-ODN. The results showed that CpG-ODN activated the innate immune system, within hours after administration, and its effect lasted over time, as metabolomics and transcriptomics profiles still varied 6 D after administration. In conclusion, through an integrated clinical omics approach, we deciphered in part the mode of action of CpG-ODN in post-hatch chicks.

Metabolomics-on-a-chip of hepatotoxicity induced by anticancer drug flutamide and Its active metabolite hydroxyflutamide using HepG2/C3a microfluidic biochips.

We used the recently introduced "metabolomics-on-a-chip" approach to test secondary drug toxicity in bioartificial organs. Bioartificial organs cultivated in microfluidic culture conditions provide a beneficial environment, in which the cellular cytoprotective mechanisms are enhanced, compared with Petri dish culture conditions. We investigated the metabolic response of HepG2/C3a cells exposed to flutamide, an anticancer prodrug, and hydroxyflutamide (HF), its active metabolite, in a microfluidic biochip. The cellular response was analyzed by (1)H nuclear magnetic resonance spectroscopy to identify cell-specific molecule-response markers. The metabolic response to flutamide results in a disruption of glucose homeostasis and in mitochondrial dysfunctions. This flutamide-specific metabolic response was illustrated by a reduction of the extracellular glucose and fructose consumptions and a general reduction of the tricarboxylic acid cycle activity leading to the reduction of the consumption of several amino acids. We also found a higher production of 3-hydroxybutyrate and lactate, and the reduction of the albumin production compared with controls. The toxic metabolic signature associated with the active metabolite HF was illustrated by a high-energy demand and an increase in several amino acid metabolism. Finally, for both molecules, the hepatotoxicity was correlated to the glutathione (GSH) metabolism illustrated by the levels of the 2-hydroxybutyrate and pyroglutamate productions and the increase of the glutamate and glycine productions. Thus, the entire set of results contributed to extract specific mechanistic toxic signatures and their relation to hepatotoxicity, which appeared consistent with literature reports. As new finding of HepG2/C3a cells hepatotoxicity, we propose a metabolic network with a related list of metabolite variations to describe the GSH depletion when followed by a cell death for the HepG2/C3a cells cultivated in our polydimethylsiloxane microfluidic biochips. Our findings illustrate the potential of metabolomics-on-a-chip as an in vitro alternative method for predictive toxicology.

Metabolomics-on-a-chip and metabolic flux analysis for label-free modeling of the internal metabolism of HepG2/C3A cells.

In vitro microfluidic systems are increasingly used as an alternative to standard Petri dishes in bioengineering and metabolomic investigations, as they are expected to provide cellular environments close to the in vivo conditions. In this work, we combined the recently developed "metabolomics-on-a-chip" approach with metabolic flux analysis to model the metabolic network of the hepatoma HepG2/C3A cell line and to infer the distribution of intracellular metabolic fluxes in standard Petri dishes and microfluidic biochips. A high pyruvate reduction to lactate was observed in both systems, suggesting that the cells operate in oxygen-limited environments. Our results also indicate that HepG2/C3A cells in the biochip are characterized by a higher consumption rate of oxygen, presumably due to a higher oxygenation rate in the microfluidic environment. This leads to a higher entry of the ultimate glycolytic product, acetyl-CoA, into the Krebs cycle. These findings are supported by the transcriptional activity of HepG2/C3A cells in both systems since we observed that genes regulated by a HIF-1 (hypoxia-regulated factor-1) transcriptional factor were over expressed under the Petri conditions, but to a lesser extent in the biochip.

Predictive toxicology using systemic biology and liver microfluidic "on chip" approaches: application to acetaminophen injury.

We have analyzed transcriptomic, proteomic and metabolomic profiles of hepatoma cells cultivated inside a microfluidic biochip with or without acetaminophen (APAP). Without APAP, the results show an adaptive cellular response to the microfluidic environment, leading to the induction of anti-oxidative stress and cytoprotective pathways. In presence of APAP, calcium homeostasis perturbation, lipid peroxidation and cell death are observed. These effects can be attributed to APAP metabolism into its highly reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI). That toxicity pathway was confirmed by the detection of GSH-APAP, the large production of 2-hydroxybutyrate and 3-hydroxybutyrate, and methionine, cystine, and histidine consumption in the treated biochips. Those metabolites have been reported as specific biomarkers of hepatotoxicity and glutathione depletion in the literature. In addition, the integration of the metabolomic, transcriptomic and proteomic collected profiles allowed a more complete reconstruction of the APAP injury pathways. To our knowledge, this work is the first example of a global integration of microfluidic biochip data in toxicity assessment. Our results demonstrate the potential of that new approach to predictive toxicology.

In vivo proton HR-MAS NMR metabolic profile of the freshwater cladoceran Daphnia magna.

The method concerning in vivo proton HR-MAS NMR metabolic profiling of the freshwater cladoceran Daphnia magna is presented. Viability tests of D. magna under different spinning rates were performed. All surviving daphnids after analysis have developed eggs and embryos like control animals. Better survival rate at the slowest rotation speed were observed. The maximum length of analysis during which the integrity of the daphnid is maintained was assessed. The recorded proton spectra of in vivo daphnia were attributed to lipids from the triglycerol category. Saturated and unsaturated omega-3 like fatty acid moieties of triacylglycerol were well identified. The relationship between physiological state of daphnids and lipid profile are discussed.

Fate of the nitrilotriacetic acid-Fe(III) complex during photodegradation and biodegradation by Rhodococcus rhodochrous.

Aminopolycarboxylic acids are ubiquitous in natural waters and wastewaters. They have the ability to form very stable water-soluble complexes with many metallic di- or trivalent ions. The iron complex nitrilotriacetic acid-Fe(III) (FeNTA) has been previously shown to increase drastically the rate of photo- and biodegradation of 2-aminobenzothiazole, an organic pollutant, by Rhodococcus rhodochrous. For this paper, the fate of FeNTA was investigated during these degradation processes. First, it was shown, using in situ (1)H nuclear magnetic resonance, that the complex FeNTA was biodegraded by Rhodococcus rhodochrous cells, but the ligand (NTA) alone was not. This result indicates that FeNTA was transported and biotransformed inside the cell. The same products, including iminodiacetic acid, glycine, and formate, were obtained during the photo- and biodegradation processes of FeNTA, likely because they both involve oxidoreduction mechanisms. When the results of the different experiments are compared, the soluble iron, measured by spectrophotometry, was decreasing when microbial cells were present. About 20% of the initial iron was found inside the cells. These results allowed us to propose detailed mechanistic schemes for FeNTA degradation by solar light and by R. rhodochrous.

Comparison of microbial and photochemical processes and their combination for degradation of 2-aminobenzothiazole.

The transformation of 2-aminobenzothiazole (ABT) was studied under various conditions: (i) a photodegradation process at a lambda of >300 nm in the presence of an Fe(III)-nitrilotriacetic acid complex (FeNTA), (ii) a biodegradation process using Rhodococcus rhodochrous OBT18 cells, and (iii) the combined processes (FeNTA plus Rhodococcus rhodochrous in the presence or absence of light). The transformation of ABT in the combined system, with or without light, was much more efficient (99% degradation after 25 h) than in the separated systems (37% photodegradation and 26% biodegradation after 125 h). No direct photolysis of ABT was observed. The main result seen is the strong positive impact of FeNTA on the photodegradation, as expected, and on the biotransformation efficiency of ABT, which was more surprising. This positive impact of FeNTA on the microbial metabolism was dependent on the FeNTA concentration. The use of UV high-performance liquid chromatography, liquid chromatography-electrospray ionization mass spectrometry, and in situ (1)H nuclear magnetic resonance provided evidence of the intermediary products and thus established transformation pathways of ABT in the different processes. These pathways were identical whether the degradation process was photo- or biotransformation. A new photoproduct was identified (4OH-ABT), corresponding to a hydroxylation reaction on position 4 of the aromatic ring of ABT.